The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M2+ 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 (FosBBc). The overall structure of FosBBc, at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosBBc co-crystallized with fosfomycin and a variety of divalent metals, including Ni2+, Mn2+, Co2+, and Zn2+, 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 FosBBc 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 FosBBc in complex with the L-cysteine-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn2+ metal in the active site have been determined. The L-cysteine 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 FosBBc indicated that the enzyme has a preference for bacillithiol over L-cysteine when activated by Mn2+ and is inhibited by Zn2+. The fact that Zn2+ is an inhibitor of FosBBc was used to obtain a ternary complex structure of the enzyme with both fosfomycin and L-cysteine bound.

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.

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 C39 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 ;5000 Å 2 . 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.

Abstract3-acetamido-3,6-didexoy-α-D-glucose or Quip3NAc is an unusual dideoxy sugar found in the Oantigens of various Gram-negative bacteria and in the S-layer glycoprotein glycans of some Grampositive 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-DQuip3N or dTDP-3-amino-3,6-didexoy-α-D-galactose (dTDP-D-Fucp3N). Each subunit of the trimeric enzyme is dominated by a left-handed β-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, 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, ten site-directed mutant proteins were constructed and their kinetic parameters measured. On the basis of these data, a catalytic mechanism is proposed for QdtC whereby 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.Unusual deoxysugars are found throughout Nature, often in the lipopolysaccharides of Gramnegative bacteria (1) and on various antibiotics (1,2), antifungals (3), anthelmintics (4), and antitumor drugs (5). One such sugar derivative, 3-acetamido-3,6-dideoxy-α-D-glucose or Quip3NAc, has been observed in the O-antigens of various Gram-negative bacteria including Escherichia coli O114 (6) and in the S-layer glycoprotein glycans of some Gram-positive bacteria (7). Nucleotide-activated sugar precursors such as dTDP-D-Quip3NAc serve as the building blocks for the formation of either the S-layer glycans or the O-antigens.In Thermoanaerobacterium thermosaccharolyticum E207-71, a Gram positive, anaerobic, thermophilic organism, five enzymes are required for the biosynthesis of dTDP-D-Quip3NAc starting from glucose-1-phosphate (Scheme 1). 3,6-dideoxyhexoses, the formation of this unusual sugar begins with the attachment of α-Dglucose-1-phosphate to a nucleotide via the action of glucose-1-phosphate thymidylyltransferase (RmlA). In the next step, the 6'-hydroxyl group is removed and the C-4' hydroxyl group is oxidized to a keto-functionality yielding dTDP-4-keto-6-deoxyglucose. This reaction is catalyzed by dTDP-glucose 4,6-dehydratase (RmlB). Both the thymidylyltransferase and the dehydratase have been well characterized with respect to structure and function (8).Three additional enzymes are ultimately required for the synthesis of dTDP-D-Quip3N...