Asparagine 23 and aspartate 305 are essential residues in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transf er of the intact enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 3'- hydroxyl group of UDP-N-acetylglucosamine (UDPNAG). This reaction constitut es the first committed step in the biosynthesis of the bacterial cell wail component peptidoglycan (murein). The transfer reaction involves the nucleo philic attack of the 3'-hydroxyl group of UDPNAG at the C-2 of PEP. The thr ee-dimensional structure of MurA complexed with UDPNAG revealed an aspartat e residue (D305 in the En. cloacae sequence) close to the 3'-hydroxyl group of UDPNAG, suggesting that it may act as an acid--base catalyst in the act ive center of the enzyme. In addition to aspartate 305, aspargaine 23 also interacts with the 3'-hydroxyl group; however, its role in catalysis or bin ding of the UDPNAG substrate is unclear. To gain information on the role of these two amino acids in the MurA-catalyzed reaction we have exchanged D30 5 for alanine, cysteine, histidine, and glutamate, and N23 for alanine and serine using site-directed mutagenesis. While the D305 alanine, cysteine, a nd histidine mutant proteins do not have detectable enzymatic activity, the D305E mutant protein exhibits a low residual activity (ca. 0.1% of the wil d-type enzyme). Unlike with wild-type MurA, no exothermic signal was obtain ed when the D305A and -E mutant proteins were titrated with UDPNAG, demonst rating that the affinity of the sugar nucleotide substrate is reduced as a result of the amino acid exchange. The reduced affinity to UDPNAG leads to a lower propensity of C115 to form either the O-phosphothioketal with PEP o r the thioether with the antibiotic fosfomycin. These findings emphasize th e dual role of D305 as a general base and an essential binding partner to U DPNAG in the active site of MurA. Similarly, the two N23 mutant proteins sh owed a much lower catalytic activity although binding of UDPNAG was not as much affected as in the case of the D305 mutant proteins. This result indic ates that this amino acid residue is mainly involved in stabilization of tr ansition states.