Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition.

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 µM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

Unraveling the effects of peroxiredoxin 2 nitration; role of C-terminal tyrosine 193.

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Novel hyperoxidation resistance motifs in 2-Cys peroxiredoxins.

2-Cys peroxiredoxins (Prxs) modulate hydrogen peroxide (H2O2)-mediated cell signaling. At high H2O2 levels, eukaryotic Prxs can be inactivated by hyperoxidation and are classified as sensitive Prxs. In contrast, prokaryotic Prxs are categorized as being resistant to hyperoxidation and lack the GGLG and C-terminal YF motifs present in the sensitive Prxs. Additional molecular determinants that account for the subtle differences in the susceptibility to hyperoxidation remain to be identified. A comparison of a new, 2.15-Å-resolution crystal structure of Prx2 in the oxidized, disulfide-bonded state with the hyperoxidized structure of Prx2 and Prx1 in complex with sulfiredoxin revealed three structural regions that rearrange during catalysis. With these regions in hand, focused sequence analyses were performed comparing sensitive and resistant Prx groups. From this combinatorial approach, we discovered two novel hyperoxidation resistance motifs, motifs A and B, which were validated using mutagenesis of sensitive human Prxs and resistant Salmonella enterica serovar Typhimurium AhpC. Introduction and removal of these motifs, respectively, resulted in drastic changes in the sensitivity to hyperoxidation with Prx1 becoming 100-fold more resistant to hyperoxidation and AhpC becoming 800-fold more sensitive to hyperoxidation. The increased sensitivity of the latter AhpC variant was also confirmed in vivo These results support the function of motifs A and B as primary drivers for tuning the sensitivity of Prxs to different levels of H2O2, thus enabling the initiation of variable signaling or antioxidant responses in cells.

Differential Kinetics of Two-Cysteine Peroxiredoxin Disulfide Formation Reveal a Novel Model for Peroxide Sensing.

Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of sulfenic acid on the enzyme, (2) condensation of the sulfenic acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluorescence, we have studied the pH dependence of reaction 2 in human peroxiredoxins 1, 2, and 5 and in Salmonella typhimurium AhpC and obtained rate constants for the reaction and p Ka values of the thiol and sulfenic acid involved for each system. The observed reaction 2 rate constant spans 2 orders of magnitude, but in all cases, reaction 2 appears to be slow compared to the same reaction in small-molecule systems, making clear the rates are limited by conformational features of the proteins. For each Prx, reaction 2 will become rate-limiting at some critical steady-state concentration of H2O2 producing the accumulation of Prx as sulfenic acid. When this happens, an alternative and faster-resolving Prx (or other peroxidase) may take over the antioxidant role. The accumulation of sulfenic acid Prx at distinct concentrations of H2O2 is embedded in the kinetic limitations of the catalytic cycle and may constitute the basis of a H2O2-mediated redox signal transduction pathway requiring neither inactivation nor posttranslational modification. The differences in the rate constants of resolution among Prx coexisting in the same compartment may partially explain their complementation in antioxidant function and stepwise sensing of H2O2 concentration.

Experimentally Dissecting the Origins of Peroxiredoxin Catalysis.

AIMS: Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases involved in oxidant defense and signal transduction. Despite much study, the precise roles of conserved residues remain poorly defined. In this study, we carried out extensive functional and structural characterization of 10 variants of such residues in a model decameric bacterial Prx. RESULTS: Three active site proximal mutations of Salmonella typhimurium AhpC, T43V, R119A, and E49Q, lowered catalytic efficiency with hydrogen peroxide by 4-5 orders of magnitude, but did not affect reactivity toward their reductant, AhpF. pKa values of the peroxidatic cysteine were also shifted up by 1-1.3 pH units for these and a decamer disruption mutant, T77I. Except for the decamer-stabilizing T77V, all mutations destabilized decamers in the reduced form. In the oxidized form, three mutants-T77V, T43A, and T43S-exhibited stabilized decamers and were more efficiently reduced by AhpF than wild-type AhpC. Crystal structures of most mutants were solved and many showed alterations in stability of the fully folded active site loop. INNOVATION: This is the first study of Prx mutants to comprehensively assess the effects of mutations on catalytic activities, the active site cysteine pKa, and the protein structure and oligomeric status. CONCLUSION: The Arg119 side chain must be properly situated for efficient catalysis, but for other debilitating variants, the functional defects could be explained by structural perturbations and/or associated decamer destabilization rather than direct effects. This underscores the importance of our comprehensive approach. A remarkable new finding was the preference of the reductant for decamers. Antioxid. Redox Signal. 28, 521-536.

