79 results on '"Bruner SD"'
Search Results
2. Correction to "Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases".
- Author
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Van Gelder K, Oliveira-Filho ER, García-García JD, Hu Y, Bruner SD, and Hanson AD
- Published
- 2024
- Full Text
- View/download PDF
3. Biosynthesis of Dolastatin 10 in Marine Cyanobacteria, a Prototype for Multiple Approved Cancer Drugs.
- Author
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Kallifidas D, Dhakal D, Chen M, Chen QY, Kokkaliari S, Colon Rosa NA, Ratnayake R, Bruner SD, Paul VJ, Ding Y, and Luesch H
- Subjects
- Oligopeptides chemistry, Depsipeptides pharmacology, Depsipeptides chemistry, Antineoplastic Agents pharmacology, Antineoplastic Agents chemistry, Cyanobacteria chemistry, Neoplasms
- Abstract
Dolastatin 10, a potent tubulin-targeting marine anticancer natural product, provided the basis for the development of six FDA-approved antibody-drug conjugates. Through the screening of cyanobacterial Caldora penicillata environmental DNA libraries and metagenome sequencing, we identified its biosynthetic gene cluster. Functional prediction of 10 enzymes encoded in the 39 kb cluster supports the dolastatin 10 biosynthesis. The nonheme diiron monooxygenase DolJ was biochemically characterized to mediate the terminal thiazole formation in dolastatin 10.
- Published
- 2024
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4. Alternative Linkage Chemistries in the Chemoenzymatic Synthesis of Microviridin-Based Cyclic Peptides.
- Author
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Patel KP, Chen WT, Delbecq L, and Bruner SD
- Subjects
- Peptide Hydrolases, Peptides, Cyclic chemistry, Peptides chemistry
- Abstract
Engineering biosynthetic pathways to ribosomally synthesized and post-translationally modified peptides (RiPPs) offers several advantages for both in vivo and in vitro applications. Here we probe the ability of peptide cyclases to generate trimacrocycle microviridin analogs with non-native cross-links. The results demonstrate that diverse chemistries are tolerated by macrocyclases in the ATP-grasp family and allow for the construction of unique cyclic peptide architectures that retain protease inhibition activity. In addition, cocomplex structures of analogs bound to a model protease were determined, illustrating how changes in functional groups maintain peptide conformation and target binding.
- Published
- 2024
- Full Text
- View/download PDF
5. Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin.
- Author
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Tripathi P, Mousa JJ, Guntaka NS, and Bruner SD
- Subjects
- Humans, Amidohydrolases, Mutagens metabolism, Escherichia coli genetics
- Abstract
Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS-PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α-aminoketone and β-ketothioester intermediates attached to separate carrier domains on the NRPS-PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier-protein substrates. Modeling protein-protein interactions supports a predicted molecular basis for enzyme-carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin., (open access.)
- Published
- 2023
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6. Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases.
- Author
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Gelder KV, Oliveira-Filho ER, García-García JD, Hu Y, Bruner SD, and Hanson AD
- Subjects
- Thiamine metabolism, Saccharomyces cerevisiae metabolism, Plants metabolism, Nitric Oxide Synthase metabolism, Sulfides metabolism, Thiazoles chemistry, Thiazoles metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes ( k
cat < 1 h-1 ). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Seven beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants.- Published
- 2023
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7. Metabolite Damage and Damage Control in a Minimal Genome.
- Author
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Haas D, Thamm AM, Sun J, Huang L, Sun L, Beaudoin GAW, Wise KS, Lerma-Ortiz C, Bruner SD, Breuer M, Luthey-Schulten Z, Lin J, Wilson MA, Brown G, Yakunin AF, Kurilyak I, Folz J, Fiehn O, Glass JI, Hanson AD, Henry CS, and de Crécy-Lagard V
- Subjects
- Oxidoreductases, Genome, Bacterial, Metabolomics methods
- Abstract
Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes known to have such functions were identified and experimentally validated, including 5-formyltetrahydrofolate cycloligase, coenzyme A (CoA) disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal. IMPORTANCE Metabolite damage and repair mechanisms are being increasingly recognized. We present here compelling genetic and biochemical evidence for the universal importance of these mechanisms by demonstrating that stripping a genome down to its barest essentials leaves metabolite damage control systems in place. Furthermore, our metabolomic and cheminformatic results point to the existence of a network of metabolite damage and damage control reactions that extends far beyond the corners of it that have been characterized so far. In sum, there can be little room left to doubt that metabolite damage and the systems that counter it are mainstream metabolic processes that cannot be separated from life itself.
- Published
- 2022
- Full Text
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8. Structural and biochemical studies of an iterative ribosomal peptide macrocyclase.
- Author
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Li G, Patel K, Zhang Y, Pugmire JK, Ding Y, and Bruner SD
- Subjects
- Amino Acid Sequence, Benchmarking, Biosynthetic Pathways, Catalysis, Crystallography, X-Ray, Cyclization, Models, Molecular, Peptide Biosynthesis, Protein Binding, Protein Conformation, Protein Processing, Post-Translational, Biological Products chemistry, Cyanobacteria metabolism, Peptides, Cyclic chemistry, Ribosomes metabolism
- Abstract
Microviridins, tricyclic peptide natural products originally isolated from cyanobacteria, function as inhibitors of diverse serine-type proteases. Here we report the structure and biochemical characterization of AMdnB, a unique iterative macrocyclase involved in a microviridin biosynthetic pathway from Anabaena sp. PCC 7120. The ATP-dependent cyclase, along with the homologous AMdnC, introduce up to nine macrocyclizations on three distinct core regions of a precursor peptide, AMdnA. The results presented here provide structural and mechanistic insight into the iterative chemistry of AMdnB. In vitro AMdnB-catalyzed cyclization reactions demonstrate the synthesis of the two predicted tricyclic products from a multi-core precursor peptide substrate, consistent with a distributive mode of catalysis. The X-ray structure of AMdnB shows a structural motif common to ATP-grasp cyclases involved in RiPPs biosynthesis. Additionally, comparison with the noniterative MdnB allows insight into the structural basis for the iterative chemistry. Overall, the presented results provide insight into the general mechanism of iterative enzymes in ribosomally synthesized and post-translationally modified peptide biosynthetic pathways., (© 2021 Wiley Periodicals LLC.)
- Published
- 2022
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9. Biochemical and structural characterization of Haemophilus influenzae nitroreductase in metabolizing nitroimidazoles.
- Author
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Liu D, Wanniarachchi TN, Jiang G, Seabra G, Cao S, Bruner SD, and Ding Y
- Abstract
Nitroheterocycle antibiotics, particularly 5-nitroimidazoles, are frequently used for treating anaerobic infections. The antimicrobial activities of these drugs heavily rely on the in vivo bioactivation, mainly mediated by widely distributed bacterial nitroreductases (NTRs). However, the bioactivation can also lead to severe toxicities and drug resistance. Mechanistic understanding of NTR-mediated 5-nitroimidazole metabolism can potentially aid addressing these issues. Here, we report the metabolism of structurally diverse nitroimidazole drug molecules by a NTR from a human pathogen Haemophilus influenzae (HiNfsB). Our detailed bioinformatic analysis uncovered that HiNfsB represents a group of unexplored oxygen-insensitive NTRs. Biochemical characterization of the recombinant enzyme revealed that HiNfsB effectively metabolizes ten clinically used nitroimidazoles. Furthermore, HiNfsB generated not only canonical nitroreduction metabolites but also stable, novel dimeric products from three nitroimidazoles, whose structures were proposed based on the results of high resolution MS and tandem MS analysis. X-ray structural analysis of the enzyme coupled with site-directed mutagenesis identified four active site residues important to its catalysis and broad substrate scope. Finally, transient expression of HiNfsB sensitized an E. coli mutant strain to 5-nitroimidazoles under anaerobic conditions. Together, these results advance our understanding of the metabolism of nitroimidazole antibiotics mediated by a new NTR group and reinforce the research on the natural antibiotic resistome for addressing the antibiotic resistance crisis., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2022
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10. Using continuous directed evolution to improve enzymes for plant applications.
