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N-Glycosylation is a fundamental protein modification found in both eukaryotes and archaea. Despite lacking N-glycans, many commensal and pathogenic bacteria have developed mechanisms to degrade these isoforms for a variety of functions, including nutrient acquisition and evasion of the immune system. Although much is known about many of the enzymes responsible for N-glycan degradation, the enzymes involved in cleaving the N-glycan core have only recently been discovered. Thus, some of the structural details have yet to be characterized, and little is known about their full distribution among bacterial strains and specifically within potential Gram-positive polysaccharide utilization loci. Here, we report crystal structures for Family 5, Subfamily 18 (GH5_18) glycoside hydrolases from the gut bacterium Bifidobacterium longum (BlGH5_18) and the soil bacterium Streptomyces cattleya (ScGH5_18), which hydrolyze the core Manβ1-4GlcNAc disaccharide. Structures of these enzymes in complex with Manβ1-4GlcNAc reveal a more complete picture of the -1 subsite. They also show that a C-terminal active site cap present in BlGH5_18 is absent in ScGH5_18. Although this C-terminal cap is not widely distributed throughout the GH5_18 family, it is important for full enzyme activity. In addition, we show that GH5_18 enzymes are found in Gram-positive polysaccharide utilization loci that share common genes, likely dedicated to importing and degrading N-glycan core structures.

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species–specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their antimalarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the salinipostins and newly identified natural A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed among many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

Methods for the focused isolation of low-abundance natural products with specific chemical substructures could expand known bioactive chemical diversity for drug discovery. Here we report the combined use of genome mining and an 15N NMR-based screening method for the targeted isolation of the low-abundance piperazic-acid-containing peptides incarnatapeptins A (1) and B (3). Incarnatapeptin B (3) shows in vitro cytotoxicity to LNCaP prostate cancer cells.

Pyridoxal 5'-phosphate (PLP) is an organic cofactor employed by ~ 4% of enzymes. The structure of the PLP cofactor allows for the stabilization of carbanions through resonance. A small number of PLP-dependent enzymes employ molecular oxygen as a cosubstrate. Here, we review the biological roles and possible mechanisms of these enzymes, and we observe that these enzymes are found in multiple protein families, suggesting that reaction with oxygen might have emerged de novo in several protein families and thus could be directed to emerge again through laboratory evolution experiments.

The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N-N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate-containing amino acid l-alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l-alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl-carrier-protein-based mechanism for activation of the precursor l-diaminopropionate, and we also show that nitric oxide can participate in non-enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N-terminal cupin domain and a C-terminal AraC-like DNA-binding domain, is required for alanosine biosynthesis.

Natural nonproteinogenic amino acids vastly outnumber the well-known 22 proteinogenic amino acids. Such amino acids are generated in specialized metabolic pathways. In these pathways, diverse biosynthetic transformations, ranging from isomerizations to the stereospecific functionalization of C-H bonds, are employed to generate structural diversity. The resulting nonproteinogenic amino acids can be integrated into more complex natural products. Here we review recently discovered biosynthetic routes to freestanding nonproteinogenic α-amino acids, with an emphasis on work reported between 2013 and mid-2019.

Significance Pyridoxal phosphate (PLP)-dependent enzymes rarely react with oxygen, but an emerging group of oxygen-, PLP-dependent enzymes oxidize l -arginine. Two types of oxidases are known: hydroxylases and desaturases. We demonstrate that arginine desaturases have a minor hydroxylase activity and then show through X-ray crystallographic, mutagenesis, spectroscopic, and computational studies that their mechanism involves two rounds of single-electron transfer to oxygen and superoxide rebound, ultimately giving a conjugated hydroperoxyl intermediate. Water can attack to give a hydroxylated product and release H 2 O 2 , but with water absent, the intermediate can be deprotonated, instead giving a desaturated product and H 2 O 2 . Our work outlines the unique mechanism and evolutionary history of these enzymes and sets the stage toward engineering these enzymes to catalyze oxidative reactions.

