SEQUENCE, STRUCTURAL, FUNCTIONAL, AND PHYLOGENETIC ANALYSES OF 3 GLYCOSIDASE FAMILIES

Glycosidases, which cleave the glycosidic bond between a carbohydrate and another moiety, have been classified into over 63 families. Here, a variety of computational techniques have been employed to examine th ree families important in normal and abnormal pathology with the aim o f developing a framework for future homology modeling, experimental an d other studies. Family 1 includes bacterial and archaeal enzymes as w ell as lactase phlorizin-hydrolase and klotho, glycosidases implicated in disaccharide intolerance II and aging respectively. A statistical model, a hidden Markov model (HMM), for the family I glycosidase domai n was trained and used as the basis for comparative examination of the conserved and variable sequence and structural features as well as th e phylogenetic relationships between family members. Although the stru ctures of four family I glycosidases have been determined, this is the first comparative examination of all these enzymes. Aspects that are unique to specific members or subfamilies (substrate binding loops) as well those common to all members (a (beta/alpha)(8) barrel fold) have been defined, Active site residues in some domains in klotho and lact ase-phlorizin hydrolases differ from other members and in one instance may bind but not cleave substrate. The four invariant and most highly conserved residues are not residues implicated in catalysis and/or su bstrate binding. Of these, a histidine may be involved in transition s tate stabilization. Glucosylceramidase (family 30) and galactosylceram idase (family 59) are mutated in the lysosomal storage disorders Gauch er disease and Krabbe disease, respectively HMM-based analysis, struct ure prediction studies and examination of disease mutations reveal a g lycosidase domain common to these two families that also occurs in som e bacterial glycosidases. Similarities in the reactions catalyzed by f amilies 30 and 59 are reflected in the presence of a structurally and functionally related (beta/alpha)(8) barrel fold related to that in fa mily 1.