Evidence that the fosfomycin target Cys115 in UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is essential for product release.
MurA (UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7) is an essential enzyme in the biosynthesis of the peptidoglycan layer of the bacterial cell. It provides an attractive template for the design of novel antibiotic drugs and is the target of the naturally occurring antibiotic fosfomycin, which covalently attaches to Cys115 in the active site of the enzyme. Mutations of Cys115 to Asp exist in pathogens such as Mycobacteria or Chlamydia rendering these organisms resistant to fosfomycin. Thus, there is a need for the elucidation of the role of this cysteine in the MurA reaction. We determined the x-ray structure of the C115S mutant of Enterobacter cloacae MurA, which was crystallized in the presence of the substrates of MurA. The structure depicts the product state of the enzyme with enolpyruvyl-UDP-N-acetylglucosamine and inorganic phosphate trapped in the active site. Kinetic analysis revealed that the Cys-to-Ser mutation results in an enzyme that appears to perform a single turnover of the reaction. Opposing the common view of Cys115 as a key residue in the chemical reaction of enolpyruvyl transfer, we now conclude that the wild-type cysteine is essential for product release only. On the basis of a detailed comparison of the product state with the intermediate state and an unliganded state of MurA, we propose that dissociation of the products is an ordered event with inorganic phosphate leaving first. Phosphate departure appears to trigger a suite of conformational changes, which finally leads to opening of the two-domain structure of MurA and the release of the second product enolpyruvyl-UDP-N-acetylglucosamine.
Eschenburg
Susanne
S
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Priestman
Melanie
M
Schönbrunn
Ernst
E
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003461:Crystallography; D003553:Cystine / Q000378:metabolism; D016972:Enterobacter cloacae / Q000235:genetics / Q000378:metabolism*; D005578:Fosfomycin / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D017434:Protein Structure, Tertiary
D000900:Anti-Bacterial Agents; D005578:Fosfomycin; D003553:Cystine; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase