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

Abstract

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 alpha-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 beta-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 beta-4 and beta-6, and the hevamine group, using two acidic residues on the C-termini of beta-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.

Looking at Enzymes from the Inside out: The Proximity of Catalytic Residues to the Molecular Centroid can be used for Detection of Active Sites and Enzyme–Ligand Interfaces

“…The relationship between protein fold and function is complex, 35 thus proteins with the same fold can perform many diverse biological functions [77][78][79][80] and conversely, the same function can be carried out by more than one protein fold. 16 These two claims are demonstrated by the diversity of functions of enzymes that fold as TIM barrels 81,82 (TIM barrel folds are associated Figure 6. Identification of active sites, which are formed by more than one chain, although the catalytic residues are located on a single chain.…”

Improved Catalytic Efficiency and Active Site Modification of 1,4-β-d-Glucan Glucohydrolase A from Thermotoga neapolitana by Directed Evolution

“…More characteristic of family 1 ␤-glycosidases (53,56), the I170T active site opening is considerably different from that of the wild type model in which aromatic residues, the sugar binding subsites (see above), protrude into the narrow cleft. At the bottom of both are the highly conserved glutamates, Glu-164 and Glu-349, the catalytic acid and nucleophile, respectively, of family 1 glycosyl hydrolases (22,23,53,57). The threonine for isoleucine substitution at residue 170, replacing a hydrophobic residue with a noncharged polar side chain, also changes the geometry of its immediate vicinity, presumably making new hydrogen bonds and "tightening" the turns of the short coil structure of which it is a part.…”

“…In the absence of a published crystal structure for WT GghA, a three-dimensional molecular model, based on sequence homology to Paenibacillus polymyxa (21), was derived for both wild type (WT) and mutant GghA proteins. Our analysis of the parent and mutant apoenzymes was facilitated by the breadth of structural data available for family 1 glycosyl hydrolases (22): the conservation of the eightstranded TIM barrel-fold (23); the known residues involved in positioning and substrate recognition (24 -26); the mechanism of hydrolysis by general acid catalysis and the condition of specific residues in the active site microenvironment (27,28). Comparing the enhanced GghA apoenzyme to known family 1 structures enabled us to suggest how its single amino acid substitution produces the observed functional improvement.…”

Bioinformatics Characterization of Potential New Beta-Glucuronidase from Streptococcus equi subsp. zooepidemicus

“…In fact, TIM domain in a form of eight b/a chains folded into barrel structure (Fig. 3) is a largely abundant structural component found in varying enzymes [23][24][25][26], and it is currently assigned to more than half of glycosyl hydrolases as a structural component. In addition, several studies of glycosyl hydrolases showed that the TIM barrel domain is responsible for the actual catalytic activity.…”