A Novel Function for the Streptococcus pneumoniae Aminopeptidase N: Inhibition of T Cell Effector Function through Regulation of TCR Signaling.

Streptococcus pneumoniae (Spn) causes a variety of disease states including fatal bacterial pneumonia. Our previous finding that introduction of Spn into an animal with ongoing influenza virus infection resulted in a CD8+ T cell population with reduced effector function gave rise to the possibility of direct regulation by pneumococcal components. Here, we show that treatment of effector T cells with lysate derived from Spn resulted in inhibition of IFNγ and tumor necrosis factor α production as well as of cytolytic granule release. Spn aminopeptidase N (PepN) was identified as the inhibitory bacterial component and surprisingly, this property was independent of the peptidase activity found in this family of proteins. Inhibitory activity was associated with reduced activation of ZAP-70, ERK1/2, c-Jun N-terminal kinase, and p38, demonstrating the ability of PepN to negatively regulate TCR signaling at multiple points in the cascade. These results reveal a novel immune regulatory function for a bacterial aminopeptidase.

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.

Endogenous, regulatory cysteine sulfenylation of ERK kinases in response to proliferative signals.

ERK-dependent signaling is key to many pathways through which extracellular signals are transduced into cell-fate decisions. One conundrum is the way in which disparate signals induce specific responses through a common, ERK-dependent kinase cascade. While studies have revealed intricate ways of controlling ERK signaling through spatiotemporal localization and phosphorylation dynamics, additional modes of ERK regulation undoubtedly remain to be discovered. We hypothesized that fine-tuning of ERK signaling could occur by cysteine oxidation. We report that ERK is actively and directly oxidized by signal-generated H2O2 during proliferative signaling, and that ERK oxidation occurs downstream of a variety of receptor classes tested in four cell lines. Furthermore, within the tested cell lines and proliferative signals, we observed that both activation loop-phosphorylated and non-phosphorylated ERK undergo sulfenylation in cells and that dynamics of ERK sulfenylation is dependent on the cell growth conditions prior to stimulation. We also tested the effect of endogenous ERK oxidation on kinase activity and report that phosphotransfer reactions are reversibly inhibited by oxidation by as much as 80-90%, underscoring the importance of considering this additional modification when assessing ERK activation in response to extracellular signals.

Peroxiredoxin Catalysis at Atomic Resolution.

Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases that guard cells against oxidative damage, are virulence factors for pathogens, and are involved in eukaryotic redox regulatory pathways. We have analyzed catalytically active crystals to capture atomic resolution snapshots of a PrxQ subfamily enzyme (from Xanthomonas campestris) proceeding through thiolate, sulfenate, and sulfinate species. These analyses provide structures of unprecedented accuracy for seeding theoretical studies, and reveal conformational intermediates giving insight into the reaction pathway. Based on a highly non-standard geometry seen for the sulfenate intermediate, we infer that the sulfenate formation itself can strongly promote local unfolding of the active site to enhance productive catalysis. Further, these structures reveal that preventing local unfolding, in this case via crystal contacts, results in facile hyperoxidative inactivation even for Prxs normally resistant to such inactivation. This supports previous proposals that conformation-specific inhibitors may be useful for achieving selective inhibition of Prxs that are drug targets.