Date Presented 04/01/2022 This panel will discuss a research study measuring the effects of intensive, group-based pediatric constraint-induced movement therapy (CIMT) for children ages 2 to 6 with hemiplegia. Results produced many statistically significant improvements in unilateral function, bimanual coordination, and occupational performance. Primary Author and Speaker: Katherine S. Ryan-Bloomer Additional Authors and Speakers: Lily B. Davis, Katelin T. Watkins, Kyra T. Woods, Bethany Tackett

Microbes produce specialized metabolites to thrive in their natural habitats. However, it is rare that a given specialized metabolite is biosynthesized via pathways with distinct intermediates and enzymes. Here, we show that the core assembly mechanism of the antibiotic indolmycin in marine gram-negative Pseudoalteromonas luteoviolacea is distinct from its counterpart in terrestrial gram-positive Streptomyces species, with a molecule that is a shunt product in the Streptomyces pathway employed as a biosynthetic substrate for a novel metal-independent N-demethylindolmycin synthase in the P. luteoviolacea pathway. To provide insight into this reaction, we solved the 1.5 A resolution structure in complex with product and identified the active site residues. Guided by our biosynthetic insights, we then engineered the Streptomyces indolmycin producer for titer improvement. This study provides a paradigm for understanding how two unique routes to a microbial specialized metabolite can emerge from convergent biosynthetic transformations. The indolmycin biosynthetic pathway in a marine gram-negative bacterium is distinct from its counterpart in terrestrial gram-positive Streptomyces species, using a Streptomyces shunt product as a substrate for an N-demethylindolmycin synthase.

Nitroimidazoles are one of the most effective ways to treat anaerobic bacterial infections. Synthetic nitroimidazoles are inspired by the structure of azomycin, isolated from Streptomyces eurocidicus in 1953. Despite its foundational role, no biosynthetic gene cluster for azomycin has been found. Guided by bioinformatics, we identified a cryptic biosynthetic gene cluster in Streptomyces cattleya and then carried out in vitro reconstitution to deduce the enzymatic steps in the pathway linking l-arginine to azomycin. The gene cluster we discovered is widely distributed among soil-dwelling actinobacteria and proteobacteria, suggesting that azomycin and related nitroimidazoles may play important ecological roles. Our work sets the stage for development of biocatalytic approaches to generate azomycin and related nitroimidazoles.

Nitric oxide (NO) has wide-ranging roles in biology, but less is known about its role in building chemical diversity. Here we report a new route to NO from the biosynthetic pathway to the N-nitroso compound streptozocin. We show that the N-nitroso group of streptozocin comes from the biosynthetic reassembly of l-arginine, with the guanidino nitrogens forming a nitrogen–nitrogen bond. To understand this biosynthetic process, we identify the biosynthetic gene cluster of streptozocin and demonstrate that free l-arginine is N-methylated by StzE to give Nω-monomethyl-l-arginine. We show that this product is then oxidized by StzF, a nonheme iron-dependent enzyme unrelated to known nitric oxide synthases, generating a urea compound and NO. Our work implies that formation and capture of NO is the likely route to N-nitroso formation in vivo. Altogether, our work unveils a new enzyme pair for the production of NO from l-arginine and sets the stage for understanding biosynthetic routes to N-nitroso natural products.

Indolmycin is an antibiotic from Streptomyces griseus ATCC 12648 with activity against Helicobacter pylori, Plasmodium falciparum, and methicillin-resistant Staphylococcus aureus. Here we describe the use of the indolmycin biosynthetic genes in E. coli to make indolmycenic acid, a chiral intermediate in indolmycin biosynthesis, which can then be converted to indolmycin through a three-step synthesis. To expand indolmycin structural diversity, we introduce a promiscuous tryptophanyl-tRNA synthetase gene (trpS) into our E. coli production system and feed halogenated indoles to generate the corresponding indolmycenic acids, ultimately allowing us to access indolmycin derivatives through synthesis. Bioactivity testing against methicillin-resistant Staphylococcus aureus showed modest antibiotic activity for 5-, 6-, and 7-fluoro-indolmycin., A semi-synthetic system for producing indolmycin, an antibiotic, was developed and used to make indole-substituted, halogenated derivatives of indolmycin, some with modest bioactivity against methicillin-resistant Staphylococcus aureus.

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their anti-malarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the saliniphostins and newly identified A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed amongst many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

Microbiome-associated bacteria can have diverse effects on health of their hosts, yet the genetic and molecular bases of these effects have largely remained elusive. This work demonstrates that a novel bacterial locus can modulate systemic plant immunity. Additionally, this work demonstrates that growth-promoting strains may have unanticipated consequences for plant immunity, and this is critical to consider when the plant microbiome is being engineered for agronomic improvement., Plant root-associated microbes promote plant growth and elicit induced systemic resistance (ISR) to foliar pathogens. In an attempt to find novel growth-promoting and ISR-inducing strains, we previously identified strains of root-associated Pseudomonas spp. that promote plant growth but unexpectedly elicited induced systemic susceptibility (ISS) rather than ISR to foliar pathogens. Here, we demonstrate that the ISS-inducing phenotype is common among root-associated Pseudomonas spp. Using comparative genomics, we identified a single Pseudomonas fluorescens locus that is unique to ISS strains. We generated a clean deletion of the 11-gene ISS locus and found that it is necessary for the ISS phenotype. Although the functions of the predicted genes in the locus are not apparent based on similarity to genes of known function, the ISS locus is present in diverse bacteria, and a subset of the genes were previously implicated in pathogenesis in animals. Collectively, these data show that a single bacterial locus contributes to modulation of systemic plant immunity.

Plant root-associated microbes promote plant growth and induce systemic resistance (ISR) to foliar pathogens. In an attempt to find novel growth-promoting and ISR-inducing strains, we previously identified strains of root-associated Pseudomonas spp. that promote plant growth but unexpectedly induced systemic susceptibility (ISS) rather than ISR to foliar pathogens. Here we demonstrate that the ISS-inducing phenotype is common among root-associated Pseudomonas spp. Using comparative genomics, we identified a single P. fluorescens locus that is unique to ISS strains. We generated a clean deletion of the 11-gene ISS locus and found that it is necessary for the ISS phenotype. Although the functions of the predicted genes in the locus are not apparent based on similarity to genes of known function, the ISS locus is present in diverse bacteria and a subset of the genes have previously been implicated in pathogenesis in animals. Collectively these data show that a single bacterial locus contributes to modulation of systemic plant immunity.ImportanceMicrobiome-associated bacteria can have diverse effects on health of their hosts, yet the genetic and molecular basis of these effects have largely remained elusive. This work demonstrates that a novel bacterial locus can modulate systemic plant immunity. Additionally, this work demonstrates that growth promoting strains may have unanticipated consequences on plant immunity and this is critical to consider when engineering the plant microbiome for agronomic improvement.

Natural products with rare functional groups are likely to be constructed by unique biosynthetic enzymes. One such rare functional group is the O-methyl nitronate, which can undergo [3 + 2] cycloaddition reactions with olefins in mild conditions. O-methyl nitronates are found in some natural products; however, how such O-methyl nitronates are assembled biosynthetically is unknown. Here we show that the assembly of the O-methyl nitronate in the natural product enteromycin carboxamide occurs via activation of glycine on a peptidyl carrier protein, followed by reaction with a diiron oxygenase to give a nitronate intermediate and then with a methyltransferase to give an O-methyl nitronate. Guided by the discovery of this pathway, we then identify related cryptic biosynthetic gene cassettes in other bacteria and show that these alternative gene cassettes can, instead, facilitate oxidative denitrification of glycine-derived nitronates. Altogether, our work reveals bifurcating pathways from a central glycine-derived nitronate intermediate in bacteria. O-methyl nitronate is a rare functional group in natural products. Now, the biosynthetic pathway to O-methyl nitronate, which involves O-methylation of a peptidyl carrier protein (PCP)-tethered nitronate, has been revealed. In some bacteria, the same PCP-tethered nitronate is shown to be oxidized by nitronate monooxygenases to provide nitrite and a PCP-tethered glyoxylate.

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts l-arginine to (S)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of H2O2. To understand this unusual chemistry, we obtain ∼1.5 A resolution structures that capture intermediates along the catalytic cycle. Our data suggest that RohP carries out a four-electron oxidation and a stereospecific alkene hydration to give the (S)-configured product. Together with our earlier studies on an O2, PLP-dependent l-arginine oxidase, our work suggests that there is a shared pathway leading to both oxidized and hydroxylated products from l-arginine.

Correction for ‘An engineered biosynthetic–synthetic platform for production of halogenated indolmycin antibiotics’ by Elesha R. Hoffarth et al., Chem. Sci., 2021, 12, 8817–8821, DOI: 10.1039/D0SC05843B.

Bifidobacterium longum subsp. infantis ATCC 15697 has emerged as a model for infant gut-associated bifidobacterial strains. Here we present a genetic system for B. longum subsp. infantis ATCC 15697 using its own DNA restriction-modification systems and create a fucose permease deletion mutant lacking the ability to use free fucose as a carbon source.

Pyrazomycin is a rare C-nucleoside antibiotic containing a naturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRL 3601, revealing that the StrR-family regulator PyrR is the cluster-situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable-isotope feeding experiments provide support for the hypothesis that PyrN is a new nitrogen-nitrogen bond-forming enzyme that catalyzes the linkage of the ϵ-NH2 nitrogen atom of l-N6 -OH-lysine and the α-NH2 nitrogen atom of l-glutamic acid. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazole ring.

