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The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde-3-phosphate (d-GAP). DXP is an essential bacteria-specific metabolite that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate (PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS in a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincides with LThDP decarboxylation. This gated mechanism distinguishes DXPS in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate, while other pyruvate decarboxylases readily activate LThDP for decarboxylation, is a long-standing question in the field. We propose that an active site network functions to prevent LThDP activation on DXPS until the cosubstrate binds. Binding of d-GAP coincides with a conformational shift and disrupts the network causing changes in the active site that promote LThDP activation. Here, we show that the substitution of putative network residues, as well as nearby residues believed to contribute to network charge distribution, predictably affects LThDP reactivity. Substitutions predicted to disrupt the network have the effect to activate LThDP for decarboxylation, resulting in CO 2 and acetate production. In contrast, a substitution predicted to strengthen the network fails to activate LThDP and has the effect to shift DXPS toward the closed state. Network-disrupting substitutions near the carboxylate of LThDP also have a pronounced effect to shift DXPS to an open state. These results offer initial insights to explain the long-lived LThDP intermediate and its activation through disruption of an active site network, which is unique to DXPS. These findings have important implications for DXPS function in bacteria and its development as an antibacterial target.

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro . Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity.

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of antimicrobial resistance related deaths worldwide. Like many pathogens with multidrug-resistant strains, S. aureus contains enzymes that confer resistance through antibiotic modification(s). One such enzyme present in S. aureus is FosB, a Mn 2+ -dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. fosB gene knockout experiments show that the minimum inhibitory concentration (MIC) of fosfomycin is significantly reduced when the FosB enzyme is not present. This suggests that inhibition of FosB could be an effective method to restore fosfomycin activity. We used high-throughput in silico -based screening to identify small-molecule analogues of fosfomycin that inhibited thiol transferase activity. Phosphonoformate (PPF) was a top hit from our approach. Herein, we have characterized PPF as a competitive inhibitor of FosB from S. aureus (FosB Sa ) and Bacillus cereus (FosB Bc ). In addition, we have determined a crystal structure of FosB Bc with PPF bound in the active site. Our results will be useful for future structure-based development of FosB inhibitors that can be delivered in combination with fosfomycin in order to increase the efficacy of this antibiotic.

A key goal of synthetic biology is to enable designed modification of peptides and proteins, both in vivo and in vitro. N- and C-Terminal modification enzymes are crucial in this regard, but there are a few enzymatic options to protect peptide termini. AgeMTPT protects the N-terminus of short peptides with isoprene and the C-terminus as a methyl ester, but its substrate scope is unknown, limiting its application. Here, we investigate the substrate selectivity of the prenyltransferase domain, revealing a requirement for N-terminal aromatic amino acids, but with tolerance for diverse uncharged amino acids in the remaining positions. To demonstrate the potential of the enzyme, substrate selectivity data were used in the enzymatic modification of leu-enkephalin at the critical N-terminal residue. AgeMTPT active site mutagenesis led to an enzyme with expanded substrate scope, including the reverse geranylation of the N-termini of peptides. These data reveal potential applications of enzymatic peptide protection in synthetic biology.

An imidazolone → triazolone replacement addressed the limited passive permeability of a series of protein arginine methyl transferase 5 (PRMT5) inhibitors. This increase in passive permeability was unexpected given the increase in the hydrogen bond acceptor (HBA) count and topological polar surface area (TPSA), two descriptors that are typically inversely correlated with permeability. Quantum mechanics (QM) calculations revealed that this unusual effect was due to an electronically driven disconnect between TPSA and 3D-PSA, which manifests in a reduction in overall HBA strength as indicated by the HBA moment descriptor from COSMO-RS (conductor-like screening model for real solvation). HBA moment was subsequently deployed as a design parameter leading to the discovery of inhibitors with not only improved passive permeability but also reduced P-glycoprotein (P-gp) transport. Our case study suggests that hidden polarity as quantified by TPSA-3DPSA can be rationally designed through QM calculations.

The bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner. In addition to its role in isoprenoid biosynthesis, DXP is required for ThDP and pyridoxal phosphate biosynthesis. Due to its function as a branch-point enzyme and its demonstrated substrate and catalytic promiscuity, we hypothesize that DXPS could be key for bacterial adaptation in the dynamic metabolic landscape during infection. Prior work in the Freel Meyers laboratory has illustrated that DXPS displays relaxed specificity toward donor and acceptor substrates and varies acceptor specificity according to the donor used. We have reported that DXPS forms dihydroxyethyl (DHE)ThDP from ketoacid or aldehyde donor substrates via decarboxylation and deprotonation, respectively. Here, we tested other DHE donors and found that DXPS cleaves d-xylulose 5-phosphate (X5P) at C2-C3, producing DHEThDP through a third mechanism involving d-GAP elimination. We interrogated DXPS-catalyzed reactions using X5P as a donor substrate and illustrated (1) production of a semi-stable enzyme-bound intermediate and (2) O 2 , H + , and d-erythrose 4-phosphate act as acceptor substrates, highlighting a new transketolase-like activity of DXPS. Furthermore, we examined X5P binding to DXPS and suggest that the d-GAP binding pocket plays a crucial role in X5P binding and turnover. Overall, this study reveals a ketose-cleavage reaction catalyzed by DXPS, highlighting the remarkable flexibility for donor substrate usage by DXPS compared to other C-C bond-forming enzymes.

