title: Lyme disease enolpyruvyl-UDP-GlcNAc synthase: fosfomycin-resistant MurA from Borrelia burgdorferi, a fosfomycin-sensitive mutant, and the catalytic role of the active site Asp.
abstract: MurAs (enolpyruvyl-UDP-GlcNAc synthases) from pathogenic bacteria such as Borrelia burgdorferi (Lyme disease) and tuberculosis are fosfomycin resistant because an Asp-for-Cys substitution prevents them from being alkylated by this epoxide antibiotic. Previous attempts to characterize naturally Asp-containing MurAs have resulted in no protein or no activity. We have expressed and characterized His-tagged Lyme disease MurA (Bb_MurA(H6)). The protein was most soluble at high salt concentrations but maximally active around physiological ionic strength. The steady-state kinetic parameters at pH 7 were k(cat) = 1.07 ± 0.03 s(-1), K(M,PEP) = 89 ± 12 μM, and K(M,UDP-GlcNAc) = 45 ± 7 μM. Mutating the active site Asp to Cys, D116C, caused a 21-fold decrease in k(cat) and rendered the enzyme fosfomycin sensitive. The pH profile of k(cat) was bell-shaped and centered around pH 5.3 for Bb_MurA(H6), with pK(a1) = 3.8 ± 0.2 and pK(a2) = 7.4 ± 0.2. There was little change in pK(a1) with the D116C mutant, 3.5 ± 0.3, but pK(a2) shifted to >11. This demonstrated that the pK(a2) of 7.4 was due to D116, almost 3 pH units above an unperturbed carboxylate, and that it must be protonated for activity. This supports D116's proposed role as a general acid/base catalyst. As fosfomycin does not react with simple thiols, nor most protein thiols, the reactivity of D116C with fosfomycin, combined with the strongly perturbed pK(a2) for D116, strongly implies an unusual active site environment and a chemical role in catalysis for Asp/Cys. There is also good evidence for C115 having a role in product release. Both roles may be operative for both Asp- and Cys-containing MurAs.
authors.affiliation: Chemical Biology Graduate Program, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
authors.forename: Shan
authors.initials: S
authors.lastname: Jiang
authors.forename: Meghann E
authors.initials: ME
authors.lastname: Gilpin
authors.forename: Menat
authors.initials: M
authors.lastname: Attia
authors.forename: Yi-Lee
authors.initials: YL
authors.lastname: Ting
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Berti
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D055162:Biocatalysis; D025065:Borrelia burgdorferi / Q000201:enzymology* / Q000502:physiology; D020134:Catalytic Domain; D018387:Codon, Initiator / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005578:Fosfomycin / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008193:Lyme Disease / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D012492:Salts / Q000494:pharmacology; D013696:Temperature
chemical_list: D018387:Codon, Initiator; D012492:Salts; D005578:Fosfomycin; D001224:Aspartic Acid; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase