Semi-rational engineering of cytochrome CYP153A from Marinobacter aquaeolei for improved ω-hydroxylation activity towards oleic acid
| Autor: | Daniel Mink, Yan Duan, René M. de Jong, Zhanglin Lin, Xianxing Gao, Lina Ba, Dunming Zhu, Iwona Kaluzna, Jianwei Gao |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Cytochrome Stereochemistry Mutant Hydroxylation Protein Engineering 01 natural sciences Applied Microbiology and Biotechnology Redox Electron Transport 03 medical and health sciences Electron transfer chemistry.chemical_compound Cytochrome P-450 Enzyme System Marinobacter NADH NADPH Oxidoreductases Saturated mutagenesis biology 010405 organic chemistry Substrate (chemistry) General Medicine 0104 chemical sciences Transformation (genetics) Oleic acid 030104 developmental biology Biochemistry chemistry Mutagenesis Site-Directed biology.protein Ferredoxins Mutant Proteins Oleic Acid Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology. 100:8779-8788 |
| ISSN: | 1432-0614 0175-7598 |
| Popis: | ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket. The in vitro-reconstituted activity of Variant II with purified Pdx and Pdr was 2.7-fold that of the template, while the whole cell transformation activity was 2.0-fold that of the template. A 96-well format-based screening scheme for CYP153A was also developed, which should be useful for engineering of other P450s with low activity. Kinetic analyses indicated that the activity improvement for CYP153A variants largely resulted from enhanced electron transfer. This further demonstrates the importance of the electron transfer between P450s and the non-native redox partners for the overall performance of hybrid P450 systems. |
| Databáze: | OpenAIRE |
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