The thiamin diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate (donor) and d-glyceraldehyde 3-phosphate (d-GAP, acceptor). DXPS is essential in bacteria but absent in human metabolism, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that enable development of selective inhibition strategies and raise interesting questions about DXPS function in bacterial pathogens. DXPS distinguishes itself within the ThDP enzyme class by its exceptionally large active site and random sequential mechanism in DXP formation. In addition, DXPS displays catalytic promiscuity and relaxed acceptor substrate specificity, yet previous studies have suggested a preference for pyruvate as the donor substrate when d-GAP is the acceptor substrate. However, such donor specificity studies are potentially hindered by a lack of knowledge about specific, alternative donor-acceptor pairs. In this study, we exploited the promiscuous oxygenase activity of DXPS to uncover alternative donor substrates for DXPS. Characterization of glycolaldehyde, hydroxypyruvate, and ketobutyrate as donor substrates revealed differences in stabilization of enzyme-bound intermediates and acceptor substrate usage, illustrating the influence of the donor substrate on reaction mechanism and acceptor specificity. In addition, we found that DXPS prevents abortive acetyl-ThDP formation from a DHEThDP carbanion/enamine intermediate, similar to transketolase, supporting the potential physiological relevance of this intermediate on DXPS. Taken together, these results offer clues toward alternative roles for DXPS in bacterial pathogen metabolism.

The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″-hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.

The synthesis and biological evaluation of analogues of uridylpeptide antibiotics were described, and the molecular interaction between the 3'-hydroxy analogue of mureidomycin A (3'-hydroxymureidomycin A) and its target enzyme, phospho-MurNAc-pentapeptide transferase (MraY), was analyzed in detail. The structure-activity relationship (SAR) involving MraY inhibition suggests that the side chain at the urea-dipeptide moiety does not affect the MraY inhibition. However, the anti- Pseudomonas aeruginosa activity is in great contrast and the urea-dipeptide motif is a key contributor. It is also suggested that the nucleoside peptide permease NppA1A2BCD is responsible for the transport of 3'-hydroxymureidomycin A into the cytoplasm. A systematic SAR analysis of the urea-dipeptide moiety of 3'-hydroxymureidomycin A was further conducted and the antibacterial activity was determined. This study provides a guide for the rational design of analogues based on uridylpeptide antibiotics.

ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely, CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (class I and class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO ( Lr PduO) homologue. In Lr PduO, these residues are associated with ATP binding and formation of an intersubunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., class II) Listeria monocytogenes EutT ( Lm EutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of Lm EutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affected access to the substrate-binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the α-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an intersubunit salt bridge with PduO family ACATs, class II EutT family ACATs utilize an unidentified mechanism for Cbl lower-ligand displacement and reduction that is different from that of PduO and CobA family ACATs.

Tricyclic carbazole is an important scaffold in many naturally occurring metabolites, as well as valuable building blocks. Here we report the reconstitution of the ring A formation of the bacterial neocarazostatin A carbazole metabolite. We provide evidence of the involvement of two unusual aromatic polyketide proteins. This finding suggests how new enzymatic activities can be recruited to specific pathways to expand biosynthetic capacities. Finally, we leveraged our bioinformatics survey to identify the untapped capacity of carbazole biosynthesis.

The product of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase, DXP, feeds into the bacterial biosynthesis of isoprenoids, thiamin diphosphate (ThDP), and pyridoxal phosphate. DXP is essential for human pathogens but not utilized by humans; thus, DXP synthase is an attractive anti-infective target. The unique ThDP-dependent mechanism and structure of DXP synthase offer ideal opportunities for selective targeting. Upon reaction with pyruvate, DXP synthase uniquely stabilizes the predecarboxylation intermediate, C2α-lactylThDP (LThDP), in a closed conformation. Subsequent binding of d-glyceraldehyde 3-phosphate induces an open conformation that is proposed to destabilize LThDP, triggering decarboxylation. Evidence for the closed and open conformations has been revealed by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography, which indicate that H49 and H299 are involved in conformational dynamics and movement of the fork and spoon motifs away from the active site is important for the closed-to-open transition. Interestingly, H49 and H299 are critical for DXP formation and interact with the predecarboxylation intermediate in the closed conformation. H299 is removed from the active site in the open conformation of the postdecarboxylation state. In this study, we show that substitution at H49 and H299 negatively impacts LThDP formation by shifting the conformational equilibrium of DXP synthase toward an open conformation. We also present a method for monitoring the dynamics of the spoon motif that uncovered a previously undetected role for H49 in coordinating the closed conformation. Overall, our results suggest that H49 and H299 are critical for the closed, predecarboxylation state providing the first direct link between catalysis and conformational dynamics.

The cyclo -l-Trp-l-Trp (cWW, 1 ) tailoring P450 GutD 2774 from Streptomyces lavendulae was characterized by expression in Streptomyces coelicolor , precursor feeding and enzyme assays. GutD 2774 catalyzes mainly the transfer of hypoxanthine to C2 and C3 of the indole ring of 1 . cWW adducts with guanine were detected as minor products. An orthologous cluster was identified in Streptomyces xanthophaeus. These results expand the spectrum of cyclodipeptide derivatives by involvement of an additional nucleobase and identification of new coupling patterns.

