Molecular Determinants of Substrate Recognition in Thermostable  -glucosidases Belonging to Glycoside Hydrolase Family 13

Abstract

Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.

Amino Acids in Conserved Region II Are Crucial to Substrate Specificity, Reaction Velocity, and Regioselectivity in the Transglucosylation of Honeybee GH-13 α-Glucosidases

“…16,18) The two following amino acid residues, equivalent to Pro226-Tyr227 of HBG-III, have been pointed out as the characteristic signature of GH-13 enzymes. 14) According to a recent subfamily classification of GH-13 a Velocity of -1,4-transglucosylation yielding erlose and maltotriose from sucrose and maltose respectively.…”

“…The residues following the two catalytic amino acids are thought to play important roles, in substrate binding at the þ1 and þ2 subsites in GH-13 enzymes. [14][15][16][17][18] In this study, to identify the amino acids responsible for the specific enzymatic properties of HBGs, we focused on the amino acid residues in region II (Fig. 1).…”

Structural elements responsible for the glucosidic linkage‐selectivity of a glycoside hydrolase family 13 exo‐glucosidase

“…The large introduced side‐chain presumably caused enough steric hindrance to prevent the substrate from binding. V195A showed higher maltase activity than wild‐type as shown in the equivalent mutant enzyme of Geobacillus thermoglucosidasius O16G [5]. In addition to V195A, V195D/G/S also showed higher maltase activity than wild‐type.…”

“…4)-glucosidic linkage, but the mutants retained hydrolytic activity toward the a-(1 ? 6)-linkage in all the cases [4,5]. This suggests that other important amino acid residues (i.e., structural elements) involved in the recognition of a-(1 ?…”

“…α‐(1 → 4)‐Specific glucosidases have Ala or Thr at this position, whereas Val is conserved in the α‐(1 → 6)‐specific glucosidases (Table 1 ). Mutant enzymes of α‐(1 → 6)‐specific glucosidases, in which the conserved Val and its neighboring amino acid residues were mutated, hydrolyzed the α‐(1 → 4)‐glucosidic linkage, but the mutants retained hydrolytic activity toward the α‐(1 → 6)‐linkage in all the cases [4,5]. This suggests that other important amino acid residues (i.e., structural elements) involved in the recognition of α‐(1 → 6)‐linkage are present.…”