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

Abstract

A gene from Streptomyces plicatus encoding a $\beta$-N-acetylhexosaminidase (SpHex) was sequenced and the encoded protein identified as a member of family 20 glycosyl hydrolases. This family includes human $\beta$-N-acetylhexosaminidases whose deficiency results in various forms of $\rm G\sb{M2}$ gangliosidosis. Based upon the X-ray structure of Serratia marcescens chitobiase (SmChb), a three-dimensional model of SpHex was generated 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, putative SpHex active site residues were selected and mutated. An Arg162His mutation increased $K\sb{m}$ 40 fold and reduced $V\sb{max}$ 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 3-dimensional model of SpHex. Glu314Gln reduced $V\sb{max}$ 296 fold, $K\sb{m}$ 7 fold and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246Asn reduced $V\sb{max}$ 2 fold and increased $K\sb{m}$ only 1.2 fold, suggesting 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.