Lipoproteins are essential for bacterial survival. Bacterial lipoprotein biosynthesis is accomplished by sequential modification by three enzymes in the inner membrane, all of which are emerging antimicrobial targets. The X-ray crystal structure of prolipoprotein diacylglyceryl transferase (Lgt) and apolipoprotein N -acyl transferase (Lnt) has been reported. However, the mechanisms of the post-translational modification catalyzed by these enzymes have not been understood. Here, we studied the mechanism of the transacylation reaction catalyzed by Lgt, the first enzyme for lipoprotein modification using molecular docking, molecular dynamics, and quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that Arg143, Arg239, and Glu202 play a critical role in stabilizing the glycerol-1-phosphate head group and activating the glycerol C3-O ester bond of the phosphatidylglycerol (PG) substrate. With PG binding, the opening of the L6-7 loop mediated by the highly conserved Arg236 residue as a gatekeeper is observed, which facilitates the release of the modified lipoprotein product, as well as the entry of another PG substrate. Further QM/MM studies revealed that His103 acts as a catalytic base to abstract a proton from the cysteine residue of the preproliprotein, initiating the diacylglyceryl transfer from PG to preprolipoprotein. This is the first study on the mechanism of lipoprotein modification catalyzed by a post-translocational processing enzyme. The transacylation mechanism of Lgt would shed light on the development of novel antimicrobial therapies targeting the challenging enzymes involved in the post-translocational modification pathway of lipoproteins.

We report the development of engineered myoglobin biocatalysts for executing asymmetric intramolecular cyclopropanations resulting in cyclopropane-fused γ-lactones, which are key motifs found in many bioactive molecules. Using this strategy, a broad range of allyl diazoacetate substrates were efficiently cyclized in high yields with up to 99% enantiomeric excess. Upon remodeling of the active site via protein engineering, myoglobin variants with stereodivergent selectivity were also obtained. In combination with whole-cell transformations, these biocatalysts enabled the gram-scale assembly of a key intermediate useful for the synthesis of the insecticide permethrin and other natural products. The enzymatically produced cyclopropyl-γ-lactones can be further elaborated to furnish a variety of enantiopure trisubstituted cyclopropanes. This work introduces a first example of biocatalytic intramolecular cyclopropanation and provides an attractive strategy for the stereodivergent preparation of fused cyclopropyl-γ-lactones of high value for medicinal chemistry and the synthesis of natural products.

Metalloenzymes are central to a wide range of essential biological activities, including nucleic acid modification, protein degradation, and many others. The role of metalloenzymes in these processes also makes them central for the progression of many diseases and, as such, makes metalloenzymes attractive targets for therapeutic intervention. Increasing awareness of the role metalloenzymes play in disease and their importance as a class of targets has amplified interest in the development of new strategies to develop inhibitors and ultimately useful drugs. In this Review, we provide a broad overview of several drug discovery efforts focused on metalloenzymes and attempt to map out the current landscape of high-value metalloenzyme targets.

Antibiotics are the cornerstone of modern healthcare. The 20th century discovery of sulfonamides and β-lactam antibiotics altered human society immensely. Simple bacterial infections were no longer a leading cause of morbidity and mortality, and antibiotic prophylaxis greatly reduced the risk of infection from surgery. The current healthcare system requires effective antibiotics to function. However, antibiotic-resistant infections are becoming increasingly prevalent, threatening the emergence of a postantibiotic era. To prevent this public health crisis, antibiotics with novel modes of action are needed. Currently available antibiotics target just a few cellular processes to exert their activity: DNA, RNA, protein, and cell wall biosynthesis. Bacterial central metabolism is underexploited, offering a wealth of potential new targets that can be pursued toward expanding the armamentarium against microbial infections. Discovered in 1997 as the first enzyme in the methylerythritol phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate (DXP) synthase is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylative condensation of pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to form DXP. This five-carbon metabolite feeds into three separate essential pathways for bacterial central metabolism: ThDP synthesis, pyridoxal phosphate (PLP) synthesis, and the MEP pathway for isoprenoid synthesis. While it has long been identified as a target for the development of antimicrobial agents, limited progress has been made toward developing selective inhibitors of the enzyme. This Account highlights advances from our lab over the past decade to understand this important and unique enzyme. Unlike all other known ThDP-dependent enzymes, DXP synthase uses a random-sequential mechanism that requires the formation of a ternary complex prior to decarboxylation of the lactyl-ThDP intermediate. Its large active site accommodates a variety of acceptor substrates, lending itself to a number of alternative activities, such as the production of α-hydroxy ketones, hydroxamates, amides, acetolactate, and peracetate. Knowledge gained from mechanistic and substrate-specificity studies has guided the development of selective inhibitors with antibacterial activity and provides a biochemical foundation toward understanding DXP synthase function in bacterial cells. Although it is a promising drug target, the centrality of DXP synthase in bacterial metabolism imparts specific challenges to assessing antibacterial activity of DXP synthase inhibitors, and the susceptibility of most bacteria to current DXP synthase inhibitors is remarkably culture-medium-dependent. Despite these challenges, the study of DXP synthase is poised to reveal the role of DXP synthase in bacterial metabolic adaptability during infection, ultimately providing a more complete picture of how inhibiting this crucial enzyme can be used to develop novel antibiotics.

