Structural Modeling and Characterization of a Thermostable Lipase from Bacillus stearothermophilus P1

• Published in: Biochemical and biophysical research communications 2001-05, Vol.283 (4), p.868-875
• Main Authors: Sinchaikul, Supachok, Sookkheo, Boonyaras, Phutrakul, Suree, Wu, Ying-Ta, Pan, Fu-Ming, Chen, Shui-Tein
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacillus stearothermophilus; Bacterial Proteins; Base Sequence; Catalysis; DNA, Bacterial; Enzyme Inhibitors - pharmacology; Enzyme Stability; Geobacillus stearothermophilus - enzymology; Lipase - antagonists & inhibitors; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Secondary; structural modeling; thermostable lipase
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2001.4854

The moderate thermophilic bacterium Bacillus stearothermophilus P1 expresses a thermostable lipase that was active and stable at the high temperature. Based on secondary structure predictions and secondary structure-driven multiple sequence alignment with the homologous lipases of known three-dimensional (3-D) structure, we constructed the 3-D structure model of this enzyme and the model reveals the topological organization of the fold, corroborating our predictions. We hypothesized for this enzyme the α/β-hydrolase fold typical of several lipases and identified Ser-113, Asp-317, and His-358 as the putative members of the catalytic triad that are located close to each other at hydrogen bond distances. In addition, the strongly inhibited enzyme by 10 mM PMSF and 1-hexadecanesulfonyl chloride was indicated that it contains a serine residue which plays a key role in the catalytic mechanism. It was also confirmed by site-directed mutagenesis that mutated Ser-113, Asp-317, and His-358 to Ala and the activity of the mutant enzyme was drastically reduced.