Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilusα‐amylase

Abstract

Site-directed mutagenesis of a thermostable a-amylase from Bacillus stearothermophilus was performed to assess the role of amino acid residues near the catalytic site in catalysis. Asn329 is presumed to be adjacent to the proposed catalytic residue Asp331. Its mutation to Lys, which is found at the corresponding position in pullulanase, resulted in the loss of 99.7% of the activity, while the mutation to Asp or Val did not drastically reduce the activity. The mutation to Val altered the temperature/activity profile so that the activity was reduced to 25% of wild-type a-amylase at 60°C but was over twofold greater at 5°C. This effect could be ascribed to a decrease in the activation enthalpy by 32%. The mutation to Asp or Lys altered the pHactivity profile concomitant with possible changes in the ionization state of the groups introduced. These results show the feasibility of altering and possibly improving the enzyme activity by mutagenesis of residues near the catalytic groups.The present study was aimed to assess the role of residues near the catalytic site in catalysis and their potential use as the targets for site-directed mutagenesis in modifying enzyme function. We have used Bacillus stearothermophilus a-amylase for this study because of its heat stability. a-amylase catalyzes the hydrolysis of a -~-1 , 4 glucosidic linkages of starch and, based on the amino acid sequence similarity of the active site, may be classified in the a-amylase family [l]. This family also includes isoamylase and pullulanase that hydrolyze a-D-1,6 linkages of amylopectin and pullulan, respectively. The differences in specificity and action patterns of the enzymes in this family must reside in their sequences, but no amino acid residues have so far been identified as crucial for such differences. . The three-dimensional structure of B. stearothermophilus a-amylase has not yet been solved; however, the sequence similarity of the active site suggests that the active-site structure closely resembles that of Taka amylase A (Aspergillus oryzae a-amylase) whose three-dimensional coordinates are available [2]. Previous mutagenesis studies on B. stearothermophilus a-amylase [3,4] have shown that the three residues, Asp234, Glu264 and Asp331, are critical for catalysis. This result coincides with those of comparable studies on other aamylases [5 -71 and supports the proposals of these residues as catalytic residues [2, 6, 81. By inspection of the threedimensional structure of Taka amylase A, Am295 (Asn329 in B. stearothennophilus a-amylase) was found to be in close proximity (atomic center distances of 0.4-0.5 nm) to Asp297 (Asp331 in B. stearothermophilus). This Asn residue does not appear to be fully exposed to solvent and, although it is almost conserved among various a-amylases (Ser in some species), no function has been assigned to it. On the other hand, Pseudomonas amyloderamosa isoamylase [9] and Klebsiella aerogenes pullulanase [lo] have Val and Lys at the corresponding position, respectively. Considering the possible roles of the ...

Protein engineering in the ?-amylase family: catalytic mechanism, substrate specificity, and stability

“…Guided by a sequence alignment, Asn-339 (TAA Asn-295) in a-amylase from B. stearotherrnophilus was replaced by lysine to mimic pullulanase, by valine to mimic isoamylase, or by aspartic acid, resulting in substantial losses of activity (N329K) and smaller losses (N329V/D) without effecting the substrate affinity [79] ( Table 4). The activity of the wild-type enzyme decreased to a greater extent with decreasing temperature (60, 37, or 15 °C) than that of the N329V mutant that at 15 °C, has twice the wild-type activity.…”

“…The activity of the wild-type enzyme decreased to a greater extent with decreasing temperature (60, 37, or 15 °C) than that of the N329V mutant that at 15 °C, has twice the wild-type activity. As an example of the engineering of the pH profile, the acid mutant (N329D) showed a loss and an increase of activity above pH 7, respectively, relative to the neutral (N329V) and the basic (N329K) mutants [79]. Although highly relevant, these mutants were not analysed for e-1,6-bond hydrolysis.…”

Electrostatics in the active site of an α‐amylase

“…The α‐amylase active site has been studied extensively, e.g. [29,37,38a,39a,40a,41a]. Mutational studies show that all three acidic residues in the active site are crucial for activity, but the details of their roles in the catalytic mechanism are not yet entirely clear.…”

An isoamylase with neutral pH optimum from a Flavobacterium species: cloning, characterization and expression of the iam gene

“…isoamylase. This is similar to the case of B. stearothermophilus mutant a-amylase, in which alteration of Asn in region 4 to Asp or Lys produces a corresponding decrease or increase in pH optimum (Takase 1993). Considering that Flavobacterium sp.…”