Use of amber suppressors to investigate the thermostability of Bacillus licheniformis alpha-amylase. Amino acid replacements at 6 histidine residues reveal a critical position at His-133.
A set of 12 Escherichia coli suppressor tRNAs, inserting different amino acids in response to an amber codon, has been used to create rapidly numerous protein variants of a thermostable amylase; by site-directed mutagenesis, amber mutations were first introduced into Bacillus licheniformis alpha-amylase gene at position His35, His133, His247, His293, His406, or His450; genes carrying one or two amber mutations were then expressed in the different suppressor strains, generating over 100 amylase variants with predicted amino acid changes that could be tested for thermostability. Within the detection limits of the assays, amino acid replacements at five histidine positions had no significant effect. In contrast, suppressed variants substituted at residue His133 clearly exhibited modified thermostability and could be either less stable or more stable than the wild-type amylase, depending on the amino acid inserted at this position; comparison of the variants indicates that the hydrophobicity of the substituting residue is an important but not a determinant factor of stabilization. The effect of the most stabilizing and destabilizing amino acid substitutions, His133 to Tyr and to Pro, respectively, were confirmed by introducing the corresponding missense mutations into the gene sequence. The advantages and limits of informational suppression in protein stability studies are discussed as well as structural features involved in the thermostability of B. licheniformis alpha-amylase.
Institut National Agronomique, Laboratoire de Génétique, Thiverval-Grignon, France.
N
N
Declerck
P
P
Joyet
C
C
Gaillardin
J M
JM
Masson
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006639:Histidine; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid; D013489:Suppression, Genetic; D013816:Thermodynamics; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D006639:Histidine; D000516:alpha-Amylases