X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action.

The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.

Structural Analysis of Streptococcus pyogenes NADH Oxidase: Conformational Dynamics Involved in Formation of the C(4a)-Peroxyflavin Intermediate.

In probing the oxygen reactivity of an Enterococcus faecalis NADH oxidase (Nox; O2 → 2H2O) C42S mutant lacking the Cys42-sulfenic acid (Cys42-SOH) redox center, we provided direct evidence of a C(4a)-peroxyflavin intermediate in the oxidative half-reaction and also described a conformational or chemical change that is rate-limiting for full reoxidation of the homodimer. In this work, the Nox from Streptococcus pyogenes (SpyNox) has been expressed and crystallized, and the overoxidized wild-type [Cys44-SOH → Cys44-sulfinic acid (Cys44-SO2H)] and C44S mutant enzyme structures have been refined at 2.0 and 2.15 Å, respectively. We show that azide binds to the two-electron reduced wild-type (EH2) enzyme and to the mutant enzyme in solution, but with a significantly higher affinity for the mutant protein. The spectral course of the titration with the SpyNox EH2 form clearly indicates progressive displacement of the Cys44-S(-) → FAD charge-transfer interaction. An azide soak with C44S Nox crystals led to the structure of the complex, as refined at 2.10 Å. The active-site N3(-) ligand is proximal to the Ser44 and His11 side chains, and a significant shift in the Ser44 side chain also appears. This provides an attractive explanation for the azide-induced loss of charge-transfer absorbance seen with the wild-type EH2 form and also permits accommodation of a C(4a)-peroxyflavin structural model. The conformation of Ser44 and the associated helical element, and the resulting steric accommodation, appear to be linked to the conformational change described in the E. faecalis C42S Nox oxidative half-reaction.

Au-ACRAMTU-PEt3 Alters Redox Balance To Inhibit T Cell Proliferation and Function.

Although T cells play a critical role in protection from viruses, bacteria, and tumors, they also cause autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. Unwanted T cell responses during organ transplant, graft-versus-host disease, and allergies are also major clinical problems. Although drugs are available to suppress unwanted immune responses, they have limited efficacy with serious side effects. Thus, new therapeutics limiting T cell activation, proliferation, and function can make an immediate clinical impact. To identify new suppressors of lymphocyte activation, proliferation, and function, we examined the immunosuppressive activity of gold(I) analogs of platinum-acridine antitumor agents. We found that the gold complex Au-ACRAMTU-PEt3 is a potent suppressor of murine and human T cell activation. Preincubation with Au-ACRAMTU-PEt3 suppresses the proliferation of CD4(+) and CD8(+) T cells at a similar concentration as pharmaceutical grade cyclosporine A. Au-ACRAMTU-PEt3 pretreatment decreases the production of IFN-γ, TNF-α, IL-2, and IL-17 by human and murine CD4(+) and CD8(+) T cells. When mice were treated with Au-ACRAMTU-PEt3 during viral infection, the expansion of virus-specific CD8(+) T cells was decreased 10-fold and viral load was elevated. Taken together, these results demonstrate that Au-ACRAMTU-PEt3 has potent immunosuppressive activity that could be used to suppress immune responses during transplantation and autoimmunity.

Peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling.

Peroxiredoxins (Prxs) are a ubiquitous family of cysteine-dependent peroxidase enzymes that play dominant roles in regulating peroxide levels within cells. These enzymes, often present at high levels and capable of rapidly clearing peroxides, display a remarkable array of variations in their oligomeric states and susceptibility to regulation by hyperoxidative inactivation and other post-translational modifications. Key conserved residues within the active site promote catalysis by stabilizing the transition state required for transferring the terminal oxygen of hydroperoxides to the active site (peroxidatic) cysteine residue. Extensive investigations continue to expand our understanding of the scope of their importance as well as the structures and forces at play within these critical defense and regulatory enzymes.