- Author
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García-García JD, Van Gelder K, Joshi J, Bathe U, Leong BJ, Bruner SD, Liu CC, and Hanson AD
- Subjects
- Directed Molecular Evolution methods, Enzymes genetics, Plant Breeding methods, Plant Proteins genetics, Saccharomyces cerevisiae genetics
- Abstract
Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme's activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the yeast (Saccharomyces cerevisiae) OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt ("plantize") enzymes from prokaryotes-especially exotic prokaryotes-to function well in mild, plant-like conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)
- Published
- 2022
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11. Epoxyqueuosine Reductase QueH in the Biosynthetic Pathway to tRNA Queuosine Is a Unique Metalloenzyme.
- Author
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Li Q, Zallot R, MacTavish BS, Montoya A, Payan DJ, Hu Y, Gerlt JA, Angerhofer A, de Crécy-Lagard V, and Bruner SD
- Subjects
- Catalytic Domain, Iron chemistry, Thermotoga maritima enzymology, Bacterial Proteins chemistry, Iron-Sulfur Proteins chemistry, Oxidoreductases Acting on CH-CH Group Donors chemistry
- Abstract
Queuosine is a structurally unique and functionally important tRNA modification, widely distributed in eukaryotes and bacteria. The final step of queuosine biosynthesis is the reduction/deoxygenation of epoxyqueuosine to form the cyclopentene motif of the nucleobase. The chemistry is performed by the structurally and functionally characterized cobalamin-dependent QueG. However, the queG gene is absent from several bacteria that otherwise retain queuosine biosynthesis machinery. Members of the IPR003828 family (previously known as DUF208) have been recently identified as nonorthologous replacements of QueG, and this family was renamed QueH. Here, we present the structural characterization of QueH from Thermotoga maritima . The structure reveals an unusual active site architecture with a [4Fe-4S] metallocluster along with an adjacent coordinated iron metal. The juxtaposition of the cofactor and coordinated metal ion predicts a unique mechanism for a two-electron reduction/deoxygenation of epoxyqueuosine. To support the structural characterization, in vitro biochemical and genomic analyses are presented. Overall, this work reveals new diversity in the chemistry of iron/sulfur-dependent enzymes and novel insight into the last step of this widely conserved tRNA modification.
- Published
- 2021
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12. Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases.
- Author
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Joshi J, Li Q, García-García JD, Leong BJ, Hu Y, Bruner SD, and Hanson AD
- Subjects
- Archaeal Proteins genetics, Biocatalysis, Catalytic Domain, Cobalt metabolism, Crystallization, Cysteine metabolism, Escherichia coli genetics, Escherichia coli Proteins genetics, Genomics methods, Iron metabolism, Microorganisms, Genetically-Modified, Oxygen metabolism, Saccharomyces cerevisiae genetics, Sulfides metabolism, Sulfur metabolism, Archaea enzymology, Archaeal Proteins chemistry, Archaeal Proteins metabolism, Bacteria enzymology, Escherichia coli enzymology, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Thiamine biosynthesis
- Abstract
Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized -- but only from archaeal methanogens that are anaerobic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared with the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance., (© 2021 The Author(s).)
- Published
- 2021
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13. Structure-Based Engineering of Peptide Macrocyclases for the Chemoenzymatic Synthesis of Microviridins.
- Author
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Patel KP, Silsby LM, Li G, and Bruner SD
- Subjects
- Ligases, Serine Proteinase Inhibitors, Cyanobacteria, Peptides
- Abstract
Microviridins are cyanobacterial tricyclic depsipeptides with unique ring architectures and function as serine protease inhibitors. In this study, we explore two strategies to probe the structure and mechanism of macrocyclases involved in microviridin biosynthesis. The results both provide approaches for in vitro chemoenzymatic synthesis and insight into the molecular interactions and function of the biosynthetic enzymes. The first strategy involves generating constitutively activated macrocyclases whereby the leader portion of the substrate peptide is covalently attached to the ATP-grasp ligases to examine leader peptide/enzyme interactions. The second strategy uses a structure-based design to create disulfide cross-linked peptide/enzyme complexes. Together, the strategies provide constitutively active enzymes and tools to study the catalysis of the macrocyclizations on synthetic core peptides.
- Published
- 2021
- Full Text
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14. Oxalate decarboxylase uses electron hole hopping for catalysis.
- Author
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Pastore AJ, Teo RD, Montoya A, Burg MJ, Twahir UT, Bruner SD, Beratan DN, and Angerhofer A
- Subjects
- Bacillus subtilis chemistry, Bacillus subtilis genetics, Binding Sites genetics, Carboxy-Lyases genetics, Carboxy-Lyases ultrastructure, Catalysis, Catalytic Domain genetics, Crystallography, X-Ray, Kinetics, Manganese chemistry, Oxygen chemistry, Tryptophan chemistry, Tryptophan genetics, Bacillus subtilis ultrastructure, Carboxy-Lyases chemistry, Electrons, Oxygen metabolism
- Abstract
The hexameric low-pH stress response enzyme oxalate decarboxylase catalyzes the decarboxylation of the oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 Å (intrasubunit) to 21.5 Å (intersubunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 Å. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, whereas replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wildtype tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multistep hole-hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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15. Structural Basis for the Interactions of the Colibactin Resistance Gene Product ClbS with DNA.
- Author
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Tripathi P and Bruner SD
- Subjects
- Alkylation, DNA chemistry, DNA Damage, DNA-Binding Proteins physiology, Escherichia coli genetics, Escherichia coli Proteins physiology, Mutagens metabolism, Peptides pharmacology, Polyketides pharmacology, RNA chemistry, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism
- Abstract
The natural product colibactin, along with its associated biosynthetic gene cluster, is an example system for the role microbially derived small molecules play in the human microbiome. This is particularly relevant in the human gut, where host microbiota is involved in various disorders, including colorectal cancer pathogenesis. Bacteria harboring the colibactin gene cluster induce alkylation of nucleobases in host DNA, forming interstrand cross-links both in vivo and in vitro . These lesions can lead to deleterious double-strand breaks and have been identified as the primary mechanism of colibactin-induced cytotoxicity. The gene product ClbS is one of several mechanisms utilized by the producing bacteria to maintain genome integrity. ClbS catalyzes hydrolytic inactivation of colibactin and has been shown to bind DNA, incurring self-resistance. Presented is the molecular basis for ClbS bound to a DNA oligonucleotide. The structure shows the interaction of the protein with the ends of a DNA duplex with terminal nucleotides flipped to the enzyme active site. The structure suggests an additional function for ClbS, the binding to damaged DNA followed by repair. Additionally, our study provides general insight into the function of the widely distributed and largely uncharacterized DUF1706 protein family.
- Published
- 2021
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16. Cyanobacterial Dihydroxyacid Dehydratases Are a Promising Growth Inhibition Target.