Pyrazomycin is a rare C-nucleoside antibiotic with a naturally occurring pyrazole ring, whose biosynthetic origin has remained obscure for decades. In this study, we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRL 3601, revealing that StrR-family regulator PyrR is the cluster-situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable-isotope feeding experiments support that PyrN is a new nitrogen-nitrogen bond forming enzyme linking the ε-NH2 nitrogen of l-N6-OH-lysine and α-NH2 nitrogen of l-glutamate. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes constructing the characteristic pyrazole ring.

Acyclic imines are unstable in aqueous conditions. For this reason, known imine reductases, which enable the synthesis of chiral amines, mainly intercept stable cyclic imines. Here we report the detailed biochemical and structural characterization of Bsp5, an imino acid reductase from the d-2-hydroxyacid dehydrogenase family that reduces acyclic imino acids produced

Covering: up to the end of 2018 Piperazic acid is a cyclic hydrazine and a non-proteinogenic amino acid found in diverse non-ribosomal peptide (NRP) and hybrid NRP-polyketide (PK) structures. Piperazic acid was first identified as a residue in the monamycins in 1959. Since then, the piperazic acid residue has been found in >30 families of natural products, representing >140 compounds. Many of these compounds have potent biological activity, ranging from anti-malarial to anti-apoptotic to anti-bacterial activity, although high toxicity often accompanies this potent biological activity. Recently, we identified a piperazate synthase, responsible for N-N bond formation to give piperazic acid. Here, we review piperazic acid-containing natural products discovered from 1959 to 2018, with an emphasis on the biosynthetic routes to these natural products.

Molecules containing a nitrogen-nitrogen (N-N) linkage have a variety of structures and biological activities; however, no enzyme has yet been demonstrated to catalyze N-N bond formation in an organic molecule. Here we report that the heme-dependent enzyme KtzT from Kutzneria sp. 744 catalyzes N-N bond formation in the biosynthesis of piperazate, a building block for nonribosomal peptides.

Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and β- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.

The Natural Product Reports themed issue on ‘Metalloenzymes in natural product biosynthetic pathways’ is introduced by the Guest Editors, Katherine Ryan and Catherine Drennan.

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts l-arginine to ( S)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of H

The bacterial tryptophanyl-tRNA synthetase inhibitor indolmycin features a unique oxazolinone heterocycle whose biogenetic origins have remained obscure for over 50 years. Here we identify and characterize the indolmycin biosynthetic pathway, using systematic in vivo gene inactivation, in vitro biochemical assays, and total enzymatic synthesis. Our work reveals that a phenylacetate-CoA ligase-like enzyme Ind3 catalyzes an unusual ATP-dependent condensation of indolmycenic acid and dehydroarginine, driving oxazolinone ring assembly. We find that Ind6, which also has chaperone-like properties, acts as a gatekeeper to direct the outcome of this reaction. With Ind6 present, the normal pathway ensues. Without Ind6, the pathway derails to an unusual shunt product. Our work reveals the complete pathway for indolmycin formation and sets the stage for using genetic and chemoenzymatic methods to generate indolmycin derivatives as potential therapeutic agents.

Cladoniamides are indolotryptoline natural products that derive from indolocarbazole precursors. Here, we present a microbial platform to artificially redirect the cladoniamide pathway to generate unnatural bisindoles for drug discovery. Specifically, we target glycosyltransferase, halogenase, and oxidoreductase genes from the phylogenetically-related indolocarbazole rebeccamycin and staurosporine pathways. We generate a series of novel compounds, reveal details about the substrate specificities of a number of enzymes, and set the stage for future efforts to develop new catalysts and compounds by engineering of bisindole genes. The strategy for structural diversification we use here could furthermore be applied to other natural product families with known biosynthetic genes.

Indolotryptolines are bisindole natural products isolated from microbial and eDNA sources. Here we report the sequence of transformations that convert an indolocarbazole to the indolotryptoline cladoniamide through reconstruction of the four-enzyme cascade in E. coli. This cascade involves, first, conversion of an indolocarbozole to a C4c-C7a cis diol by ClaX1; second, N-methylation by ClaM1; third, rearrangement to the indolotryptoline scaffold by ClaX2; and fourth, installation of an O-methyl group by ClaM3. We furthermore elucidate the origins of minor cladoniamides D-G as the products of non-enzymatic, base-catalyzed opening of the succinimide ring of cladonimiades A-B. Overall, this work reveals the precarious pathway indolocarbazole-derived metabolites must traverse as they are converted into indolotryptoline products and highlights the importance of non-enzymatic chemistry in generating bisindole diversity.

