STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF STREPTOMYCES-PLICATUS BETA-N-ACETYLHEXOSAMINIDASE BY COMPARATIVE MOLECULAR MODELING AND SITE-DIRECTED MUTAGENESIS

We have sequenced the Streptomyces plicatus beta-N-acetylhexosaminidas e (SpHex) gene and identified the encoded protein as a member of famil y 20 glycosyl hydrolases, This family includes human beta-N-acetylhexo saminidases whose deficiency results in various forms of G(M2) ganglio sidosis, Based upon the x-ray structure of Serratia marcescens chitobi ase (SmChb), we generated a three-dimensional model of SpHex by compar ative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human beta-N-acetyl hexosaminidases, with differences being confined mainly to loop region s. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb x-ray structure, we selec ted 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) i n human beta-N-acetylhexosaminidase A (HexA) and Arg(349) in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consiste nt 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, consi stent with it being the catalytic acid residue as suggested by our mod el and other studies. Asp(246) --> Asn reduced V-max 2-fold and increa sed 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 xr ay structure of SmChb, these studies suggest a common catalytic mechan ism for family 20 glycosyl hydrolases.