- Author
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Zhang P, MacTavish BS, Yang G, Chen M, Roh J, Newsome KR, Bruner SD, and Ding Y
- Subjects
- Catalytic Domain, Escherichia coli genetics, Escherichia coli growth & development, Iron-Sulfur Proteins metabolism, Mutation, Oxygen metabolism, Hydro-Lyases metabolism, Synechocystis enzymology, Synechocystis growth & development
- Abstract
Microbes are essential to the global ecosystem, but undesirable microbial growth causes issues ranging from food spoilage and infectious diseases to harmful cyanobacterial blooms. The use of chemicals to control microbial growth has achieved significant success, while specific roles for a majority of essential genes in growth control remain unexplored. Here, we show the growth inhibition of cyanobacterial species by targeting an essential enzyme for the biosynthesis of branched-chain amino acids. Specifically, we report the biochemical, genetic, and structural characterization of dihydroxyacid dehydratase from the model cyanobacterium Synechocystis sp. PCC 6803 (SnDHAD). Our studies suggest that SnDHAD is an oxygen-stable enzyme containing a [2Fe-2S] cluster. Furthermore, we demonstrate that SnDHAD is selectively inhibited in vitro and in vivo by the natural product aspterric acid, which also inhibits the growth of representative bloom-forming Microcystis and Anabaena strains but has minimal effects on microbial pathogens with [4Fe-4S] containing DHADs. This study suggests DHADs as a promising target for the precise growth control of microbes and highlights the exploration of other untargeted essential genes for microbial management.
- Published
- 2020
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17. Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli.
- Author
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Patterson JA, He H, Folz JS, Li Q, Wilson MA, Fiehn O, Bruner SD, Bar-Even A, and Hanson AD
- Subjects
- Aldehyde Oxidoreductases metabolism, Bacterial Proteins metabolism, Cysteine metabolism, Formaldehyde toxicity, Genes, Bacterial, Genome, Bacterial, Thiazolidines metabolism, Endopeptidases genetics, Endopeptidases metabolism, Escherichia coli genetics, Escherichia coli metabolism, Formaldehyde metabolism, Proline metabolism
- Abstract
Formaldehyde (HCHO) is a reactive carbonyl compound that formylates and cross-links proteins, DNA, and small molecules. It is of specific concern as a toxic intermediate in the design of engineered pathways involving methanol oxidation or formate reduction. The interest in engineering these pathways is not, however, matched by engineering-relevant information on precisely why HCHO is toxic or on what damage-control mechanisms cells deploy to manage HCHO toxicity. The only well-defined mechanism for managing HCHO toxicity is formaldehyde dehydrogenase-mediated oxidation to formate, which is counterproductive if HCHO is a desired pathway intermediate. We therefore sought alternative HCHO damage-control mechanisms via comparative genomic analysis. This analysis associated homologs of the Escherichia coli pepP gene with HCHO-related one-carbon metabolism. Furthermore, deleting pepP increased the sensitivity of E. coli to supplied HCHO but not other carbonyl compounds. PepP is a proline aminopeptidase that cleaves peptides of the general formula X-Pro-Y, yielding X + Pro-Y. HCHO is known to react spontaneously with cysteine to form the close proline analog thioproline (thiazolidine-4-carboxylate), which is incorporated into proteins and hence into proteolytic peptides. We therefore hypothesized that certain thioproline-containing peptides are toxic and that PepP cleaves these aberrant peptides. Supporting this hypothesis, PepP cleaved the model peptide Ala-thioproline-Ala as efficiently as Ala-Pro-Ala in vitro and in vivo, and deleting pepP increased sensitivity to supplied thioproline. Our data thus (i) provide biochemical genetic evidence that thioproline formation contributes substantially to HCHO toxicity and (ii) make PepP a candidate damage-control enzyme for engineered pathways having HCHO as an intermediate., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2020
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18. The metabolite repair enzyme Nit1 is a dual-targeted amidase that disposes of damaged glutathione in Arabidopsis .
- Author
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Niehaus TD, Patterson JA, Alexander DC, Folz JS, Pyc M, MacTavish BS, Bruner SD, Mullen RT, Fiehn O, and Hanson AD
- Subjects
- Cytoplasm enzymology, Cytoplasm genetics, Plastids enzymology, Plastids genetics, Amidohydrolases genetics, Amidohydrolases metabolism, Aminohydrolases genetics, Aminohydrolases metabolism, Arabidopsis enzymology, Arabidopsis genetics, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Glutathione metabolism
- Abstract
The tripeptide glutathione (GSH) is implicated in various crucial physiological processes including redox buffering and protection against heavy metal toxicity. GSH is abundant in plants, with reported intracellular concentrations typically in the 1-10 mM range. Various aminotransferases can inadvertently transaminate the amino group of the γ-glutamyl moiety of GSH to produce deaminated glutathione (dGSH), a metabolite damage product. It was recently reported that an amidase known as Nit1 participates in dGSH breakdown in mammals and yeast. Plants have a hitherto uncharacterized homolog of the Nit1 amidase. We show that recombinant Arabidopsis Nit1 (At4g08790) has high and specific amidase activity towards dGSH. Ablating the Arabidopsis Nit1 gene causes a massive accumulation of dGSH and other marked changes to the metabolome. All plant Nit1 sequences examined had predicted plastidial targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays show that both potential translation start codons in Arabidopsis Nit1 were used and confocal microscopy of Nit1-GFP fusions in plant cells confirmed both cytoplasmic and plastidial localization. Furthermore, we show that Arabidopsis enzymes present in leaf extracts convert GSH to dGSH at a rate of 2.8 pmol min
-1 mg-1 in the presence of glyoxalate as an amino acceptor. Our data demonstrate that plants have a dGSH repair system that is directed to at least two cellular compartments via the use of alternative translation start sites., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)- Published
- 2019
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19. Heterologous Production of Microbial Ribosomally Synthesized and Post-translationally Modified Peptides.
- Author
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Zhang Y, Chen M, Bruner SD, and Ding Y
- Abstract
Ribosomally synthesized and post-translationally modified peptides, or RiPPs, which have mainly isolated from microbes as well as plants and animals, are an ever-expanding group of peptidic natural products with diverse chemical structures and biological activities. They have emerged as a major category of secondary metabolites partly due to a myriad of microbial genome sequencing endeavors and the availability of genome mining software in the past two decades. Heterologous expression of RiPP gene clusters mined from microbial genomes, which are often silent in native producers, in surrogate hosts such as Escherichia coli and Streptomyces strains can be an effective way to elucidate encoded peptides and produce novel derivatives. Emerging strategies have been developed to facilitate the success of the heterologous expression by targeting multiple synthetic biology levels, including individual proteins, pathways, metabolic flux and hosts. This review describes recent advances in heterologous production of RiPPs, mainly from microbes, with a focus on E. coli and Streptomyces strains as the surrogate hosts.
- Published
- 2018
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20. Salvage of the 5-deoxyribose byproduct of radical SAM enzymes.
