Role of the Glutamate 332 Residue in the Transglycosylation Activity of Thermus Maltogenic Amylase

Abstract

A sequence alignment shows that residue 332 is conserved as glutamate in maltogenic amylases (MAases) and in other related enzymes such as cyclodextrinase and neopullulanase, while the corresponding position is conserved as histidine in alpha-amylases. We analyzed the role of Glu332 in the hydrolysis and the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation activity significantly, but enhanced hydrolysis activity on alpha-(1,3)-, alpha-(1,4)-, and alpha-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chain at position 332 of MAase plays an important role in the formation and accumulation of transfer products by modulating the relative rates of hydrolysis and transglycosylation. From the structure, we propose that an acidic side chain at position 332, which is located in a pocket, is involved in aligning the acceptor molecule to compete with water molecules in the nucleophilic attack of the glycosyl-enzyme intermediate.

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

“…The similarity between the two groups implies an evolutionary relationship, as observed previously for a cluster of bacterial enzymes with similarity to animal aamylases, suggested to be a result of horizontal gene transfer (Da Lage et al, 2004). The bacterial a-amylases belonging to subfamily GH13_5 include enzymes from Bacillus and Cytophaga species (Yuuki et al, 1985;Jeang et al, 2002;Kanai et al, 2004), some of which are thermostable (Kim et al, 2000) and used in industry (Guzman-Maldonado & Paredes-Lopez, 1995). Several of these bacterial enzymes are known to produce oligosaccharides of specific lengths from starch, including, for example, a maltohexaose-forming a-amylase from Bacillus (Kanai et al, 2004).…”

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

“…The A231/F260/F184 residues at the +1/+2 subsites are completely conserved in CGTases but are replaced by other amino acids at the same position in R-amylases (36) ( Table 1). Mutations at these subsites have previously highlighted this acceptor region to be essential in the determination of the reaction specificities of GH clan H enzymes (19,(37)(38)(39)(40)(41)(42). The drastic reduction of cyclization activity by these moderately bulkier substitutes A231T/V, A231V/F260W, may be explained by the inefficiency of the final step of the transglycosylation reaction.…”

Site-Directed Mutagenesis as Applied to Biocatalysts

“…6) supported our hypothesis of the relationship between the presence of an aromatic residue at the position equivalent to Y377 in neopullulanase and the transglycosylation activity of the enzyme and a saccharifying profile. We identified other residues in subsite +1 that are involved in the transglycosylation activity of other glycosyl hydrolases (Kim et al, 2000;Leemhuis et al, 2004;van der Veen et al, 2001). One of these (H222) is part of the second highly conserved region among glycosyl hydrolases and has also been implicated in calcium ion coordination.…”