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J Biol Chem, Vol. 273, Issue 31, 19618-19624, July 31, 1998

Structural and Functional Characterization of Streptomyces plicatus -N-Acetylhexosaminidase by Comparative Molecular Modeling and Site-directed Mutagenesis

Brian L. Mark, Gregory A. Wasney, Tim J. S. Salo, Amir R. Khan^¶, Zhimin Cao, Phillips W. Robbins^**, Michael N. G. James^¶, and Barbara L. Triggs-Raine

From the Departments of  Biochemistry and Molecular Biology and  Human Genetics, University of Manitoba, Winnipeg, Manitoba, R3E 0W3, Canada, the ^¶ Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada, and the ^** Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

We have sequenced the Streptomyces plicatus -N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human -N-acetylhexosaminidases whose deficiency results in various forms of G_M2 gangliosidosis. Based upon the x-ray structure of Serratia marcescens chitobiase (SmChb), we generated a three-dimensional model of SpHex by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human -N-acetylhexosaminidases, with differences being confined mainly to loop regions. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb x-ray structure, we selected and mutated putative SpHex active site residues. Arg¹⁶²  His mutation increased K_m 40-fold and reduced V_max 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg¹⁷⁸ in human -N-acetylhexosaminidase A (HexA) and Arg³⁴⁹ in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consistent with our three-dimensional model of SpHex. Glu³¹⁴  Gln reduced V_max 296-fold, reduced K_m 7-fold, and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp²⁴⁶  Asn reduced V_max 2-fold and increased K_m only 1.2-fold, suggesting that Asp²⁴⁶ may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the x-ray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.

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