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

• Published in: Biochemistry (Easton) 2011-03, Vol.50 (12), p.2205-2212
• Main Authors: Jiang, Shan, Gilpin, Meghann E, Attia, Menat, Ting, Yi-Lee, Berti, Paul J
• Format: Article
• Language: English
• Subjects: Alkyl and Aryl Transferases - chemistry; Alkyl and Aryl Transferases - genetics; Alkyl and Aryl Transferases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Aspartic Acid; Biocatalysis; Borrelia burgdorferi - enzymology; Borrelia burgdorferi - physiology; Catalytic Domain; Codon, Initiator - genetics; Drug Resistance, Bacterial - genetics; Fosfomycin - pharmacology; Hydrogen-Ion Concentration; Kinetics; Lyme Disease - enzymology; Molecular Sequence Data; Mutation; Salts - pharmacology; Temperature
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi1017842

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_MurAH6). 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_MurAH6, 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.