Abstract
α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13.
Highlights
Several α-amylases and related enzymes composed of a (β/α)7-barrel were classified into the family GH57; more recently the family GH119 was established[9,10]
The first determined 3D structure of GH57 was that of the 4-α-glucanotransferase from Thermococcus litoralis (TLGT)
Isolate NL3 showed the largest clearing zone, indicating a relatively high α-amylase activity and was selected for further study. 16S rDNA sequence analysis showed that strain NL3 was most closely related to Bacillus megaterium
Summary
Screening of Kakaban lake isolates producing extracellular amylases. Eight of twenty bacterial isolates from Kakaban landlocked marine lake tested positive for the hydrolysis of starch by producing a clear halo around their colonies on red-dyed amylopectin agar plates. There is a strictly conserved aspartic acid residue succeeding the “strange” lysine, which corresponds with the position of the catalytic nucleophile (Fig. 2) The sequences of both these groups, proposed here to constitute a novel GH13 subfamily xy around the α-amylase from B. megaterium BmaN1, are all highly similar to those of α-amylases around the α-amylase from B. aquimaris BaqA (Nos 35–39 in Fig. 1) suggested recently to define a new and independent GH13 subfamily xx[19]. The evolutionary relatedness of the α-amylase from B. megaterium BmaN1, representing all its homologues with lysine and histidine in positions of the catalytic nucleophile and transition state stabilizer, respectively (Fig. 2; Nos 1–27 in Fig. 1), to members of the recently proposed GH13 subfamily around the BaqA (Nos 35–39 in Fig. 1) as well as to representatives of remaining well-established GH13 subfamilies with α-amylase specificity (Nos 40–63 in Fig. 1), is shown in the evolutionary tree (Fig. 4). Further experiments are needed to demonstrate whether the His residue is one of the catalytic residues of α-amylases
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