Recent developments in next-generation sequencing technologies have brought recognition of microbial genomes as a rich resource for novel natural product discovery. However, owing to the scarcity of efficient procedures to connect genes to molecules, only a small fraction of secondary metabolomes have been investigated to date. Transformation-associated recombination (TAR) cloning takes advantage of the natural in vivo homologous recombination of Saccharomyces cerevisiae to directly capture large genomic loci. Here we report a TAR-based genetic platform that allows us to directly clone, refactor, and heterologously express a silent biosynthetic pathway to yield a new antibiotic. With this method, which involves regulatory gene remodeling, we successfully expressed a 67-kb nonribosomal peptide synthetase biosynthetic gene cluster from the marine actinomycete Saccharomonospora sp. CNQ-490 and produced the dichlorinated lipopeptide antibiotic taromycin A in the model expression host Streptomyces coelicolor. The taromycin gene cluster (tar) is highly similar to the clinically approved antibiotic daptomycin from Streptomyces roseosporus, but has notable structural differences in three amino acid residues and the lipid side chain. With the activation of the tar gene cluster and production of taromycin A, this study highlights a unique "plug-and-play" approach to efficiently gaining access to orphan pathways that may open avenues for novel natural product discoveries and drug development.

The isolation and structure elucidation of indolotryptoline alkaloid xenocladoniamide F (8) from a cladoniamide heterologous production system is reported. The structure of 8 is determined by NMR and X-ray crystallography. Relative to the parent cladoniamides, 8 lacks any remnant of an N-methylsuccinimide ring. This pentacyclic alkaloid is the most minimal indolotryptoline natural product yet reported.

GTPases are critical molecular switches involved in a wide range of biological functions. Recent phylogenetic and genomic analyses of the large, mostly uncharacterized COG0523 subfamily of GTPases revealed a link between some COG0523 proteins and metal homeostasis pathways. In this report, we detail the bioinorganic characterization of YjiA, a representative member of COG0523 subgroup 9 and the only COG0523 protein to date with high-resolution structural information. We find that YjiA is capable of binding several types of transition metals with dissociation constants in the low micromolar range and that metal binding affects both the oligomeric structure and GTPase activity of the enzyme. Using a combination of X-ray crystallography and site-directed mutagenesis, we identify, among others, a metal-binding site adjacent to the nucleotide-binding site in the GTPase domain that involves a conserved cysteine and several glutamate residues. Mutations of the coordinating residues decrease the impact of metal, suggesting that metal binding to this site is responsible for modulating the GTPase activity of the protein. These findings point toward a regulatory function for these COG0523 GTPases that is responsive to their metal-bound state.

Bisindoles are a class of natural products derived from oxidative dimerization of tryptophan, and many of these molecules have potential use as anticancer agents. The recent isolation of new bisindoles and their corresponding gene clusters has greatly expanded the repertoire of biosynthetic genes available to synthetic biologists. This chapter describes methods to exploit the biosynthetic pathways leading to bisindoles, using cladoniamides as a representative example. Specifically, we describe how to identify and heterologously express gene clusters and how to manipulate pathways in order to generate new bisindoles. We also discuss methods for cultivating, extracting, purifying, and characterizing these new metabolites.

SummaryThe indolocarbazole biosynthetic enzymes StaC, InkE, RebC, and AtmC mediate the degree of oxidation of chromopyrrolic acid on route to the natural products staurosporine, K252a, rebeccamycin, and AT2433-A1, respectively. Here, we show that StaC and InkE, which mediate a net 4-electron oxidation, bind FAD with a micromolar Kd, whereas RebC and AtmC, which mediate a net 8-electron oxidation, bind FAD with a nanomolar Kd while displaying the same FAD redox properties. We further create RebC-10x, a RebC protein with ten StaC-like amino acid substitutions outside of previously characterized FAD-binding motifs and the complementary StaC-10x. We find that these mutations mediate both FAD affinity and product specificity, with RebC-10x displaying higher StaC activity than StaC itself. X-ray structures of this StaC catalyst identify the substrate of StaC as 7-carboxy-K252c and suggest a unique mechanism for this FAD-dependent enzyme.