- Author
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Beaudoin GAW, Li Q, Folz J, Fiehn O, Goodsell JL, Angerhofer A, Bruner SD, and Hanson AD
- Subjects
- Aldehyde-Lyases chemistry, Aldehydes chemistry, Biological Transport, Crystallography, X-Ray, Deoxyadenosines chemistry, Escherichia coli metabolism, Gene Deletion, Isomerases chemistry, Metabolomics, Phenotype, Phosphotransferases chemistry, Protein Conformation, Ribosemonophosphates chemistry, Bacillus thuringiensis enzymology, Deoxyribose chemistry, S-Adenosylmethionine chemistry
- Abstract
5-Deoxyribose is formed from 5'-deoxyadenosine, a toxic byproduct of radical S-adenosylmethionine (SAM) enzymes. The degradative fate of 5-deoxyribose is unknown. Here, we define a salvage pathway for 5-deoxyribose in bacteria, consisting of phosphorylation, isomerization, and aldol cleavage steps. Analysis of bacterial genomes uncovers widespread, unassigned three-gene clusters specifying a putative kinase, isomerase, and sugar phosphate aldolase. We show that the enzymes encoded by the Bacillus thuringiensis cluster, acting together in vitro, convert 5-deoxyribose successively to 5-deoxyribose 1-phosphate, 5-deoxyribulose 1-phosphate, and dihydroxyacetone phosphate plus acetaldehyde. Deleting the isomerase decreases the 5-deoxyribulose 1-phosphate pool size, and deleting either the isomerase or the aldolase increases susceptibility to 5-deoxyribose. The substrate preference of the aldolase is unique among family members, and the X-ray structure reveals an unusual manganese-dependent enzyme. This work defines a salvage pathway for 5-deoxyribose, a near-universal metabolite.
- Published
- 2018
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21. Redesigning thiamin synthesis: Prospects and potential payoffs.
- Author
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Hanson AD, Amthor JS, Sun J, Niehaus TD, Gregory JF 3rd, Bruner SD, and Ding Y
- Subjects
- Plants genetics, Pyrimidines chemistry, Pyrimidines metabolism, Thiamine chemistry, Thiazoles chemistry, Thiazoles metabolism, Metabolic Engineering, Metabolic Networks and Pathways, Oxygen metabolism, Plants metabolism, Synthetic Biology, Thiamine metabolism
- Abstract
Thiamin is essential for plant growth but is short-lived in vivo and energetically very costly to produce - a combination that makes thiamin biosynthesis a prime target for improvement by redesign. Thiamin consists of thiazole and pyrimidine moieties. Its high biosynthetic cost stems from use of the suicide enzyme THI4 to form the thiazole and the near-suicide enzyme THIC to form the pyrimidine. These energetic costs lower biomass yield potential and are likely compounded by environmental stresses that destroy thiamin and hence increase the rate at which it must be made. The energy costs could be slashed by refactoring the thiamin biosynthesis pathway to eliminate the suicidal THI4 and THIC reactions. To substantiate this design concept, we first document the energetic costs of the THI4 and THIC steps in the pathway and explain how cutting these costs could substantially increase crop biomass and grain yields. We then show that a refactored pathway must produce thiamin itself rather than a stripped-down analog because the thiamin molecule cannot be simplified without losing biological activity. Lastly, we consider possible energy-efficient alternatives to the inefficient natural THI4- and THIC-mediated steps., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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22. A distributive peptide cyclase processes multiple microviridin core peptides within a single polypeptide substrate.
- Author
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Zhang Y, Li K, Yang G, McBride JL, Bruner SD, and Ding Y
- Subjects
- Amino Acid Sequence, Catalysis, Cyclization, Kinetics, Lactones chemistry, Protein Conformation, Protein Sorting Signals, Ribosomes metabolism, Substrate Specificity, Depsipeptides chemistry, Enzymes chemistry
- Abstract
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an important family of natural products. Their biosynthesis follows a common scheme in which the leader peptide of a precursor peptide guides the modifications of a single core peptide. Here we describe biochemical studies of the processing of multiple core peptides within a precursor peptide, rare in RiPP biosynthesis. In a cyanobacterial microviridin pathway, an ATP-grasp ligase, AMdnC, installs up to two macrolactones on each of the three core peptides within AMdnA. The enzyme catalysis occurs in a distributive fashion and follows an unstrict N-to-C overall directionality, but a strict order in macrolactonizing each core peptide. Furthermore, AMdnC is catalytically versatile to process unnatural substrates carrying one to four core peptides, and kinetic studies provide insights into its catalytic properties. Collectively, our results reveal a distinct biosynthetic logic of RiPPs, opening up the possibility of modular production via synthetic biology approaches.
- Published
- 2018
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23. An unusual diphosphatase from the PhnP family cleaves reactive FAD photoproducts.
- Author
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Beaudoin GAW, Li Q, Bruner SD, and Hanson AD
- Subjects
- Acidobacteria genetics, Adenosine Diphosphate Ribose metabolism, Amino Acid Motifs, Bacterial Proteins genetics, Bacterial Proteins metabolism, Catalytic Domain, Cloning, Molecular, Crystallography, X-Ray, Escherichia coli genetics, Escherichia coli metabolism, Flavin-Adenine Dinucleotide metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Kinetics, Models, Molecular, Phosphoric Diester Hydrolases genetics, Phosphoric Diester Hydrolases metabolism, Photochemical Processes, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Acidobacteria enzymology, Adenosine Diphosphate Ribose chemistry, Bacterial Proteins chemistry, Flavin-Adenine Dinucleotide chemistry, Phosphoric Diester Hydrolases chemistry
- Abstract
Flavins are notoriously photolabile, but while the photoproducts derived from the iso -alloxazine ring are well known the other photoproducts are not. In the case of FAD, typically the main cellular flavin, the other photoproducts are predicted to include four- and five-carbon sugars linked to ADP. These FAD photoproducts were shown to be potent glycating agents, more so than ADP-ribose. Such toxic compounds would require disposal via an ADP-sugar diphosphatase or other route. Comparative analysis of bacterial genomes uncovered a candidate disposal gene that is chromosomally clustered with genes for FAD synthesis or transport and is predicted to encode a protein of the PhnP cyclic phosphodiesterase family. The representative PhnP family enzyme from Koribacter versatilis (here named Fpd, F AD p hotoproduct d iphosphatase) was found to have high, Mn
2+ -dependent diphosphatase activity against FAD photoproducts, FAD, and ADP-ribose, but almost no phosphodiesterase activity against riboflavin 4',5'-cyclic phosphate, a chemical breakdown product of FAD. To provide a structural basis of the unique Fpd activity, the crystal structure of K. versatilis Fpd was determined. The results place Fpd in the broad metallo-β-lactamase-like family of hydrolases, a diverse family commonly using two metals for hydrolytic catalysis. The active site of Fpd contains two Mn2+ ions and a bound phosphate, consistent with a diphosphatase mechanism. Our results characterize the first PhnP family member that is a diphosphatase rather than a cyclic phosphodiesterase and suggest its involvement in a cellular damage-control system that efficiently hydrolyzes the reactive, ADP-ribose-like products of FAD photodegradation., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)- Published
- 2018
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24. ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.
- Author
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Tripathi P, Shine EE, Healy AR, Kim CS, Herzon SB, Bruner SD, and Crawford JM
- Subjects
- Binding Sites, Crystallography, X-Ray, Cyclopropanes chemistry, Escherichia coli drug effects, Escherichia coli genetics, Hydrolases chemistry, Microbial Viability drug effects, Peptides chemistry, Peptides pharmacology, Peptides toxicity, Polyketides chemistry, Polyketides pharmacology, Polyketides toxicity, Cyclopropanes metabolism, Drug Resistance drug effects, Escherichia coli enzymology, Escherichia coli metabolism, Hydrolases metabolism, Peptides metabolism, Polyketides metabolism
- Abstract
Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.
- Published
- 2017
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25. Metabolite damage and repair in metabolic engineering design.
- Author
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Sun J, Jeffryes JG, Henry CS, Bruner SD, and Hanson AD
- Subjects
- Metabolic Engineering methods, Metabolome
- Abstract
The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects., (Copyright © 2017 International Metabolic Engineering Society. All rights reserved.)
