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

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

Brian L. MarkDagger , Gregory A. WasneyDagger , Tim J. S. SaloDagger , Amir R. Khan, Zhimin CaoDagger , Phillips W. Robbins**, Michael N. G. James, and Barbara L. Triggs-RaineDagger Dagger Dagger

From the Departments of Dagger  Biochemistry and Molecular Biology and Dagger Dagger  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 beta -N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human beta -N-acetylhexosaminidases whose deficiency results in various forms of GM2 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 beta -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 right-arrow His mutation increased Km 40-fold and reduced Vmax 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg178 in human beta -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 right-arrow Gln reduced Vmax 296-fold, reduced Km 7-fold, and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246 right-arrow Asn reduced Vmax 2-fold and increased Km 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.


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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