Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essentialfor the catalysis

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosam ine mutase was depleted. When expressed in Escherichia coli as fusion prote ins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins display ed phosphoacetylglucosamine mutase activities, demonstrating that they inde ed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases o f divergent organisms. Mutations of the conserved amino acids found in thes e domains, which were designated region I, II, and III, respectively, demon strated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), ASp(278), and Arg(281) in region II of HsAgm1p severely dimi nished the enzyme activity and the ability to rescue the S. cerevisiae agm1 Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) di d not. These results indicate that Ser(64), Asp(278), and Arg(281) Of HsAgm 1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser (64). Furthermore, alanine substitution for Arg(496) in region III signific antly increased the K-m value for N-acetylglucosamine-6-phosphate, demonstr ating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phos phate. (C) 2000 Elsevier Science B.V. All rights reserved.