- Published
- 2017
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26. Structure and Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the Colibactin Biosynthetic Pathway.
- Author
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Guntaka NS, Healy AR, Crawford JM, Herzon SB, and Bruner SD
- Subjects
- Bacterial Proteins genetics, Crystallography, X-Ray, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Models, Molecular, Protein Conformation, Thiolester Hydrolases genetics, Bacterial Proteins metabolism, Peptides metabolism, Polyketides metabolism, Thiolester Hydrolases metabolism
- Abstract
Colibactin is a genotoxic hybrid nonribosomal peptide/polyketide secondary metabolite produced by various pathogenic and probiotic bacteria residing in the human gut. The presence of colibactin metabolites has been correlated to colorectal cancer formation in several studies. The specific function of many gene products in the colibactin gene cluster can be predicted. However, the role of ClbQ, a type II editing thioesterase, has not been established. The importance of ClbQ has been demonstrated by genetic deletions that abolish colibactin cytotoxic activity, and recent studies suggest an atypical role in releasing pathway intermediates from the assembly line. Here we report the 2.0 Å crystal structure and biochemical characterization of ClbQ. Our data reveal that ClbQ exhibits greater catalytic efficiency toward acyl-thioester substrates as compared to precolibactin intermediates and does not discriminate among carrier proteins. Cyclized pyridone-containing colibactins, which are off-pathway derivatives, are not viable substrates for ClbQ, while linear precursors are, supporting a role of ClbQ in facilitating the promiscuous off-loading of premature precolibactin metabolites and novel insights into colibactin biosynthesis.
- Published
- 2017
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27. Cytotoxic protein from the mushroom Coprinus comatus possesses a unique mode for glycan binding and specificity.
- Author
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Zhang P, Li K, Yang G, Xia C, Polston JE, Li G, Li S, Lin Z, Yang LJ, Bruner SD, and Ding Y
- Subjects
- Amino Acid Sequence, Apoptosis drug effects, Cell Survival drug effects, Crystallography, X-Ray, Fungal Proteins chemistry, Fungal Proteins pharmacology, HEK293 Cells, Humans, Jurkat Cells, Models, Molecular, Protein Binding, Protein Conformation, Protein Multimerization, Sequence Homology, Amino Acid, Agaricales metabolism, Coprinus metabolism, Fungal Proteins metabolism, Polysaccharides metabolism
- Abstract
Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment., Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
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28. Probing the structural basis of oxygen binding in a cofactor-independent dioxygenase.
- Author
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Li K, Fielding EN, Condurso HL, and Bruner SD
- Subjects
- Binding Sites, Crystallography, X-Ray, Dioxygenases chemistry, Dioxygenases genetics, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Protein Conformation, Streptomyces chemistry, Streptomyces genetics, Streptomyces metabolism, Dioxygenases metabolism, Oxygen metabolism, Streptomyces enzymology
- Abstract
The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.
- Published
- 2017
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29. Engineered P450 biocatalysts show improved activity and regio-promiscuity in aromatic nitration.
- Author
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Zuo R, Zhang Y, Jiang C, Hackett JC, Loria R, Bruner SD, and Ding Y
- Abstract
Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-DL-Trp. The production of both 4-Me-5-NO
2 -L-Trp and 4-Me-7-NO2 -L-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.- Published
- 2017
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30. Identification of a Novel Epoxyqueuosine Reductase Family by Comparative Genomics.
- Author
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Zallot R, Ross R, Chen WH, Bruner SD, Limbach PA, and de Crécy-Lagard V
- Subjects
- Lactobacillus enzymology, Lactobacillus genetics, Nucleoside Q metabolism, Genomics, Nucleoside Q analogs & derivatives, Oxidoreductases metabolism
- Abstract
The reduction of epoxyqueuosine (oQ) is the last step in the synthesis of the tRNA modification queuosine (Q). While the epoxyqueuosine reductase (EC 1.17.99.6) enzymatic activity was first described 30 years ago, the encoding gene queG was only identified in Escherichia coli in 2011. Interestingly, queG is absent from a large number of sequenced genomes that harbor Q synthesis or salvage genes, suggesting the existence of an alternative epoxyqueuosine reductase in these organisms. By analyzing phylogenetic distributions, physical gene clustering, and fusions, members of the Domain of Unknown Function 208 (DUF208) family were predicted to encode for an alternative epoxyqueuosine reductase. This prediction was validated with genetic methods. The Q modification is present in Lactobacillus salivarius, an organism missing queG but harboring the duf208 gene. Acinetobacter baylyi ADP1 is one of the few organisms that harbor both QueG and DUF208, and deletion of both corresponding genes was required to observe the absence of Q and the accumulation of oQ in tRNA. Finally, the conversion oQ to Q was restored in an E. coli queG mutant by complementation with plasmids harboring duf208 genes from different bacteria. Members of the DUF208 family are not homologous to QueG enzymes, and thus, duf208 is a non-orthologous replacement of queG. We propose to name DUF208 encoding genes as queH. While QueH contains conserved cysteines that could be involved in the coordination of a Fe/S center in a similar fashion to what has been identified in QueG, no cobalamin was identified associated with recombinant QueH protein.
- Published
- 2017
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31. The PacC transcription factor regulates secondary metabolite production and stress response, but has only minor effects on virulence in the insect pathogenic fungus Beauveria bassiana.
- Author
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Luo Z, Ren H, Mousa JJ, Rangel DE, Zhang Y, Bruner SD, and Keyhani NO
- Subjects
- Animals, Bacterial Proteins genetics, Beauveria genetics, Beauveria growth & development, Larva growth & development, Larva microbiology, Lepidoptera growth & development, Secondary Metabolism, Sequence Deletion, Spores, Fungal genetics, Spores, Fungal growth & development, Spores, Fungal metabolism, Transcription Factors genetics, Virulence, Bacterial Proteins metabolism, Beauveria metabolism, Beauveria pathogenicity, Lepidoptera microbiology, Tenebrio microbiology, Transcription Factors metabolism
- Abstract
The PacC transcription factor is an important component of the fungal ambient pH-responsive regulatory system. Loss of pacC in the insect pathogenic fungus Beauveria bassiana resulted in an alkaline pH-dependent decrease in growth and pH-dependent increased susceptibility to osmotic (salt, sorbitol) stress and SDS. Extreme susceptibility to Congo Red was noted irrespective of pH, and ΔBbpacC conidia showed subtle increases in UV susceptibility. The ΔBbPacC mutant showed a reduced ability to acidify media during growth due to failure to produce oxalic acid. The ΔBbPacC mutant also did not produce the insecticidal compound dipicolinic acid, however, production of a yellow-colored compound was noted. The compound, named bassianolone B, was purified and its structure determined. Despite defects in growth, stress resistance, and oxalate/insecticidal compound production, only a small decrease in virulence was seen for the ΔBbpacC strain in topical insect bioassays using larvae from the greater waxmoth, Galleria mellonella or adults of the beetle, Tenebrio molitor. However, slightly more pronounced decreases were seen in virulence via intrahemcoel injection assays (G. mellonella) and in assays using T. molitor larvae. These data suggest important roles for BbpacC in mediating growth at alkaline pH, regulating secondary metabolite production, and in targeting specific insect stages., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)
