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

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 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 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. Arg(162) --> His mutation increased K-m 40-fold and reduced V-max 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg(178) in human beta-N-acetylhexosaminidase A (HexA) and Arg(349) in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consistent with our three-dimensional model of SpHex, Glu(314) --> 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. Asp(246) --> Asn reduced V-max 2-fold and increased K-m only 1.2-fold, suggesting that Asp(246) may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the xray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.