Role of the Loop Containing Residue 115 in the Induced-Fit Mechanism of the Bacterial Cell Wall Biosynthetic Enzyme MurA
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- 作者：Ernst Schö ; nbrunn ; Susanne Eschenburg ; Florian Krekel ; Karolin Luger ; and Nikolaus Amrhein
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：March 7, 2000
- 年：2000
- 卷：39
- 期：9
- 页码：2164 - 2173
- 全文大小：638K
- 年卷期：v.39,no.9(March 7, 2000)
- ISSN：1520-4995
文摘
The induced-fit mechanism in Enterobacter cloacae MurA has been investigated by kineticstudies and X-ray crystallography. The antibiotic fosfomycin, an irreversible inhibitor of MurA, induceda structural change in UDP-N-acetylglucosamine (UDPGlcNAc)-liganded enzyme with a time dependencesimilar to that observed for the inactivation progress. The mechanism of action of fosfomycin on MurAappeared to be of the bimolecular type, the overall rate constants of inactivation and structural changebeing 
nals/bichaw/39/i09/eqn/bi991091je10001.gif"> = 104 M-1 s-1 and nals/bichaw/39/i09/eqn/bi991091je10002.gif"> = 85 M-1 s-1, respectively. Fosfomycin as well as the second MurAsubstrate, phosphoenolpyruvate (PEP), are known to interact with the side chain of Cys115. Like wild-type MurA, the catalytically inactive single-site mutant protein Cys115Ser structurally interacted withUDPGlcNAc in a rapidly reversible reaction. However, in contrast to wild-type enzyme, binding of PEPto mutant protein induced a rate-limited, biphasic structural change. Fosfomycin did not affect the structureof the mutant protein. The crystal structure of unliganded Cys115Ser MurA at 1.9 Å resolution revealedthat the overall conformation of the loop comprising residues 112-121 is not influenced by the mutation.However, other than Cys115 in wild-type MurA, Ser115 exhibits two distinct side-chain conformations.A detailed view on the loop revealed the existence of an elaborate hydrogen-bonding network mainlysupplied by water molecules, presumably stabilizing its conformation in the unliganded state. Thecomparison between the known crystal structures of MurA, together with the kinetic data obtained, suggestintermediate conformational states in the MurA reaction, in which the loop undergoes multiple structuralchanges upon ligand binding.