Structure of BrxA from Staphylococcus aureus, a bacilliredoxin involved in redox homeostasis in Firmicutes.

Bacilliredoxins are small proteins that are involved in redox homeostasis in bacillithiol-producing bacteria. They reduce mixed bacillithiol disulfides on protected proteins through a disulfide-exchange reaction, restoring the thiol group on the target protein. Bacilliredoxins contain an unusual conserved CGC motif, and their exact catalytic mechanism remains unclear. Here, a 1.6 Šresolution X-ray crystallographic structure of the bacilliredoxin BrxA (YphP) from Staphylococcus aureus is presented. The structure contains bacillithiol in a mixed disulfide with Cys54, as well as a disulfide linkage at Cys56, which may play a role in dimer stabilization. The structure presented here will provide insight into the function of BrxA and other bacilliredoxins.

X-ray crystallographic structure of BshB, the zinc-dependent deacetylase involved in bacillithiol biosynthesis.

Many gram-positive bacteria produce bacillithiol to aid in the maintenance of redox homeostasis and degradation of toxic compounds, including the antibiotic fosfomycin. Bacillithiol is produced via a three-enzyme pathway that includes the action of the zinc-dependent deacetylase BshB. Previous studies identified conserved aspartate and histidine residues within the active site that are involved in metal binding and catalysis, but the enzymatic mechanism is not fully understood. Here we report two X-ray crystallographic structures of BshB from Bacillus subtilis that provide insight into the BshB catalytic mechanism.

A structural and functional analysis of the glycosyltransferase BshA from Staphylococcus aureus: Insights into the reaction mechanism and regulation of bacillithiol production.

Bacillithiol is a glucosamine-derived antioxidant found in several pathogenic Gram-positive bacteria. The compound is involved in maintaining the appropriate redox state within the cell as well as detoxifying foreign agents like the antibiotic fosfomycin. Bacillithiol is produced via the action of three enzymes, including BshA, a retaining GT-B glycosyltransferase that utilizes UDP-N-acetylglucosamine and l-malate to produce N-acetylglucosaminyl-malate. Recent studies suggest that retaining GT-B glycosyltransferases like BshA utilize a substrate-assisted mechanism that goes through an SN i-like transition state. In a previous study, we relied on X-ray crystallography as well as computational simulations to hypothesize the manner in which substrates would bind the enzyme, but several questions about substrate binding and the role of one of the amino acid residues persisted. Another study demonstrated that BshA might be subject to feedback inhibition by bacillithiol, but this phenomenon was not analyzed further to determine the exact mechanism of inhibition. Here we present X-ray crystallographic structures and steady-state kinetics results that help elucidate both of these issues. Our ligand-bound crystal structures demonstrate that the active site provides an appropriate steric and geometric arrangement of ligands to facilitate the substrate-assisted mechanism. Finally, we show that bacillithiol is competitive for UDP-N-acetylglucosamine with a Ki value near 120-130 µM and likely binds within the BshA active site, suggesting that bacillithiol modulates BshA activity via feedback inhibition. The work presented here furthers our understanding of bacillithiol metabolism and can aid in the development of inhibitors to counteract resistance to antibiotics such as fosfomycin.

Structure and function of the bacillithiol-S-transferase BstA from Staphylococcus aureus.

Bacillithiol is a low-molecular weight thiol produced by many gram-positive organisms, including Staphylococcus aureus and Bacillus anthracis. It is the major thiol responsible for maintaining redox homeostasis and cellular detoxification, including inactivation of the antibiotic fosfomycin. The metal-dependent bacillithiol transferase BstA is likely involved in these sorts of detoxification processes, but the exact substrates and enzyme mechanism have not been identified. Here we report the 1.34 Å resolution X-ray crystallographic structure of BstA from S. aureus. Our structure confirms that BstA belongs to the YfiT-like metal-dependent hydrolase superfamily. Like YfiT, our structure contains nickel within its active site, but our functional data suggest that BstA utilizes zinc for activity. Although BstA and YfiT both contain a core four helix bundle and coordinate their metal ions in the same fashion, significant differences between the protein structures are described here.

