The (βα)8 glycosidases: sequence and structure analyses suggest distant evolutionary relationships

There are currently at least nine distinct glycosidase sequence families which are all known to adopt a TIM barrel fold [Henrissat,B. and Davies,G. (1997) Curr. Opin. Struct. Biol., 7, 637–644]. To explore the relationships between these enzymes and their evolution, comprehensive sequence and structure comparisons were performed, generating four distinct clusters. The first cluster, S1, comprises the α-amylase related enzymes, all with the retention mechanism (axial→axial). The second cluster, S2, included two functional subgroups, one composed of various kinds of glucosidases all with the retention mechanism (equatorial→equatorial) (the so-called 4/7 superfamily), and the other subgroup including the β-amylases with the inversion mechanism (axial→ equatorial). The third cluster, S3, with the retention mechanism (equatorial→equatorial), could be subdivided, based on the catalytic residues and mechanisms, into two functional subgroups: the chitinase group, catalysed by two acidic residues on the C-termini of β-4 and β-6, and the hevamine group, using two acidic residues on the C-termini of β-4 for catalysis. The fourth cluster, S4, is composed of chitobiase with the retention mechanism (equatorial→ equatorial). These clusters are compared with the sequence families derived by Henrissat and coworkers. PSI-BLAST profiles and multiple-alignments of tertiary structures suggest that S1 and S2 are distantly related, as are S3 and S4, which have N-acetylated substrates. This work highlights the difficulties of untangling distant evolutionary relationships in ubiquitous folds such as the TIM barrel.