Semi-rational Engineering Of Cytochrome P450BM-3

P450BM-3is a kind of P450monooxygenase which can catalyze the hydroxylation of long-chain saturated fatty and epoxidation of unsaturated fatty acids. Because of the protein engineering, the substrate spectrum of P450BM-3has expanded to alkane, cycloalkane and so on. A mutant of P450BM-3(A74G/F87V/L188Q) has been engineered by rational design which can hydroxylate indole. In this paper, in order to improve its capability in the hydroxylation of indole to indigo as well as expand its substrate spectrum, the P450BM-3mutants have been founded. At the same time, in order to explore the relationship between the structure and the function of the enzyme, G46was conducted by site-saturation mutagenesis on the G46site based on the triple variant P450BM-3(A74G/F87V/L188Q).First, the mutant enzyme P450BM-3(A74G/F87V/L188Q/D422G/G46S) based on the four sites substitution variant P450BM-3(A74G/F87V/L188Q/D422G) with higher catalytic activity and better thermostability was performed by semi-rational engineering. The mutant enzyme was called G46S mutant in the followed paper. The semi-rational engineering the paper mentioned firstly screened out the sites with the higher value of b-factor which were measured by the B-FITTER software, then the site was determined after considering the sites with the higher B-factor and the sites among the entrance to the substrate channel. The mutant enzyme G46S was investigated further about the enzymatic activity properties. Compared with the parent enzyme, half inactivation (T5010) temperature is5℃higher than that of the parent enzyme, its half-life(t1/2) under50℃is more than double of the parent enzyme. Its km reduced22percent while kcat has increased by90percent. The catalytic efficiency (kcat/Km) was enhanced by2.48fold over that of the parent enzyme.Second, in order to understand the relation between the structure and the function and as well as prove the G46site is actually important to the enzyme, G46was conducted by site-saturation mutagenesis on the G46site based on the three sites substitution variant P450BM-3(A74G/F87V/L188Q). Six mutants enzyme were identified to hydroxylating indole to indigo more efficiently than other mutants and the parent enzyme. Among the six mutants, G46W performs the highest capability, almost7.03fold of the parent enzyme. Moreover, three mutants enzyme (G46E, G46Y, and G46W) performed not only better capability but also better thermostability than the patent enzyme.Third, the study attempted to expand the substrate spectrum of P450BM-3to get the mutant enzyme which can hydroxylate steroid substances. The method must base on the crystal structure of P450BM-3and the study about the relationship between structure and function of P450BM-3. The site was screened out by analyzing the sites which the software has obtained and the sites may change the structure of the entrance to the substrate channel. Finally, the mutant enzyme which was cut off the twenty amino acids at the N-terminal based on the triple variant P450BM-3(A74G/F87V/L188Q). The mutant enzyme was called-20mutant. The-20mutant was investigated further about its enzymatic activity properties. The mutant cannot hydroxylate steriod substance. The failure may be attributed to the poor understanding of the relation between the structure and the function at face and just considered the structure not the real binding force between the mutant enzyme and the substance.In brief, the paper used semi-rational engineering to improve the catalytic activity of P450BM-3. Mutants with higher activity and the better thermostability have been gained. These mutants enzyme the paper gained can serve as the parent enzyme for further study. Not only this, the paper provide some hints for further study of the catalytic mechanism of P450BM-3and for the semi-rational or rational engineering to improve the capability of the P450BM-3.