A Structural, Functional, and Computational Analysis of BshA, the First Enzyme in the Bacillithiol Biosynthesis Pathway.

Bacillithiol is a compound produced by several Gram-positive bacterial species, including the human pathogens Staphylococcus aureus and Bacillus anthracis. It is involved in maintaining cellular redox balance as well as the destruction of reactive oxygen species and harmful xenobiotic agents, including the antibiotic fosfomycin. BshA, BshB, and BshC are the enzymes involved in bacillithiol biosynthesis. BshA is a retaining glycosyltransferase responsible for the first committed step in bacillithiol production, namely the addition of N-acetylglucosamine to l-malate. Retaining glycosyltransferases like BshA are proposed to utilize an SNi-like reaction mechanism in which leaving group departure and nucleophilic attack occur on the same face of the hexose. However, significant questions regarding the details of how BshA and similar enzymes accommodate their substrates and facilitate catalysis persist. Here we report X-ray crystallographic structures of BshA from Bacillus subtilis 168 bound with UMP and/or GlcNAc-mal at resolutions of 2.15 and 2.02 Å, respectively. These ligand-bound structures, along with our functional and computational studies, provide clearer insight into how BshA and other retaining GT-B glycosyltransferases operate, corroborating the substrate-assisted, SNi-like reaction mechanism. The analyses presented herein can serve as the basis for the design of inhibitors capable of preventing bacillithiol production and, subsequently, help combat resistance to fosfomycin in various pathogenic Gram-positive microorganisms.

X-ray crystallographic structure of BshC, a unique enzyme involved in bacillithiol biosynthesis.

Bacillithiol is produced by many Gram-positive bacteria via a pathway utilizing the enzymes BshA, BshB, and BshC. Here we report the 1.77 Å resolution crystal structure of BshC, the putative cysteine ligase in bacillithiol production. The structure reveals that BshC contains a core Rossmann fold with connecting peptide motifs (CP1 and CP2) and a unique α-helical coiled-coil domain that facilitates dimerization. The model contains citrate and glycerol in the canonical active site and ADP in a second binding pocket. The overall structure and bound ligands give insight into the function of this unique enzyme.

Structure and function of the genomically encoded fosfomycin resistance enzyme, FosB, from Staphylococcus aureus.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of global morbidity and mortality. Like many multi-drug-resistant organisms, S. aureus contains antibiotic-modifying enzymes that facilitate resistance to a multitude of antimicrobial compounds. FosB is a Mn(2+)-dependent fosfomycin-inactivating enzyme found in S. aureus that catalyzes nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bactericidal properties. The three-dimensional X-ray crystal structure of FosB from S. aureus (FosB(Sa)) has been determined to a resolution of 1.15 Å. Cocrystallization of FosB(Sa) with either l-Cys or BSH results in a disulfide bond between the exogenous thiol and the active site Cys9 of the enzyme. An analysis of the structures suggests that a highly conserved loop region of the FosB enzymes must change conformation to bind fosfomycin. While two crystals of FosB(Sa) contain Zn(2+) in the active site, kinetic analyses of FosB(Sa) indicated that the enzyme is inhibited by Zn(2+) for l-Cys transferase activity and only marginally active for BSH transferase activity. Fosfomycin-treated disk diffusion assays involving S. aureus Newman and the USA300 JE2 methicillin-resistant S. aureus demonstrate a marked increase in the sensitivity of the organism to the antibiotic in either the BSH or FosB null strains, indicating that both are required for survival of the organism in the presence of the antibiotic. This work identifies FosB as a primary fosfomycin-modifying pathway of S. aureus and establishes the enzyme as a potential therapeutic target for increased efficacy of fosfomycin against the pathogen.

Structural and chemical aspects of resistance to the antibiotic fosfomycin conferred by FosB from Bacillus cereus.

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.

Synthesis of bacillithiol and the catalytic selectivity of FosB-type fosfomycin resistance proteins.

Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, D-glucosamine hydrochloride, in 11 steps and 8-9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gram-positive microorganisms associated with the deactivation of the antibiotic fosfomycin. The in vitro results indicate that the preferred thiol substrate and metal ion for the FosB from Staphylococcus aureus are BSH and Ni(II), respectively. However, the metal-ion selectivity is less distinct with FosB from Bacillus subtilis, Bacillus anthracis, or Bacillus cereus.

Catalytic mechanism of perosamine N-acetyltransferase revealed by high-resolution X-ray crystallographic studies and kinetic analyses.

N-Acetylperosamine is an unusual dideoxysugar found in the O-antigens of some Gram-negative bacteria, including the pathogenic Escherichia coli strain O157:H7. The last step in its biosynthesis is catalyzed by PerB, an N-acetyltransferase belonging to the left-handed ß-helix superfamily of proteins. Here we describe a combined structural and functional investigation of PerB from Caulobacter crescentus. For this study, three structures were determined to 1.0 Å resolution or better: the enzyme in complex with CoA and GDP-perosamine, the protein with bound CoA and GDP-N-acetylperosamine, and the enzyme containing a tetrahedral transition state mimic bound in the active site. Each subunit of the trimeric enzyme folds into two distinct regions. The N-terminal domain is globular and dominated by a six-stranded mainly parallel ß-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed ß-helix, which has nearly seven turns. This region provides the scaffold for CoA binding. On the basis of these high-resolution structures, site-directed mutant proteins were constructed to test the roles of His 141 and Asp 142 in the catalytic mechanism. Kinetic data and pH-rate profiles are indicative of His 141 serving as a general base. In addition, the backbone amide group of Gly 159 provides an oxyanion hole for stabilization of the tetrahedral transition state. The pH-rate profiles are also consistent with the GDP-linked amino sugar substrate entering the active site in its unprotonated form. Finally, for this investigation, we show that PerB can accept GDP-3-deoxyperosamine as an alternative substrate, thus representing the production of a novel trideoxysugar.

Biosynthetic enzymes of unusual microbial sugars.

The biological importance of proteins and nucleic acids in the natural world is undeniable, and research efforts on these macromolecules have often overshadowed those directed at carbohydrates. It is now known, however, that carbohydrates not only play roles in energy storage and plant cell wall structure, but are also intimately involved in such processes as fertilization, the immune response, and cell adhesion. Indeed, recent years have seen an explosion in research efforts directed at uncovering and understanding new sugar moieties. The dideoxysugars and trideoxysugars, which are synthesized by a variety of bacteria, fungi, and plants, represent an especially intriguing class of carbohydrates. They are found, for example, on the lipopolysaccharides of some Gram-negative bacteria or on antibacterial agents such as erythromycin. Many of them are formed from simple monosaccharides such as glucose-6-phosphate or fructose-6-phosphate via a myriad of enzymatic reactions including acetylations, aminations, dehydrations, epimerizations, reductions, and methylations. In this review we focus on the recent structural investigations of the bacterial N-acetyltransferases and the PLP-dependent aminotransferases that function on nucleotide-linked sugar substrates.

Two site-directed mutations are required for the conversion of a sugar dehydratase into an aminotransferase.

L-colitose and d-perosamine are unusual sugars found in the O-antigens of some Gram-negative bacteria such as Escherichia coli, Vibrio cholerae, and Salmonella enterica, among others. The biosynthetic pathways for these two sugars begin with the formation of GDP-mannose from d-mannose 1-phosphate and GTP followed by the subsequent dehydration and oxidation of GDP-mannose to yield GDP-4-keto-6-deoxymannose. Following the production of GDP-4-keto-6-deoxymannose, the two pathways diverge. In the case of GDP-perosamine biosynthesis, the next step involves an amination reaction at the C-4' position of the sugar, whereas in GDP-colitose production, the 3'-hydroxyl group is removed. The enzymes catalyzing these reactions are GDP-perosamine synthase and GDP-4-keto-6-deoxymannose-3-dehydratase (ColD), respectively. Both of these enzymes are pyridoxal 5'-phosphate (PLP) dependent, and their three-dimensional structures place them into the well-characterized aspartate aminotransferase superfamily. A comparison of the active site architecture of ColD from E. coli (strain 5a, type O55:H7) to that of GDP-perosamine synthase from Caulobacter crescentus CB15 suggested that only two mutations would be required to convert ColD into an aminotransferase. Here we present a combined structural and functional analysis of the ColD S187N/H188K mutant protein that, indeed, has been converted from a sugar dehydratase into an aminotransferase.

Structural and functional studies of QdtC: an N-acetyltransferase required for the biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose.

3-Acetamido-3,6-dideoxy-alpha-D-glucose or Quip3NAc is an unusual dideoxy sugar found in the O-antigens of various Gram-negative bacteria and in the S-layer glycoprotein glycans of some Gram-positive bacteria. It is produced in these organisms as a dTDP-linked sugar, with five enzymes ultimately required for its biosynthesis. The focus of this investigation is on the enzyme QdtC, a CoA-dependent N-acetyltransferase that catalyzes the last step in the Quip3NAc biosynthetic pathway. For this analysis, three crystal structures were determined: the wild-type enzyme in the presence of acetyl-CoA and two ternary complexes of the enzyme with CoA and either dTDP-D-Quip3N or dTDP-3-amino-3,6-didexoy-alpha-D-galactose (dTDP-D-Fucp3N). Each subunit of the trimeric enzyme is dominated by a left-handed beta-helix motif with 11 turns. The three active sites are located at the subunit-subunit interfaces, and the two dTDP-sugar ligands employed in this study bind to the protein in nearly identical manners. Those residues responsible for anchoring the hexose moieties of the dTDP-sugars to the protein include Glu 141, Asn 159, and Asp 160 from one subunit and His 134 from another subunit. To probe the roles of various amino acid residues in the catalytic mechanism of the enzyme, 10 site-directed mutant proteins were constructed and their kinetic parameters measured. On the basis of these data, a catalytic mechanism is proposed for QdtC in which the acetylation of the sugar amino group does not require a catalytic base provided by the protein. Rather, the sulfur of CoA functions as the ultimate proton acceptor.

Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase.

Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.

GDP-perosamine synthase: structural analysis and production of a novel trideoxysugar.

Perosamine or 4-amino-4,6-dideoxy- d-mannose is an unusual sugar found in the O-antigens of some Gram-negative bacteria such as Vibrio cholerae O1 (the causative agent of cholera) or Escherichia coli O157:H7 (the leading cause of food-borne illnesses). It and similar deoxysugars are added to the O-antigens of bacteria via the action of glycosyltransferases that employ nucleotide-linked sugars as their substrates. The focus of this report is GDP-perosamine synthase, a PLP-dependent enzyme that catalyzes the last step in the formation of GDP-perosamine, namely, the amination of the sugar C-4'. Here we describe the three-dimensional structure of the enzyme from Caulobacter crescentus determined to a nominal resolution of 1.8 A and refined to an R-factor of 17.9%. The overall fold of the enzyme places it into the well-characterized aspartate aminotransferase superfamily. Each subunit of the dimeric enzyme contains a seven-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet, and 12 alpha-helices. Amino acid residues from both subunits form the active sites of the GDP-perosamine synthase dimer. Recently, the structure of another PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose-3-dehydratase (or ColD), was determined in our laboratory, and this enzyme employs the same substrate as GDP-perosamine synthase. Unlike GDP-perosamine synthase, however, ColD functions as a dehydratase that removes the sugar C-3' hydroxyl group. By purifying the ColD product and reacting it with purified GDP-perosamine synthase, we have produced a novel GDP-linked sugar, GDP-4-amino-3,4,6-trideoxy- d-mannose. Details describing the X-ray structural investigation of GDP-perosamine synthase and the enzymatic synthesis of GDP-4-amino-3,4,6-trideoxy- d-mannose are presented.

GDP-4-keto-6-deoxy-D-mannose 3-dehydratase, accommodating a sugar substrate in the active site.

