Deduced amino-acid sequence of a calcium-free alpha-amylase from a strain of Bacillus: implications from molecular modeling of high oxidation stability and chelator resistance of the enzyme.
Alkaline alpha-amylase (AmyK38) from the alkaliphilic Bacillus sp. strain KSM-K38 is a unique enzyme in that it is highly chelator-resistant and oxidatively stable [Hagihara, H., Igarashi, K., Hayashi, Y., Endo, K., Ikawa-Kitayama, K., Ozaki, K., Kawai, S. & Ito, S. (2001) Appl. Environ. Microbiol. 67, 1744-1750]. This enzyme was found to contain no Ca and require Na (or monovalent cations) for manifestation of activity. The nucleotide sequence of the gene for the novel enzyme was determined, and it harbored an ORF of 1503 bp encoding the enzyme of 501 amino acids, including a 21-amino-acid signal peptide. The deduced amino-acid sequence of the mature enzyme (55 097 Da) showed moderate homology to those of alpha-amylases from Bacillus licheniformis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, with approximately 63% identity. A methionine residue, which is conserved and susceptible to chemical oxidation, was replaced with leucine in AmyK38. Moreover, many conserved residues that are crucial ligands for Ca were replaced with other amino acids, thereby leading to loss of the Ca coordination geometries. By building a molecular model, we showed the calcium-independent, oxidatively stable active-site topology and structural integrity of AmyK38.
Hagihara
H
H
Tochigi Research Laboratories of Kao Corporation, Ichikai, Haga, Japan.
Hayashi
Y
Y
Endo
K
K
Igarashi
K
K
Ozawa
T
T
Kawai
S
S
Ozaki
K
K
Ito
S
S
D000468:Alkalies; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000032:analysis; D002412:Cations / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000737:chemistry* / Q000187:drug effects
D000468:Alkalies; D002412:Cations; D002614:Chelating Agents; D000516:alpha-Amylases; D002118:Calcium