Roles of the aromatic residues conserved in the active center of Saccharomycopsis .alpha.-amylase for transglycosylation and hydrolysis activity

Abstract

The molecular structure of Saccharomycopsis fibuligera alpha-amylase was predicted by a homology-based modeling technique, and the amino acid residues composing the active site were displayed with color codes according to their order of conservation. We noticed two highly conserved aromatic residues located in the active center, tyrosine 83 (Y83) and tryptophan 84 (W84), and examined their roles in catalytic activity by site-directed mutagenesis. The W, leucine (L), and asparagine (N) mutants at Y83 and the L mutant at W84 showed remarkable enhancement of transglycosylation activity and complementary decreases in native hydrolysis activity. The phenylalanine (F) mutant at Y83 and the F and Y mutants at W84 only decreased hydrolysis activity. Mechanistic and kinetic studies of these mutants using a reducing-end-blocked substrate and a hydrolysis-specific substrate revealed a probable transglycosylation mechanism and critical contributions of the 83rd and 84th aromatic residues to efficient hydrolysis. Given that aromatic residues stack against the faces of sugars, we assumed that Y83 and, presumably, W84 play roles in the binding of oligosaccharide substrates through the stacking interaction and in the indirect fixation of the catalytic water molecule through hydrogen bonding with the hydroxyl of the bound substrates. Mutations to nonaromatic residues could cause slight changes in the binding topology of substrates to favor transglycosylation over hydrolysis.

Proteolysis of α-amylase from Saccharomycopsis fibuligera: characterization of digestion products

“…A structural study of Sfamy was performed based on the similarity to Taka-amylase from A. niger (PDB entry code 7TAA) by Matsui et al (1994). The amino acids at the equal position were predicted.…”

“…The catalytic domain of an α-amylase is formed by a (β/α) 8 -barrel (TIM barrel) which has a rigid conformation. According to Matsui et al (1994), this rigidity is maintained by hydrophobic interactions in the core, which is built mainly by aromatic residues, and is strengthened by three disulfide bridges (Matsui et al 1994), and may be responsible for the resistance of the p39 fragment against proteolytic digestion.…”

“…The X-ray structure of the latter enzyme has been elucidated (Matsuura et al 1984) and consists of an N-terminal catalytic domain of 356 residues and a C-terminal one of 92 residues. The structure of Sfamy is yet to be elucidated; nevertheless Matsui et al (1994) have shown that the currently available Taka-amylase structure can be employed as a structural model for Sfamy. Based on the sequence homology, the N-terminal part of the sequence of Sfamy resembles the catalytic domain of Taka-amylase (Matsuura et al 1984).…”

Glycosyl hydrolases from Bifidobacterium adolescentis DSM20083. An overview

“…The literature shows that transglycosylation can be improved by site-directed mutagenesis of the amino acids in the vicinity of the catalytic amino acids [7,13,17]. However, the α-galactosidase of B. adolescentis belongs to GH-family 36 of which no 3D structure is currently available, and information on its catalytic residues is lacking.…”

“…The difference between lysine and arginine lies in the pK values of their basic side-chains, 10.5 and 12.5, respectively. The mutant Y500L was based on results of Matsui et al [17]. They hypothesized that, in the case of α-amylase, the aromatic ring of tyrosine was involved in the binding of oligomeric substrates through a so-called "stacking interaction", whereas the hydroxyl group of tyrosine played a role in the fixation of the catalytic water molecule.…”