Colitose is a dideoxysugar found in the O-antigen of the lipopolysaccharide that coats the outer membrane of some Gram-negative bacteria. Four enzymes are required for its production starting from D-mannose-1-phosphate and GTP. The focus of this investigation is GDP-4-keto-6-deoxy-D-mannose 3-dehydratase or ColD, which catalyzes the removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. The enzyme is pyridoxal 5'-phosphate-dependent, but unlike most of these proteins, the conserved lysine residue that covalently holds the cofactor in the active site is replaced with a histidine residue. Here we describe the three-dimensional structure of ColD, determined to 1.7A resolution, whereby the active site histidine has been replaced with an asparagine residue. For this investigation, crystals of the site-directed mutant protein were grown in the presence of GDP-4-amino-4,6-dideoxy-D-mannose (GDP-perosamine). The electron density map clearly reveals the presence of the sugar analog trapped in the active site as an external aldimine. The active site is positioned between the two subunits of the dimer. Whereas the pyrophosphoryl groups of the ligand are anchored to the protein via Arg-219 and Arg-331, the hydroxyl groups of the hexose only lie within hydrogen bonding distance to ordered water molecules. Interestingly, the hexose moiety of the ligand adopts a boat rather than the typically observed chair conformation. Activity assays demonstrate that this mutant protein cannot catalyze the dehydration step. Additionally, we report data revealing that wild-type ColD is able to catalyze the production of GDP-4-keto-3,6-dideoxymannose using GDP-perosamine instead of GDP-4-keto-6-deoxymannose as a substrate.

A structural study of GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: caught in the act of geminal diamine formation.

Di- and trideoxysugars are an important class of carbohydrates synthesized by certain plants, fungi, and bacteria. Colitose, for example, is a 3,6-dideoxysugar found in the O-antigens of Gram-negative bacteria such as Escherichia coli, Salmonella enterica, Yersinia pseudotuberculosis, and Vibrio cholerae, among others. These types of dideoxysugars are thought to serve as antigenic determinants and to play key roles in bacterial defense and survival. Four enzymes are required for the biochemical synthesis of colitose starting from mannose-1-phosphate. The focus of this investigation, GDP-4-keto-6-deoxy-d-mannose-3-dehydratase (ColD), catalyzes the third step in the pathway, namely the PLP-dependent removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. Whereas most PLP-dependent enzymes contain an active site lysine, ColD utilizes a histidine as its catalytic acid/base. The ping-pong mechanism of the enzyme first involves the conversion of PLP to PMP followed by the dehydration step. Here we present the three-dimensional structure of a site-directed mutant form of ColD whereby the active site histidine has been replaced with a lysine. The electron density reveals that the geminal diamine, a tetrahedral intermediate in the formation of PMP from PLP, has been trapped within the active site region. Functional assays further demonstrate that this mutant form of ColD cannot catalyze the dehydration reaction.

The structure of GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: a unique coenzyme B6-dependent enzyme.

L-colitose is a 3,6-dideoxysugar found in the O-antigens of some Gram-negative bacteria such as Escherichia coli and in marine bacteria such as Pseudoalteromonas tetraodonis. The focus of this investigation, GDP-4-keto-6-deoxy-D-mannose-3-dehydratase, catalyzes the third step in colitose production, which is the removal of the hydroxyl group at C3' of GDP-4-keto-6-deoxymannose. It is an especially intriguing PLP-dependent enzyme in that it acts as both a transaminase and a dehydratase. Here we present the first X-ray structure of this enzyme isolated from E. coli Strain 5a, type O55:H7. The two subunits of the protein form a tight dimer with a buried surface area of approximately 5000 A2. This is a characteristic feature of the aspartate aminotransferase superfamily. Although the PLP-binding pocket is formed primarily by one subunit, there is a loop, delineated by Phe 240 to Glu 253 in the second subunit, that completes the active site architecture. The hydrated form of PLP was observed in one of the enzyme/cofactor complexes described here. Amino acid residues involved in anchoring the cofactor to the protein include Gly 56, Ser 57, Asp 159, Glu 162, and Ser 183 from one subunit and Asn 248 from the second monomer. In the second enzyme/cofactor complex reported, a glutamate ketimine intermediate was found trapped in the active site. Taken together, these two structures, along with previously reported biochemical data, support the role of His 188 as the active site base required for catalysis.