Designing proteins with novel folds remains a major challenge, as the biophysical properties of the target fold are not known a priori and no sequence profile exists to describe its features. Therefore, most computational design efforts so far have been directed toward creating proteins that recapitulate existing folds. Here we present a strategy centered upon the design of novel intramolecular interfaces that enables the construction of a target fold from a set of starting fragments. This strategy effectively reduces the amount of computational sampling necessary to achieve an optimal sequence, without compromising the level of topological control. The solenoid architecture has been a target of extensive protein design efforts, as it provides a highly modular platform of low topological complexity. However, none of the previous efforts have attempted to depart from the natural form, which is characterized by a uniformly handed superhelical architecture. Here we aimed to design a more complex platform, abolishing the superhelicity by introducing internally alternating handedness, resulting in a novel, corrugated architecture. We employed our interface-driven strategy, designing three proteins and confirming the design by solving the structure of two examples.

Assimilation of one-carbon compounds presents a key biochemical challenge that limits their use as sustainable feedstocks for microbial growth and production. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compounds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO 2 . Importantly, the biosynthesis of serine from formate and CO 2 does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.

The bacterial metabolite 1-deoxy-d-xyulose 5-phosphate (DXP) is essential in bacterial central metabolism feeding into isoprenoid, thiamin diphosphate (ThDP), and pyridoxal phosphate de novo biosynthesis. Halting its production through the inhibition of DXP synthase is an attractive strategy for the development of novel antibiotics. Recent work has revealed that DXP synthase utilizes a unique random sequential mechanism that requires formation of a ternary complex among pyruvate-derived C2α-lactylthiamin diphosphate (LThDP), d-glyceraldehyde 3-phosphate (d-GAP), and enzyme, setting it apart from all other known ThDP-dependent enzymes. Herein, we describe the development of bisubstrate inhibitors bearing an acetylphosphonate (AP) pyruvate mimic and a distal negative charge mimicking the phosphoryl group of d-GAP, designed to target the unique form of DXP synthase that binds LThDP and d-GAP in a ternary complex. A d-phenylalanine-derived triazole acetylphosphonate (d-PheTrAP) emerged as the most potent inhibitor in this series, displaying slow, tight-binding inhibition with a K i * of 90 ± 10 nM, forward ( k 1 ) and reverse ( k 2 ) isomerization rates of 1.1 and 0.14 min -1 , respectively, and exquisite selectivity (>15000-fold) for DXP synthase over mammalian pyruvate dehydrogenase. d-PheTrAP is the most potent, selective DXP synthase inhibitor described to date and represents the first inhibitor class designed specifically to exploit the unique E-LThDP-GAP ternary complex in ThDP enzymology.

Hemoproteins have recently emerged as promising biocatalysts for promoting a variety of carbene transfer reactions including cyclopropanation and Y-H insertion (Y = N, S, Si, B). For these and synthetic carbene transfer catalysts alike, achieving high chemoselectivity toward cyclopropanation in olefin substrates bearing unprotected Y-H groups has proven remarkably challenging due to competition from the more facile carbene Y-H insertion reaction. In this report, we describe the development of a novel artificial metalloenzyme based on an engineered myoglobin incorporating a serine-ligated Co-porphyrin cofactor that is capable of offering high selectivity toward olefin cyclopropanation over N-H and Si-H insertion. Intramolecular competition experiments revealed a distinct and dramatically altered chemoselectivity of the Mb(H64V,V68A,H93S)[Co(ppIX)] variant in carbene transfer reactions compared to myoglobin-based variants containing the native histidine-ligated heme cofactor or other metal/proximal ligand substitutions. These studies highlight the functional plasticity of myoglobin as a "carbene transferase" and illustrate how modulation of the cofactor environment within this metalloprotein scaffold represents a valuable strategy for accessing carbene transfer reactivity not exhibited by naturally occurring hemoproteins or transition metal catalysts.

The field of protein conjugation most commonly refers to the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between two polypeptides, or between a single polypeptide and a new molecule (polymer, small molecule, nucleic acid, carbohydrate, etc.). Due to the modest selectivity of chemical methods for protein conjugation, there are increased efforts to develop biocatalysts that confer regioselectivity for site-specific modification, thereby complementing the existing toolbox of chemical conjugation strategies. This review summarizes key advances in the use of enzymes to functionalize proteins with commercial relevance. The examples put forth have demonstrated value at the industrial level or show promising industrial potential in the laboratory.

Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

In mammalian cells, the incorporation of the 21st amino acid, selenocysteine, into proteins is guided by the Sec machinery. The function of this protein complex requires several protein-protein and protein-RNA interactions, leading to the incorporation of selenocysteine at UGA codons. It is guided by stem-loop structures localized in the 3' untranslated regions of the selenoprotein-encoding genes. Here, we conducted a global analysis of interactions between the Sec biosynthesis and incorporation components using a bioluminescence resonance energy transfer assay in mammalian cells that showed that selenocysteine synthase (SEPSECS), SECp43, and selenophosphate synthetases SEPHS1 and SEPHS2 form oligomers in eukaryotic cells. We also showed that SEPHS2 interacts with SEPSECS and SEPHS1; these interactions were confirmed by co-immunoprecipitation. To further analyze the interactions of SECp43, the protein was expressed in Escherichia coli, and small-angle X-ray scattering analysis revealed that it is a globular protein comprising two RNA-binding domains. Using phage display, we identified potential interaction sites and highlighted two residues (K166 and P167) required for its dimerization. The SECp43 structural model presented here constitutes the basis of future exploration of the protein-protein interactions among early components of the selenocysteine biosynthesis and incorporation pathway.