Dissecting peroxiredoxin catalysis: separating binding, peroxidation, and resolution for a bacterial AhpC.

Peroxiredoxins make up a ubiquitous family of cysteine-dependent peroxidases that reduce hydroperoxide or peroxynitrite substrates through formation of a cysteine sulfenic acid (R-SOH) at the active site. In the 2-Cys peroxiredoxins, a second (resolving) cysteine reacts with the sulfenic acid to form a disulfide bond. For all peroxiredoxins, structural rearrangements in the vicinity of the active site cysteine(s) are necessary to allow disulfide bond formation and subsequent reductive recycling. In this study, we evaluated the rate constants for individual steps in the catalytic cycle of Salmonella typhimurium AhpC. Conserved Trp residues situated close to both peroxidatic and resolving cysteines in AhpC give rise to large changes in fluorescence during the catalytic cycle. For recycling, AhpF very efficiently reduces the AhpC disulfide, with a single discernible step and a rate constant of 2.3 × 10(7) M(-1) s(-1). Peroxide reduction was more complex and could be modeled as three steps, beginning with a reversible binding of H2O2 to the enzyme (k1 = 1.36 × 10(8) M(-1) s(-1), and k-1 = 53 s(-1)), followed by rapid sulfenic acid generation (620 s(-1)) and then rate-limiting disulfide bond formation (75 s(-1)). Using bulkier hydroperoxide substrates with higher Km values, we found that different efficiencies (kcat/Km) for turnover of AhpC with these substrates are primarily caused by their slower rates of binding. Our findings indicate that this bacterial peroxiredoxin exhibits rates for both reducing and oxidizing parts of the catalytic cycle that are among the fastest observed so far for this diverse family of enzymes.

The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin.

To reduce peroxides, peroxiredoxins (Prxs) require a key "peroxidatic" Cys that, in a substrate-ready fully folded (FF) conformation, is oxidized to sulfenic acid and then, after a local unfolding (LU) of the active site, forms a disulfide bond with a second "resolving" Cys. For Salmonella typhimurium alkyl hydroperoxide reductase C (StAhpC) and some other Prxs, the FF structure is only known for a peroxidatic Cys→Ser variant, which may not accurately represent the wild-type enzyme. Here, we obtain the structure of authentic reduced wild-type StAhpC by dithiothreitol treatment of disulfide form crystals that fortuitously accommodate both the LU and FF conformations. The unique environment of one molecule in the crystal reveals a thermodynamic linkage between the folding of the active site loop and C-terminal regions, and comparisons with the Ser variant show structural and mobility differences from which we infer that the Cys→Ser mutation stabilizes the FF active site. A structure for the C165A variant (a resolving Cys to Ala mutant) in the same crystal form reveals that this mutation destabilizes the folding of the C-terminal region. These structures prove that subtle modifications to Prx structures can substantially influence enzymatic properties. We also present a simple thermodynamic framework for understanding the various mixtures of FF and LU conformations seen in these structures. On the basis of this framework, we rationalize how physiologically relevant regulatory post-translational modifications may modulate activity, and we propose a nonconventional strategy for designing selective Prx inhibitors.

Evaluating peroxiredoxin sensitivity toward inactivation by peroxide substrates.

Peroxiredoxins (Prxs) not only are very effective peroxide-reducing enzymes but also are susceptible to being oxidatively inactivated by their own substrates. The level of sensitivity to such hyperoxidation varies depending on both the enzyme involved and the type of peroxide substrate. For some Prxs, the hyperoxidation has physiological relevance, so it is important to define approaches that can be used to quantify sensitivity. Here, we describe three distinct approaches that can be used to obtain quantitative or semiquantitative estimates of Prx sensitivity and define C(hyp1%) as a simple way of quantifying sensitivity so that values can easily be compared.

