Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase.
Mutations that cover the sequence of Bacillus stearothermophilus alpha-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant alpha-amylases were expressed in Escherichia coli, which also secreted the product. Ninety-eight mutants were identified by sequencing and their enzyme activities were classified into three classes: wild-type, reduced or null. A molecular model of the enzyme was constructed using the coordinates of Takaamylase A and a consensus alignment of mammalian, plant, and bacterial alpha-amylases. The location of mutant amino acids on the model indicate that mutations which destroy or decrease the catalytic activity are particularly clustered: (i) around the active site and along the substrate-binding groove and (ii) in the interface between the central alpha/beta barrel and the C-terminal domain. Exposed loops are typically tolerant towards mutations.
Holm
L
L
Biotechnical Laboratory VTT, Espoo, Finland.
Koivula
A K
AK
Lehtovaara
P M
PM
Hemminki
A
A
Knowles
J K
JK
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D000516:alpha-Amylases; D002118:Calcium