Background Malaria remains a challenging and fatal infectious disease in developing nations and the urgency for the development of new drugs is even greater due to the rapid spread of anti-malarial drug resistance. While numerous parasite genetic, protein and metabolite biomarkers have been proposed for testing emerging anti-malarial compounds, they do not universally correspond with drug efficacy. The biophysical character of parasitized cells is a compelling alternative to these conventional biomarkers because parasitized erythrocytes become specifically rigidified and this effect is potentiated by anti-malarial compounds, such as chloroquine and artesunate. This biophysical biomarker is particularly relevant because of the mechanistic link between cell deformability and enhanced splenic clearance of parasitized erythrocytes. Methods Recently a microfluidic mechanism, called the multiplexed fluidic plunger that provides sensitive and rapid measurement of single red blood cell deformability was developed. Here it was systematically used to evaluate the deformability changes of late-stage trophozoite-infected red blood cells (iRBCs) after treatment with established clinical and pre-clinical anti-malarial compounds. Results It was found that rapid and specific iRBC rigidification was a universal outcome of all but one of these drug treatments. The greatest change in iRBC rigidity was observed for (+)-SJ733 and NITD246 spiroindolone compounds, which target the Plasmodium falciparum cation-transporting ATPase ATP4. As a proof-of-principle, compounds of the bisindole alkaloid class were screened, where cladoniamide A was identified based on rigidification of iRBCs and was found to have previously unreported anti-malarial activity with an IC50 lower than chloroquine. Conclusion These results demonstrate that rigidification of iRBCs may be used as a biomarker for anti-malarial drug efficacy, as well as for new drug screening. The novel anti-malarial properties of cladoniamide A were revealed in a proof-of-principle drug screen. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0957-z) contains supplementary material, which is available to authorized users.

RebC is a putative flavin hydroxylase functioning together with RebP to carry out a key step in the biosynthesis of rebeccamycin. To probe the mechanism of flavin-based chemistry in RebC, we solved the structure of RebC with reduced flavin. Upon flavin reduction, the RebC crystal undergoes a change in its unit cell dimension concurrent with a 5 A movement of the isoalloxazine ring, positioning the flavin ring adjacent to the substrate-binding pocket. Additionally, a disordered helix becomes ordered upon flavin reduction, closing off one side of the substrate-binding pocket. This structure, along with previously reported structures, increases our understanding of the RebC enzyme mechanism, indicating that either the reduction of the flavin itself or binding of substrate is sufficient to drive major conformational changes in RebC to generate a closed active site. Our finding that flavin reduction seals the active site such that substrate cannot enter suggests that our reduced flavin RebC structure is off-pathway and that substrate binding is likely to precede flavin reduction during catalysis. Along with kinetic data presented here, these structures suggest that the first cycle of catalysis in RebC may resemble that of p-hydroxybenzoate hydroxylase, with substrate binding promoting flavin reduction.

VioE, an unusual enzyme with no characterized homologues, plays a key role in the biosynthesis of violacein, a purple pigment with antibacterial and cytotoxic properties. Without bound cofactors or metals, VioE, from the bacterium Chromobacterium violaceum, mediates a 1,2 shift of an indole ring and oxidative chemistry to generate prodeoxyviolacein, a precursor to violacein. Our 1.21 Å resolution structure of VioE shows that the enzyme shares a core fold previously described for lipoprotein transporter proteins LolA and LolB. For both LolB and VioE, a bound polyethylene glycol molecule suggests the location of the binding and/or active site of the protein. Mutations of residues near the bound polyethylene glycol molecule in VioE have identified the active site and five residues important for binding or catalysis. This structural and mutagenesis study suggests that VioE acts as a catalytic chaperone, using a fold previously associated with lipoprotein transporters to catalyze the production of its prodeoxyviolacein product.

The biosynthesis of rebeccamycin, an antitumor compound, involves the remarkable eight-electron oxidation of chlorinated chromopyrrolic acid. Although one rebeccamycin biosynthetic enzyme is capable of generating low levels of the eight-electron oxidation product on its own, a second protein, RebC, is required to accelerate product formation and eliminate side reactions. However, the mode of action of RebC was largely unknown. Using crystallography, we have determined a likely function for RebC as a flavin hydroxylase, captured two snapshots of its dynamic catalytic cycle, and trapped a reactive molecule, a putative substrate, in its binding pocket. These studies strongly suggest that the role of RebC is to sequester a reactive intermediate produced by its partner protein and to react with it enzymatically, preventing its conversion to a suite of degradation products that includes, at low levels, the desired product.