Sulfur plays a critical role for the development and maintenance of life on earth, which is reflected by the wealth of primary metabolites, macromolecules, and cofactors bearing this element. Whereas a large body of knowledge has existed for sulfur trafficking in primary metabolism, the secondary metabolism involving sulfur has long been neglected. Yet, diverse sulfur functionalities have a major impact on the biological activities of natural products. Recent research at the genetic, biochemical, and chemical levels has unearthed a broad range of enzymes, sulfur shuttles, and chemical mechanisms for generating carbon-sulfur bonds. This Review will give the first systematic overview on enzymes catalyzing the formation of organosulfur natural products.

A bacterial flavin transferase (ApbE) was recently employed for flavin mononucleotide (FMN) modification on the Na + -translocating NADH:quinone oxidoreductase C (NqrC) protein in the pathogenic Gram-negative bacterium Vibrio cholerae. We employed this unique post-translational modification in mammalian cells and found that the FMN transfer reaction robustly occurred when NqrC and ApbE were genetically targeted in the cytosol of live mammalian cells. Moreover, NqrC expression in the endoplasmic reticulum (NqrC-ER) induced the retro-translocation of NqrC to the cytosol, leading to the proteasome-mediated ER-associated degradation of NqrC, which is considered to be an innate immunological response toward the bacterial protein. This unexpected cellular process of NqrC-ER could be exploited for the construction of an in cellulo proteasome inhibitor screening system, and our proposed approach yielded substantially improved results compared to a previous method. In addition, a truncated version of RnfG (half-RnfG) was found to be potentially useful as a genetically encoded tag for monitoring protein-protein interactions in a specific compartment, even in the ER, in a live cell according to its fluorogenic post-translational modification via ApbE. This new genetically encoded system in mammalian cells should serve as a valuable tool for anticancer drug screening and other applications in molecular and synthetic biology.

Recent innovations in peptide natural product biosynthesis reveal a surprising wealth of previously uncharacterized biochemical reactions that have potential applications in synthetic biology. Among these, the cyanobactins are noteworthy because these peptides are protected at their N- and C-termini by macrocyclization. Here, we use a novel bifunctional enzyme AgeMTPT to protect linear peptides by attaching prenyl and methyl groups at their free N- and C-termini. Using this peptide protectase in combination with other modular biosynthetic enzymes, we describe the total synthesis of the natural product aeruginosamide B and the biosynthesis of linear cyanobactin natural products. Our studies help to define the enzymatic mechanism of macrocyclization, providing evidence against the water exclusion hypothesis of transpeptidation and favoring the kinetic lability hypothesis.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4'-aminopyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4'-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ∼10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes induced by conformational variations in the active site. Additionally, mutagenesis studies reveal DXS to be an evolutionarily resilient enzyme. Particularly, we hypothesize that residues H82 and H304 may act in a compensatory fashion if the other is lost due to mutation. Further, nucleus-independent chemical shifts (NICSs) and aromatic stabilization energy (ASE) calculations suggest that reduction in TDP aromaticity also serves as a factor for regulating ylide formation and controlling reactivity.

The β-galactosidases from Lactobacillus reuteri L103 (Lreuβgal), Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (Lbulβgal), and Bifidobacterium breve DSM 20281 (Bbreβgal-I and Bbreβgal-II) were investigated in detail with respect to their propensity to transfer galactosyl moieties onto lactose, its hydrolysis products D-glucose and D-galactose, and certain sugar acceptors such as N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc), and L-fucose (Fuc) under defined, initial velocity conditions. The rate constants or partitioning ratios (kNu/kwater) determined for these different acceptors (termed nucleophiles, Nu) were used as a measure for the ability of a certain substance to act as a galactosyl acceptor of these β-galactosidases. When using Lbulβgal or Bbreβgal-II, the galactosyl transfer to GlcNAc was 6 and 10 times higher than that to lactose, respectively. With lactose and GlcNAc used in equimolar substrate concentrations, Lbulβgal and Bbreβgal-II catalyzed the formation of N-acetyl-allolactosamine with the highest yields of 41 and 24%, respectively, as calculated from the initial GlcNAc concentration.

The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (ΔG(⧧) = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from (4)C1 → [(4)E](⧧) → (1,4)B/(4)E to (4)C1 → [(4)H3](⧧) → (4)C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.

The present article comprehensively reviews the macromolecular synthesis using enzymes as catalysts. Among the six main classes of enzymes, the three classes, oxidoreductases, transferases, and hydrolases, have been employed as catalysts for the in vitro macromolecular synthesis and modification reactions. Appropriate design of reaction including monomer and enzyme catalyst produces macromolecules with precisely controlled structure, similarly as in vivo enzymatic reactions. The reaction controls the product structure with respect to substrate selectivity, chemo-selectivity, regio-selectivity, stereoselectivity, and choro-selectivity. Oxidoreductases catalyze various oxidation polymerizations of aromatic compounds as well as vinyl polymerizations. Transferases are effective catalysts for producing polysaccharide having a variety of structure and polyesters. Hydrolases catalyzing the bond-cleaving of macromolecules in vivo, catalyze the reverse reaction for bond forming in vitro to give various polysaccharides and functionalized polyesters. The enzymatic polymerizations allowed the first in vitro synthesis of natural polysaccharides having complicated structures like cellulose, amylose, xylan, chitin, hyaluronan, and chondroitin. These polymerizations are "green" with several respects; nontoxicity of enzyme, high catalyst efficiency, selective reactions under mild conditions using green solvents and renewable starting materials, and producing minimal byproducts. Thus, the enzymatic polymerization is desirable for the environment and contributes to "green polymer chemistry" for maintaining sustainable society.

