Lysine 22 in UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae is crucial for enzymatic activity and the formation of covalent adducts with the substrate phosphoenolpyruvate and the antibiotic fosfomycin.
UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first committed step in the biosynthesis of the bacterial cell wall component peptidoglycan. The enzyme is the target of the antibiotic fosfomycin. A lysine residue (K22), strictly conserved in MurAs and the structurally and mechanistically related 5-enolpyruvylshikimate 3-phosphate synthases (EPSPS), is located near the active center of the enzyme. This residue is thought to be involved directly in the binding of the substrate phosphoenolpyruvate (PEP) and also to participate in the conformational change leading to the formation of the catalytically competent enzyme complex. Using site-directed mutagenesis, we have replaced this lysine with arginine (K22R), valine (K22V), and glutamate (K22E). These mutant proteins were expressed, purified, and characterized in comparison to wild-type MurA and a previously described inactive C115S mutant protein. It was found that all three K22 mutant proteins had less than 0.5% of the wild-type activity. Using isothermal titration calorimetry, it could be shown that the binding parameters for the UDP-sugar nucleotide substrate are not affected by the mutations, except for the K22E mutant protein. Similarly, binding of PEP was found to be unaffected in the K22 mutant proteins as demonstrated by tryptophan fluorescence quench titrations. On the other hand, the level of formation of a covalent adduct with either PEP or fosfomycin with the thiol group of cysteine 115 was diminished. The propensity to form an adduct with PEP decreased in the following order: wild type >> K22R > K22V > K22E. A comparable effect was found on the formation of the inhibitory covalent adduct of MurA and the antibiotic fosfomycin. These results are discussed in terms of an involvement of lysine 22 in a conformational change of MurA.
Samland
A K
AK
Eidgenössische Technische Hochschule Zürich, Institut für Pflanzenwissenschaften, Switzerland.
Amrhein
N
N
Macheroux
P
P
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D016972:Enterobacter cloacae / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D005578:Fosfomycin / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics
D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D005578:Fosfomycin; D010728:Phosphoenolpyruvate; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase; D008239:Lysine