Kinetic and thermodynamic features reveal that Escherichia coli BCP is an unusually versatile peroxiredoxin.

In Escherichia coli, bacterioferritin comigratory protein (BCP) is a peroxiredoxin (Prx) that catalyzes the reduction of H(2)O(2) and organic hydroperoxides. This protein, along with plant PrxQ, is a founding member of one of the least studied subfamilies of Prxs. Recent structural data have suggested that proteins in the BCP/PrxQ group can exist as monomers or dimers; we report here that, by analytical ultracentrifugation, both oxidized and reduced E. coli BCP behave as monomers in solution at concentrations as high as 200 µM. Unexpectedly, thioredoxin (Trx1)-dependent peroxidase assays conducted by stopped-flow spectroscopy demonstrated that V(max,app) increases with increasing Trx1 concentrations, indicating a nonsaturable interaction (K(m) > 100 µM). At a physiologically reasonable Trx1 concentration of 10 µM, the apparent K(m) value for H(2)O(2) is ~80 µM, and overall, the V(max)/K(m) for H(2)O(2), which remains constant at the various Trx1 concentrations (consistent with a ping-pong mechanism), is ~1.3 × 10(4) M(-1) s(-1). Our kinetic analyses demonstrated that BCP can utilize a variety of reducing substrates, including Trx1, Trx2, Grx1, and Grx3. BCP exhibited a high redox potential of -145.9 ± 3.2 mV, the highest to date observed for a Prx. Moreover, BCP exhibited a broad peroxide specificity, with comparable rates for H(2)O(2) and cumene hydroperoxide. We determined a pK(a) of ~5.8 for the peroxidatic cysteine (Cys45) using both spectroscopic and activity titration data. These findings support an important role for BCP in interacting with multiple substrates and remaining active under highly oxidizing cellular conditions, potentially serving as a defense enzyme of last resort.

Measurement of peroxiredoxin activity.

Peroxiredoxins are cysteine-dependent peroxidases that react with hydrogen peroxide, larger hydroperoxide substrates, and peroxynitrite. Protocols are provided to measure Prx activity with peroxide by (1) a coupled reaction with NADPH, thioredoxin reductase, and thioredoxin, (2) the direct monitoring of thioredoxin oxidation, (3) competition with horseradish peroxidase, and (4) peroxide consumption using the FOX assay.

Conformational studies of the robust 2-Cys peroxiredoxin Salmonella typhimurium AhpC by solution phase hydrogen/deuterium (H/D) exchange monitored by electrospray ionization mass spectrometry.

This is the first comprehensive HX-MS study of a "robust" 2-Cys peroxiredoxin (Prx), namely Salmonella typhimurium AhpC (StAhpC). Prx proteins control intracellular peroxide levels and are abundant antioxidant proteins in eukaryotes, archaea and bacteria. Crystal structural analyses and structure/activity studies of several bacterial and mammalian 2-Cys Prxs have revealed that the activity of 2-Cys Prxs is regulated by redox-dependent oligmerization and a sensitivity of the active site cysteine residue to overoxidation. The propensity to overoxidation is linked to the conformational flexibility of the peroxidatic active site loop. The HX-MS results emphasize the modulation of the conformational motility of the active site loop by disulfide formation. To obtain information on the conformational impact of decamer formation on the active site loop motility, mutants with Thr77 substituted by Ile, a decamer-disrupting mutation or by Val, a decamer-stabilizing mutation, were studied. For the isoleucine mutant, enhanced mobility was observed for regions encompassing the α4 helix located in the dimer-dimer interface and regions surrounding the peroxidatic loop. In contrast, the T77V mutation resulted in an increase in conformational stability in most regions of the protein except for the active site loop and the region encompassing the resolving cysteine.

Characterization of the N-acetyl-α-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis .

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-d-glucosaminyl l-malate (GlcN-malate) from UDP-GlcNAc and l-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-d-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.