- Published
- 2017
- Full Text
- View/download PDF
32. ClbM is a versatile, cation-promiscuous MATE transporter found in the colibactin biosynthetic gene cluster.
- Author
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Mousa JJ, Newsome RC, Yang Y, Jobin C, and Bruner SD
- Subjects
- Antiporters metabolism, Bacterial Proteins metabolism, Biological Products chemistry, Biological Transport, Biological Transport, Active, Cations, Crystallography, X-Ray, Cytoplasm metabolism, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Hydrogen Bonding, Hydrogen-Ion Concentration, Ion Transport, Klebsiella pneumoniae metabolism, Microbiota, Multigene Family, Mutation, Organic Cation Transport Proteins metabolism, Peptide Hydrolases metabolism, Potassium chemistry, Prodrugs, Protein Structure, Secondary, Rhodamines chemistry, Rubidium chemistry, Sodium chemistry, Water chemistry, Escherichia coli genetics, Escherichia coli Proteins genetics, Organic Cation Transport Proteins genetics, Peptides genetics, Peptides metabolism, Polyketides metabolism
- Abstract
Multidrug transporters play key roles in cellular drug resistance to toxic molecules, yet these transporters are also involved in natural product transport as part of biosynthetic clusters in bacteria and fungi. The genotoxic molecule colibactin is produced by strains of virulent and pathobiont Escherichia coli and Klebsiella pneumoniae. In the biosynthetic cluster is a multidrug and toxic compound extrusion protein (MATE) proposed to transport the prodrug molecule precolibactin across the cytoplasmic membrane, for subsequent cleavage by the peptidase ClbP and cellular export. We recently determined the X-ray structure of ClbM, and showed preliminary data suggesting its specific role in precolibactin transport. Here, we define a functional role of ClbM by examining transport capabilities under various biochemical conditions. Our data indicate ClbM responds to sodium, potassium, and rubidium ion gradients, while also having substantial transport activity in the absence of alkali cations., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
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33. A strictly monofunctional bacterial hydroxymethylpyrimidine phosphate kinase precludes damaging errors in thiamin biosynthesis.
- Author
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Thamm AM, Li G, Taja-Moreno M, Gerdes SY, de Crécy-Lagard V, Bruner SD, and Hanson AD
- Abstract
The canonical kinase (ThiD) that converts the thiamin biosynthesis intermediate hydroxymethylpyrimidine (HMP) monophosphate to the diphosphate can also very efficiently convert free HMP to the monophosphate in prokaryotes, plants, and fungi. This HMP kinase activity enables salvage of HMP, but it is not substrate-specific and so allows toxic HMP analogs and damage products to infiltrate the thiamin biosynthesis pathway. Comparative analysis of bacterial genomes uncovered a gene, thiD2 , that is often fused to the thiamin synthesis gene thiE and could potentially encode a replacement for ThiD. Standalone ThiD2 proteins and ThiD2 fusion domains are small (~130-residues) and do not belong to any previously known protein family. Genetic and biochemical analyses showed that representative standalone and fused ThiD2 proteins catalyze phosphorylation of HMP monophosphate, but not of HMP or its toxic analogs and damage products such as bacimethrin and 5-(hydroxymethyl)-2-methylpyrimidin-4-ol. As strictly monofunctional HMP monophosphate kinases, ThiD2 proteins eliminate a potentially fatal vulnerability of canonical ThiD, at the cost of the ability to reclaim HMP formed by thiamin turnover., (©2017 The Author(s).)
- Published
- 2017
- Full Text
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34. Structural basis for precursor protein-directed ribosomal peptide macrocyclization.
- Author
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Li K, Condurso HL, Li G, Ding Y, and Bruner SD
- Subjects
- Cyclization, Protein Conformation, Ribosomal Proteins metabolism, Ribosomes metabolism, Ribosomal Proteins biosynthesis, Ribosomal Proteins chemistry
- Abstract
Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein-protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.
- Published
- 2016
- Full Text
- View/download PDF
35. Structural and mechanistic diversity of multidrug transporters.
- Author
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Mousa JJ and Bruner SD
- Subjects
- Bacterial Proteins metabolism, Humans, Molecular Structure, Drug Resistance, Multiple, Membrane Transport Proteins
- Abstract
Covering: 2009 to mid 2016Multidrug transporters are common and prevalent in all orders of life, having diverse functions from the removal of toxins, resistance to cytotoxins, and the transport of specific eluents. In addition, multidrug transporters pose a significant threat to modern medicine. Able to transport structurally diverse small molecule drugs, these transporters are implicated in antibiotic resistant strains of bacteria, as well as chemotherapeutic-resistance cancer cells. Although important in such resistance, a relatively small number of multidrug transporters have been structurally characterized, primarily due to the difficulty in purifying and crystallizing active membrane proteins and protein complexes. This review will cover recent structural breakthroughs in the past six years that have led to increased knowledge of the mechanisms of multidrug transporter chemistry, and the role of these transporters in exporting secondary metabolites.
- Published
- 2016
- Full Text
- View/download PDF
36. Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans.
- Author
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Zhang X, Li K, Jones RA, Bruner SD, and Butcher RA
- Subjects
- Acyl-CoA Oxidase genetics, Acyl-CoA Oxidase metabolism, Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Amino Acid Sequence, Animals, Binding Sites, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Crystallography, X-Ray, Flavin-Adenine Dinucleotide chemistry, Flavin-Adenine Dinucleotide metabolism, Gene Expression, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Kinetics, Models, Molecular, Mutation, Oxidation-Reduction, Pheromones biosynthesis, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Alignment, Substrate Specificity, Acyl-CoA Oxidase chemistry, Caenorhabditis elegans chemistry, Caenorhabditis elegans Proteins chemistry, Pheromones chemistry
- Abstract
Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state., Competing Interests: The authors declare no conflict of interest.
- Published
- 2016
- Full Text
- View/download PDF
37. Interdomain and Intermodule Organization in Epimerization Domain Containing Nonribosomal Peptide Synthetases.
- Author
-
Chen WH, Li K, Guntaka NS, and Bruner SD
- Subjects
- Catalytic Domain, Crystallography, X-Ray, Diketopiperazines chemistry, Isomerism, Mutagenesis, Site-Directed, Peptide Synthases chemistry, Peptide Synthases genetics, Protein Conformation, Peptide Synthases metabolism
- Abstract
Nonribosomal peptide synthetases are large, complex multidomain enzymes responsible for the biosynthesis of a wide range of peptidic natural products. Inherent to synthetase chemistry is the thioester templated mechanism that relies on protein/protein interactions and interdomain dynamics. Several questions related to structure and mechanism remain to be addressed, including the incorporation of accessory domains and intermodule interactions. The inclusion of nonproteinogenic d-amino acids into peptide frameworks is a common and important modification for bioactive nonribosomal peptides. Epimerization domains, embedded in nonribosomal peptide synthetases assembly lines, catalyze the l- to d-amino acid conversion. Here we report the structure of the epimerization domain/peptidyl carrier protein didomain construct from the first module of the cyclic peptide antibiotic gramicidin synthetase. Both holo (phosphopantethiene post-translationally modified) and apo structures were determined, each representing catalytically relevant conformations of the two domains. The structures provide insight into domain-domain recognition, substrate delivery during the assembly line process, in addition to the structural organization of homologous condensation domains, canonical players in all synthetase modules.