Bacimethrin (4-amino-5-hydroxymethyl-2-methoxypyrimidine), a natural product isolated from some bacteria, has been implicated as an inhibitor of bacterial and yeast growth, as well as in inhibition of thiamin biosynthesis. Given that thiamin biosynthetic enzymes could convert bacimethrin to 2'-methoxythiamin diphosphate (MeOThDP), it is important to evaluate the effect of this coenzyme analogue on thiamin diphosphate (ThDP)-dependent enzymes. The potential functions of MeOThDP were explored on five ThDP-dependent enzymes: the human and Escherichia coli pyruvate dehydrogenase complexes (PDHc-h and PDHc-ec, respectively), the E. coli 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), and the human and E. coli 2-oxoglutarate dehydrogenase complexes (OGDHc-h and OGDHc-ec, respectively). Using several mechanistic tools (fluorescence, circular dichroism, kinetics, and mass spectrometry), it was demonstrated that MeOThDP binds in the active centers of ThDP-dependent enzymes, however, with a binding mode different from that of ThDP. While modest activities resulted from addition of MeOThDP to E. coli PDHc (6-11%) and DXPS (9-14%), suggesting that MeOThDP-derived covalent intermediates are converted to the corresponding products (albeit with rates slower than that with ThDP), remarkably strong activity (up to 75%) resulted upon addition of the coenzyme analogue to PDHc-h. With PDHc-ec and PDHc-h, the coenzyme analogue could support all reactions, including communication between components in the complex. No functional substitution of MeOThDP for ThDP was in evidence with either OGDH-h or OGDH-ec, shown to be due to tight binding of ThDP.

Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis.

A practical C-H functionalization method for the methylation of heteroarenes is presented. Inspiration from Nature's methylating agent, S-adenosylmethionine (SAM), allowed for the design and development of zinc bis(phenylsulfonylmethanesulfinate), or PSMS. The action of PSMS on a heteroarene generates a (phenylsulfonyl)methylated intermediate that can be easily separated from unreacted starting material. This intermediate can then be desulfonylated to the methylated product or elaborated to a deuteriomethylated product, and can divergently access medicinally important motifs. This mild, operationally simple protocol that can be conducted in open air at room temperature is compatible with sensitive functional groups for the late-stage functionalization of pharmacologically relevant substrates.

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M(2+)-dependent thiol tranferases that catalyze nucleophilic addition of either L-cysteine (L-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosB(Bc)). The overall structure of FosB(Bc), at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosB(Bc) cocrystallized with fosfomycin and a variety of divalent metals, including Ni(2+), Mn(2+), Co(2+), and Zn(2+), indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosB(Bc) structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate L-Cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel, positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosB(Bc) in complex with the L-Cys-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn(2+) metal in the active site have been determined. The L-Cys moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosB(Bc) indicated that the enzyme has a preference for BSH over L-Cys when activated by Mn(2+) and is inhibited by Zn(2+). The fact that Zn(2+) is an inhibitor of FosB(Bc) was used to obtain a ternary complex structure of the enzyme with both fosfomycin and L-Cys bound.

ATP:co(I)rrinoid adenosyltransferases (ACATs) are enzymes that catalyze the formation of adenosylcobalamin (AdoCbl, coenzyme B(12)) from cobalamin and ATP. There are three families of ACATs, namely, CobA, EutT, and PduO. In Salmonella enterica, CobA is the housekeeping enzyme that is required for de novo AdoCbl synthesis and for salvaging incomplete precursors and cobalamin from the environment. Here, we report the crystal structure of CobA in complex with ATP, four-coordinate cobalamin, and five-coordinate cobalamin. This provides the first crystallographic evidence of the existence of cob(II)alamin in the active site of CobA. The structure suggests a mechanism in which the enzyme adopts a closed conformation and two residues, Phe91 and Trp93, displace 5,6-dimethylbenzimidazole, the lower nucleotide ligand base of cobalamin, to generate a transient four-coordinate cobalamin, which is critical in the formation of the AdoCbl Co-C bond. In vivo and in vitro mutational analyses of Phe91 and Trp93 emphasize the important role of bulky hydrophobic side chains in the active site. The proposed manner in which CobA increases the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond appears to be analogous to that utilized by the PduO-type ACATs, where in both cases the polar coordination of the lower ligand to the cobalt ion is eliminated by placing that face of the corrin ring adjacent to a cluster of bulky hydrophobic side chains.

The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-hydroxyethyl donor with d-glyceraldehyde-3-phosphate (d-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of D-GAP, while addition of D-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and D-GAP bound, and the central enzyme-bound enamine reacts with D-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of D-GAP suggests a new approach to inhibitor design.

Many signaling proteins such as the members of the Ras superfamily of GTPases are posttranslationally modified by covalent attachment of lipid groups, which is crucial for the correct localization and function of these proteins. Numerous lipidated proteins are oncogens often found mutated in several human cancers. Therefore, several therapeutic strategies have been developed based on the inhibition of the enzymes involved in these lipidation steps. Here, we will summarize the results on protein lipidation inhibition, mainly focusing on the small molecules targeting the isoprenylation and acylation of proteins.

