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- Title
Enhanced Substrate Specificity of Thermostable Cytochrome P450 CYP175A1 by Site Saturation Mutation on Tyrosine 68.
- Authors
Karmakar, Srabani; Nag, Sudip Kumar; Taher, Mohd; Kansara, Bharat T.; Mazumdar, Shyamalava
- Abstract
Thermostable cytochrome P450 (CYP175A1) cloned from Thermus thermophilus shows mid-point unfolding temperature (Tm) of 88 °C (361 K) along with high thermodynamic stability making it a potential industrially viable biocatalyst. Molecular docking analyses, and structural superposition with steroidogenic and fatty acid metabolizing cytochrome P450 s suggested that the tyrosine 68 may have important role in binding as well as metabolism of substrates by the enzyme. Site-saturation mutation of the tyrosine 68 residue was carried out and several unique mutations were obtained that were properly folded and showed high thermostability. We investigated the effects of variation of the single residue, Tyr68 at the substrate binding pocket of the enzyme on the substrate specificity of CYP175A1. Screening of the mutant colonies of CYP175A1 obtained after saturation mutagenesis of Tyr68 using saturated fatty acid, myristic acid and poly unsaturated fatty acids showed that the Y68K had notable binding and catalytic activity for mono-oxygenation of the saturated fatty acid (myristic acid), which had no major detectable binding affinity towards the WT enzyme. The Y68R mutant of CYP175A1, on the other hand was found to selectively bind and catalyse reaction of cholesterol. The wild type as well as both the mutants of the enzyme however bind poly unsaturated fatty acids. The results thus show that saturation mutation of a single amino acid at the substrate binding pocket of the thermostable cytochrome P450 could induce sufficient changes in the substrate binding pocket of the enzyme that can efficiently change substrate specificity of the enzyme.
- Subjects
CYTOCHROME P-450; ENZYME specificity; UNSATURATED fatty acids; SATURATED fatty acids; THERMUS thermophilus; TYROSINE
- Publication
Protein Journal, 2022, Vol 41, Issue 6, p659
- ISSN
1572-3887
- Publication type
Article
- DOI
10.1007/s10930-022-10084-3