- Published
- 2016
- Full Text
- View/download PDF
38. Microbial siderophore-based iron assimilation and therapeutic applications.
- Author
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Li K, Chen WH, and Bruner SD
- Subjects
- Animals, Bacteria chemistry, Humans, Iron chemistry, Siderophores biosynthesis, Siderophores chemistry, Bacteria metabolism, Iron metabolism, Siderophores therapeutic use
- Abstract
Siderophores are structurally diverse, complex natural products that bind metals with extraordinary specificity and affinity. The acquisition of iron is critical for the survival and virulence of many pathogenic microbes and diverse strategies have evolved to synthesize, import and utilize iron. There has been a substantial increase of known siderophore scaffolds isolated and characterized in the past decade and the corresponding biosynthetic gene clusters have provided insight into the varied pathways involved in siderophore biosynthesis, delivery and utilization. Additionally, therapeutic applications of siderophores and related compounds are actively being developed. The study of biosynthetic pathways to natural siderophores augments the understanding of the complex mechanisms of bacterial iron acquisition, and enables a complimentary approach to address virulence through the interruption of siderophore biosynthesis or utilization by targeting the key enzymes to the siderophore pathways.
- Published
- 2016
- Full Text
- View/download PDF
39. An artificial self-sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio-selectivity.
- Author
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Zuo R, Zhang Y, Huguet-Tapia JC, Mehta M, Dedic E, Bruner SD, Loria R, and Ding Y
- Subjects
- Binding Sites, Biocatalysis, Cytochrome P-450 Enzyme System chemistry, Indoles chemistry, Protein Domains, Protein Engineering methods, Recombinant Fusion Proteins metabolism, Tandem Mass Spectrometry, Tryptophan analogs & derivatives, Cytochrome P-450 Enzyme System metabolism, Fluorine chemistry, Nitrates chemistry, Tryptophan chemistry, Tryptophan isolation & purification
- Abstract
Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l-tryptophan. To develop cost-efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self-sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4-F-dl-tryptophan and 5-F-l-tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE-based biocatalysts for the production of valuable nitroaromatics., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
- Full Text
- View/download PDF
40. Crystal structure of the homocysteine methyltransferase MmuM from Escherichia coli.
- Author
-
Li K, Li G, Bradbury LM, Hanson AD, and Bruner SD
- Subjects
- Amino Acid Sequence, Binding Sites, Crystallography, X-Ray, Escherichia coli chemistry, Escherichia coli genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Homocysteine metabolism, Homocysteine S-Methyltransferase genetics, Homocysteine S-Methyltransferase metabolism, Methionine metabolism, Models, Molecular, Molecular Sequence Data, Sequence Alignment, Escherichia coli enzymology, Escherichia coli Proteins chemistry, Homocysteine S-Methyltransferase chemistry
- Abstract
Homocysteine S-methyltransferases (HMTs, EC 2.1.1.0) catalyse the conversion of homocysteine to methionine using S-methylmethionine or S-adenosylmethionine as the methyl donor. HMTs play an important role in methionine biosynthesis and are widely distributed among micro-organisms, plants and animals. Additionally, HMTs play a role in metabolite repair of S-adenosylmethionine by removing an inactive diastereomer from the pool. The mmuM gene product from Escherichia coli is an archetypal HMT family protein and contains a predicted zinc-binding motif in the enzyme active site. In the present study, we demonstrate X-ray structures for MmuM in oxidized, apo and metallated forms, representing the first such structures for any member of the HMT family. The structures reveal a metal/substrate-binding pocket distinct from those in related enzymes. The presented structure analysis and modelling of co-substrate interactions provide valuable insight into the function of MmuM in both methionine biosynthesis and cofactor repair., (© 2016 Authors; published by Portland Press Limited.)
- Published
- 2016
- Full Text
- View/download PDF
41. MATE transport of the E. coli-derived genotoxin colibactin.
- Author
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Mousa JJ, Yang Y, Tomkovich S, Shima A, Newsome RC, Tripathi P, Oswald E, Bruner SD, and Jobin C
- Subjects
- Animals, Crystallography, X-Ray, DNA Damage drug effects, Disease Models, Animal, Escherichia coli Infections microbiology, Escherichia coli Infections pathology, Escherichia coli Proteins chemistry, Ilex, Mice, Models, Molecular, Organic Cation Transport Proteins chemistry, Protein Conformation, Protein Transport, Zebrafish, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Mutagens metabolism, Organic Cation Transport Proteins metabolism, Peptides metabolism, Polyketides metabolism
- Abstract
Various forms of cancer have been linked to the carcinogenic activities of microorganisms(1-3). The virulent gene island polyketide synthase (pks) produces the secondary metabolite colibactin, a genotoxic molecule(s) causing double-stranded DNA breaks(4) and enhanced colorectal cancer development(5,6). Colibactin biosynthesis involves a prodrug resistance strategy where an N-terminal prodrug scaffold (precolibactin) is assembled, transported into the periplasm and cleaved to release the mature product(7-10). Here, we show that ClbM, a multidrug and toxic compound extrusion (MATE) transporter, is a key component involved in colibactin activity and transport. Disruption of clbM attenuated pks+ E. coli-induced DNA damage in vitro and significantly decreased the DNA damage response in gnotobiotic Il10(-/-) mice. Colonization experiments performed in mice or zebrafish animal models indicate that clbM is not implicated in E. coli niche establishment. The X-ray structure of ClbM shows a structural motif common to the recently described MATE family. The 12-transmembrane ClbM is characterized as a cation-coupled antiporter, and residues important to the cation-binding site are identified. Our data identify ClbM as a precolibactin transporter and provide the first structure of a MATE transporter with a defined and specific biological function.
- Published
- 2016
- Full Text
- View/download PDF
42. Structure and functional analysis of the siderophore periplasmic binding protein from the fuscachelin gene cluster of Thermobifida fusca.
- Author
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Li K and Bruner SD
- Subjects
- Actinobacteria genetics, Amino Acid Sequence, Bacterial Proteins genetics, Crystallography, X-Ray, Molecular Docking Simulation, Molecular Sequence Data, Multigene Family, Periplasmic Binding Proteins genetics, Protein Conformation, Actinobacteria chemistry, Actinobacteria metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Periplasmic Binding Proteins chemistry, Periplasmic Binding Proteins metabolism, Siderophores metabolism
- Abstract
Iron acquisition is a complex, multicomponent process critical for most organisms' survival and virulence. Small iron chelating molecules, siderophores, mediate transport as key components of common pathways for iron assimilation in many microorganisms. The chemistry and biology of the extraordinary tight and specific metal binding siderophores is of general interest in terms of host/guest chemistry and is a potential target toward the development of therapeutic treatments for microbial virulence. The siderophore pathway of the moderate thermophile, Thermobifida fusca, is an excellent model system to study the process in Gram-positive bacteria. Here we describe the structure and characterization of the siderophore periplasmic binding protein, FscJ from the fuscachelin gene cluster of T. fusca. The structure shows a di-domain arrangement connected with a long α-helix hinge. Several X-ray structures detail ligand-free conformational changes at different pH values, illustrating complex interdomain flexibility of the siderophore receptors. We demonstrated that FscJ has a unique recognition mechanism and details the binding interaction with ferric-fuscachelin A through ITC and docking analysis. The presented work provides a structural basis for the complex molecular mechanisms of siderophore recognition and transportation., (© 2015 Wiley Periodicals, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
43. Oxygen diffusion pathways in a cofactor-independent dioxygenase.
- Author
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Di Russo NV, Condurso HL, Li K, Bruner SD, and Roitberg AE
- Abstract
Molecular oxygen plays an important role in a wide variety of enzymatic reactions. Through recent research efforts combining computational and experimental methods a new view of O
2 diffusion is emerging, where specific channels guide O2 to the active site. The focus of this work is DpgC, a cofactor-independent oxygenase. Molecular dynamics simulations, together with mutagenesis experiments and xenon-binding data, reveal that O2 reaches the active site of this enzyme using three main pathways and four different access points. These pathways connect a series of dynamic hydrophobic pockets, concentrating O2 at a specific face of the enzyme substrate. Extensive molecular dynamics simulations provide information about which pathways are more frequently used. This data is consistent with the results of kinetic measurements on mutants and is difficult to obtain using computational cavity-location methods. Taken together, our results reveal that although DpgC is rare in its ability of activating O2 in the absence of cofactors or metals, the way O2 reaches the active site is similar to that reported for other O2 -using proteins: multiple access channels are available, and the architecture of the pathway network can provide regio- and stereoselectivity. Our results point to the existence of common themes in O2 access that are conserved among very different types of proteins.- Published
- 2015
- Full Text
- View/download PDF
44. Structure and Mechanism of the Siderophore-Interacting Protein from the Fuscachelin Gene Cluster of Thermobifida fusca.