Carbohydrates play a central role in a wide range of biological processes. As with nucleic acids and proteins, modifications of specific sites within the glycan chain can modulate a carbohydrate's overall biological function. For example, acylation, methylation, sulfation, epimerization, and phosphorylation can occur at various positions within a carbohydrate to modulate bioactivity. Therefore, there is significant interest in identifying discrete carbohydrate modifications and understanding their biological effects. Additionally, enzymes that catalyze those modifications and proteins that bind modified glycans provide numerous targets for therapeutic intervention. This review will focus on modifications of glycans that occur after the oligomer/polymer has been assembled, generally referred to as post-glycosylational modifications.

Peptidyl nucleoside antibiotics are a group of natural products targeting MraY, a bacterial translocase involved in the lipid-linked cycle in peptidoglycan biosynthesis. In this Perspective, we explore how Nature builds complex peptidyl nucleoside antibiotics scaffolds from simple nucleoside and amino acid building blocks. We discuss the current stage of research on biosynthetic pathways for peptidyl nucleoside antibiotics, primarily focusing on chemical logic and enzymatic machinery for uridine transformation and coupling to peptides. We further survey the nonribosomal biosynthetic paradigm for a subgroup of uridyl peptide antibiotics represented by pacidamycins, concluded by diversification opportunities for antibiotic optimization.

Heparan sulfate is a highly sulfated polysaccharide that exhibits important physiological and pathological functions. The glucosamine residue of heparan sulfate can carry sulfo groups at the 2-N, 3-O, and 6-O positions, leading to diverse polysaccharide structures. 6-O-Sulfation at the glucosamine residue contributes to a wide range of biological functions. Here, we report a method for controlling the positioning of 6-O-sulfo groups in oligosaccharides. This was achieved by synthesizing oligosaccharide backbones from a disaccharide building block utilizing glycosyltransferases followed by modifications using heparan sulfate N-sulfotransferase and 6-O-sulfotransferases. This method offers a viable approach for preparing heparan sulfate oligosaccharides with precisely located 6-O-sulfo groups.

THI6 is a bifunctional enzyme found in the thiamin biosynthetic pathway in eukaryotes. The N-terminal domain of THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate, and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole in a salvage pathway. In prokaryotes, thiamin phosphate synthase and 4-methyl-5-hydroxyethylthiazole kinase are separate gene products. Here we report the first crystal structure of a eukaryotic THI6 along with several complexes that characterize the active sites responsible for the two chemical reactions. THI6 from Candida glabrata is a homohexamer in which the six protomers form a cage-like structure. Each protomer is composed of two domains, which are structurally homologous to their monofunctional bacterial counterparts. Two loop regions not found in the bacterial enzymes provide interactions between the two domains. The structures of different protein-ligand complexes define the thiazole and ATP binding sites of the 4-methyl-5-hydroxyethylthiazole kinase domain and the thiazole phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate binding sites of the thiamin phosphate synthase domain. Our structural studies reveal that the active sites of the two domains are 40 Å apart and are not connected by an obvious channel. Biochemical studies show 4-methyl-5-hydroxyethylthiazole phosphate is a substrate for THI6; however, adenosine diphospho-5β-ethyl-4-methylthiazole-2-carboxylic acid, the product of THI4, is not a substrate for THI6. This suggests that an unidentified enzyme is necessary to produce the substrate for THI6 from the THI4 product.

Kdo(2)-lipid A, a conserved substructure of lipopolysaccharide, plays critical roles in Gram-negative bacterial survival and interaction with host organisms. Inhibition of Kdo biosynthesis in Escherichia coli results in cell death and accumulation of the tetra-acylated precursor lipid IV(A). E. coli KdtA (EcKdtA) is a bifunctional enzyme that transfers two Kdo units from two CMP-Kdo molecules to lipid IV(A). In contrast, Haemophilus influenzae KdtA (HiKdtA) transfers only one Kdo unit. E. coli CMR300, which lacks Kdo transferase because of a deletion in kdtA, can be rescued to grow in broth at 37 degrees C if multiple copies of msbA are provided in trans. MsbA, the inner membrane transporter for nascent lipopolysaccharide, prefers hexa-acylated to tetra-acylated lipid A, but with the excess MsbA present in CMR300, lipid IV(A) is efficiently exported to the outer membrane. CMR300 is hypersensitive to hydrophobic antibiotics and bile salts and does not grow at 42 degrees C. Expressing HiKdtA in CMR300 results in the accumulation of Kdo-lipid IV(A) in place of lipid IV(A) without suppression of its growth phenotypes at 30 degrees C. EcKdtA restores intact lipopolysaccharide, together with normal antibiotic resistance, detergent resistance, and growth at 42 degrees C. To determine which residues are important for the mono- or bifunctional character of KdtA, protein chimeras were constructed using EcKdtA and HiKdtA. These chimeras, which are catalytically active, were characterized by in vitro assays and in vivo complementation. The N-terminal half of KdtA, especially the first 30 amino acid residues, specifies whether one or two Kdo units are transferred to lipid IV(A).

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

A study of DXP synthase has revealed flexibility in the acceptor substrate binding pocket for nonpolar substrates and has uncovered new details of the catalytic mechanism to show that pyruvate can act as both donor and acceptor substrate.

