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

• Published in: Biochimica et biophysica acta Vol. 1492; no. 2; pp. 369 - 376
• Main Authors: Mio, Toshiyuki, Yamada-Okabe, Toshiko, Arisawa, Mikio, Yamada-Okabe, Hisafumi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 24.07.2000
• Subjects: Active site; Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Candida albicans - enzymology; Candida albicans - genetics; Catalysis; Cloning; Cloning, Molecular; DNA Mutational Analysis; DNA, Complementary - analysis; Genomic Library; Human; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; N-Acetylglucosamine; Phosphoacetylglucosamine mutase; Phosphotransferases (Phosphomutases) - genetics; Phosphotransferases (Phosphomutases) - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sequence Homology, Amino Acid; Yeast; Human; Phosphoacetylglucosamine mutase; Yeast; N-Acetylglucosamine; Cloning; Active site
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(00)00120-2

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 phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated 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 diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Δ null mutant. Conservative mutations of His 65 and Asp 276 restored detectable activities, whereas those of Ser 64, Asp 278, and Arg 281 did not. These results indicate that Ser 64, Asp 278, and Arg 281 of HsAgm1p 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 significantly increased the K m value for N-acetylglucosamine-6-phosphate, demonstrating that Arg 496 serves as a binding site for N-acetylglucosamine-6-phosphate.