Structural and functional characterization of Streptomyces plicatus β- N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis

Abstract

We have sequenced the Streptomyces plicatus β-N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human β-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 β-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 β-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.