Adenosyltransferases (ATRs) catalyze the synthesis of the reactive cobalt-carbon bond found in coenzyme B(12) or 5'-deoxyadenosylcobalamin (AdoCbl), which serves as a cofactor for a number of isomerases. The reaction involves a reductive adenosylation of cob(II)alamin in which an electron delivered by a reductase reduces cob(II)alamin to cob(I)alamin, which attacks the 5'-carbon of ATP to form AdoCbl and inorganic triphosphate. Of the three classes of ATRs found in nature, the PduO type, which is also the only one found in mammals, is the most extensively studied. The crystal structures of a number of PduO-type ATRs are available and reveal a trimeric organization with the active sites located at the subunit interfaces. We have previously demonstrated that the ATR from Methylobacterium extorquens, which supports methylmalonyl-CoA mutase activity, serves dual functions; i.e., it tailors the active AdoCbl form of the cofactor and then transfers it directly to the dependent mutase (Padovani et al. (2008) Nat. Chem. Biol. 4, 194). Only two of the three active sites in ATR are simultaneously occupied by AdoCbl. In this study, we demonstrate that binding of the substrate ATP to ATR that is fully loaded with AdoCbl leads to the ejection of 1 equivalent of the cofactor into solution. In the presence of methylmalonyl-CoA mutase and ATP, AdoCbl is transferred from ATR to the acceptor protein in a process that exhibits an approximately 3.5-fold lower K(act) for ATP compared to the one in which cofactor is released into solution. Furthermore, ATP favorably influences cofactor transfer in the forward direction by reducing the ratio of apo-methylmalonyl-CoA mutase/holo-ATR required for delivery of 1 equivalent of AdoCbl, from 4 to 1. These results lead us to propose a rotary mechanism for ATR function in which, at any given time, only two of its active sites are used for AdoCbl synthesis and where binding of ATP to the vacant site leads to the transfer of the high value AdoCbl product to the acceptor mutase.

Two classes of bacteriophages, the single-stranded DNA Microviridae and the single-stranded RNA Alloleviviridae, accomplish lysis by expressing "protein antibiotics", or polypeptides that inhibit cell wall biosynthesis. Previously, we have provided genetic and physiological evidence that E, a 91-amino acid membrane protein encoded by the prototype microvirus, varphiX174, is a specific inhibitor of the translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I, from UDP-N-acetylmuramic acid-pentapeptide and the lipid carrier, undecaprenol phosphate. Here we report the first purification of E, which has been refractory to overexpression because of its lethality to Escherichia coli. Moreover, using a fluorescently labeled analogue of the sugar-nucleotide substrate, we demonstrate that E acts as a noncompetitive inhibitor of detergent-solubilized MraY, with respect to both soluble and lipid substrates. In addition, we show that the E sensitivity of five MraY mutant proteins, produced from alleles selected for resistance to E, can be correlated to the apparent affinities determined by in vivo multicopy suppression experiments. These results are inconsistent with previous reports that E inhibited membrane-embedded MraY but not the detergent-solubilized enzyme, which led to a model in which E functions by binding MraY and blocking the formation of an essential heteromultimeric complex involving MraY and other murein biosynthesis enzymes. We discuss a new model in which E binds to MraY at a site composed of the two transmembrane domains within which the E resistance mutations map and the fact that the result of this binding is a conformational change that inactivates the enzyme.

Monotopic membrane proteins bind tightly to cell membranes but do not generally span the lipid bilayer. Their interactions with lipid bilayers may be studied via coarse-grained molecular dynamics (CG-MD) simulations. Understanding such interactions is important as monotopic enzymes frequently act on hydrophobic substrates, while X-ray structures rarely provide direct information about their interactions with membranes. CG-MD self-assembly simulations enable prediction of the orientation and depth of insertion into a lipid bilayer of a monotopic protein, and also of the interactions of individual protein residues with lipid molecules. The CG-MD method has been evaluated via comparison with extended (>30 ns) atomistic simulations of monoamine oxidase, revealing good agreement between the results of coarse-grained and atomistic simulations. CG-MD simulations have been applied to a set of 11 monotopic proteins for which three-dimensional structures are available. These proteins may be divided into two groups on the basis of the results of the simulations. One group consists of those proteins which are inserted into the lipid bilayer to a limited extent, interacting mainly at the phospholipid-water interface. The second group consists of those which are inserted more deeply into the bilayer. Those monotopic proteins which are inserted more deeply cause significant local perturbation of bilayer properties such as bilayer thickness. Deeper insertion seems to correlate with a greater number of basic residues in the "foot" whereby a monotopic protein interacts with the membrane.

Aminoacyl-tRNA protein transferases catalyze the post-translational addition of amino acids to proteins. The eubacterial leucyl/phenylalanyl-tRNA-protein transferase (L/F transferase) catalyzes the transfer of leucine or phenylalanine from their respective aminoacylated tRNAs to the N-termini of substrate proteins possessing an N-terminal lysine or arginine amino acid. Conventional assays to quantify L/F transferase activity involve measuring radioactive amino acid incorporation into substrate proteins. We have developed a quantitative matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure to measure the enzymatic activity of L/F transferase. The procedure utilizes stable isotope labeled substrate and internal standard peptides. The method is used to determine the kinetic parameters of k(cat) and K(m) for the enzymatic transfer of phenylalanine and three unnatural amino acid derivatives from an aminoacyl-tRNA to a peptide substrate.