- Author
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Li K, Chen WH, and Bruner SD
- Subjects
- Actinobacteria chemistry, Actinobacteria genetics, Amino Acid Sequence, Bacterial Proteins genetics, Models, Molecular, Molecular Sequence Data, Protein Binding, Sequence Alignment, Siderophores chemistry, Actinobacteria metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Multigene Family, Siderophores metabolism
- Abstract
Microbial iron acquisition is a complex process and frequently a key and necessary step for survival. Among the several paths for iron assimilation, small molecule siderophore-mediated transport is a commonly employed strategy of many microorganisms. The chemistry and biology of the extraordinary tight and specific binding of siderophores to metal is also exploited in therapeutic treatments for microbial virulence and metal toxicity. The intracellular fate of iron acquired via the siderophore pathway is one of the least understood steps in the complex process at the molecular level. A common route to cellular incorporation is the single-electron reduction of ferric to ferrous iron catalyzed by specific and/or nonspecific reducing agents. The biosynthetic gene clusters for siderophores often contain representatives of one or two families of redox-active enzymes: the flavin-containing "siderophore-interacting protein" and iron-sulfur ferric siderophore reductases. Here we present the structure and characterization of the siderophore-interacting protein, FscN, from the fuscachelin siderophore gene cluster of Thermobifida fusca. The structure shows a flavoreductase fold with a noncovalently bound FAD cofactor along with an unexpected metal bound adjacent to the flavin site. We demonstrated that FscN is redox-active and measured the binding and reduction of ferric fuscachelin. This work provides a structural basis for the activity of a siderophore-interacting protein and further insight into the complex and important process of iron acquisition and utilization.
- Published
- 2015
- Full Text
- View/download PDF
45. Applicability of fluorescence-based sensors to the determination of kinetic parameters for O₂ in oxygenases.
- Author
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Di Russo NV, Bruner SD, and Roitberg AE
- Subjects
- Electrodes, Kinetics, Fluorescent Dyes chemistry, Oxygen chemistry, Oxygenases chemistry
- Abstract
Optical methods for O2 determination based on dynamic fluorescence quenching have been applied to measure oxygen uptake rates in cell culture and to determine intracellular oxygen levels. Here we demonstrate the applicability of fluorescence-based probes in determining kinetic parameters for O2 using as an example catalysis by a cofactor-independent oxygenase (DpgC). Fluorescence-based sensors provide a direct assessment of enzyme-catalyzed O2 consumption using commercially available, low-cost instrumentation that is easily customizable and, thus, constitutes a convenient alternative to the widely used Clark-type electrode, especially in cases where chemical interference is expected to be problematic., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
46. Structure and noncanonical chemistry of nonribosomal peptide biosynthetic machinery.
- Author
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Condurso HL and Bruner SD
- Subjects
- Biological Products isolation & purification, Biological Products metabolism, Molecular Structure, Peptides metabolism, Protein Conformation, Biological Products chemistry, Peptide Synthases chemistry, Peptide Synthases metabolism, Peptides chemistry
- Abstract
Structural biology has provided significant insights into the complex chemistry and macromolecular organization of nonribosomal peptide synthetases. In addition, novel pathways are continually described, expanding the knowledge of known biosynthetic chemistry.
- Published
- 2012
- Full Text
- View/download PDF
47. Synthesis and structure confirmation of fuscachelins A and B, structurally unique natural product siderophores from Thermobifida fusca.
- Author
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Dimise EJ, Condurso HL, Stoker GE, and Bruner SD
- Subjects
- Biological Products chemistry, Chelating Agents chemistry, Siderophores chemistry, Actinomycetales chemistry, Biological Products chemical synthesis, Chelating Agents chemical synthesis, Iron chemistry, Siderophores chemical synthesis
- Abstract
The fuscachelin siderophores have been prepared synthetically as have their metal chelation complexes. The heterodimeric nature of the fuscachelin decamer lends itself to a convergent synthetic strategy. Synthetic access to the natural products and intermediates will provide readily adaptable tools in future studies examining iron-sequestration and the biosynthetic machinery.
- Published
- 2012
- Full Text
- View/download PDF
48. Structure guided approaches toward exploiting and manipulating nonribosomal peptide and polyketide biosynthetic pathways.
- Author
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Condurso HL and Bruner SD
- Subjects
- Biosynthetic Pathways, Cyclization, Peptides metabolism, Polyketides metabolism, Ribosomes metabolism, Peptides chemistry, Polyketides chemistry
- Abstract
Nonribosomal peptide and polyketide natural products are structurally diverse small molecules synthesized on complex enzyme assemblies. The ability to rationally engineer secondary metabolic pathways is a promising approach to novel therapeutics. Atomic resolution structures of biosynthetic enzymes provide information on active site architecture and macromolecular assembly that can aid in the engineering of new compounds. This review surveys recent applications toward biosynthetic engineering of natural products guided by structural biology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
49. Structural basis for phosphopantetheinyl carrier domain interactions in the terminal module of nonribosomal peptide synthetases.
- Author
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Liu Y, Zheng T, and Bruner SD
- Subjects
- Catalytic Domain, Crystallography, X-Ray, Escherichia coli metabolism, Models, Molecular, Peptide Biosynthesis, Nucleic Acid-Independent, Protein Structure, Tertiary, Peptide Synthases chemistry, Peptide Synthases metabolism
- Abstract
Phosphopantetheine-modified carrier domains play a central role in the template-directed, biosynthesis of several classes of primary and secondary metabolites. Fatty acids, polyketides, and nonribosomal peptides are constructed on multidomain enzyme assemblies using phosphopantetheinyl thioester-linked carrier domains to traffic and activate building blocks. The carrier domain is a dynamic component of the process, shuttling pathway intermediates to sequential enzyme active sites. Here, we report an approach to structurally fix carrier domain/enzyme constructs suitable for X-ray crystallographic analysis. The structure of a two-domain construct of Escherichia coli EntF was determined with a conjugated phosphopantetheinyl-based inhibitor. The didomain structure is locked in an active orientation relevant to the chemistry of nonribosomal peptide biosynthesis. This structure provides details into the interaction of phosphopantetheine arm with the carrier domain and the active site of the thioesterase domain., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
50. Enzyme catalysis: C-H activation is a Reiske business.
- Author
-
Bruner SD
- Subjects
- Biocatalysis, Biological Products biosynthesis, Carbon metabolism, Cyclization, Hydrogen metabolism, Hydrogen Bonding, Enzymes metabolism
- Published
- 2011
- Full Text
- View/download PDF
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