Directed Evolution Of Cytochrome P450 BM-3

P450 BM-3 is a kind of P450 monooxygenase which can catalyze the hydroxylation of long-chain saturated fatty at theω-1,ω-2, orω-3 position and epoxidation of unsaturated fatty acids. Owing to protein engineering, the substrate spectrum of mutants of P450 BM-3 has expanded to middle-chain fatty acid, alkane, cycloalkane, polycyclic aromatic hydrocarbons, among of which a mutant of cytochrome P450 BM-3(F87V/A74G/L188Q) engineered by rational design can hydroxylate indole into indigo. In order to explore the relationship between structure and function of cytochrome P450 BM-3 further and discover its other potential application, a mutation library holding amino acid substitutions in one sites critical to P450 BM-3 activity toward indole, D168 was constructed by saturation mutagenesis on the D168 site based on the four sites substitution variant P450 BM-3(A74G/F87V/L188Q/E435T) along with another mutant library of error-prone PCR focusing on the monooxygenase domain of P450 BM-3 (A74G/F87V/L188Q/E435T). Based on the characteristics of production of indole hydroxylation, two mutant exhibiting different catalytic properties from the parent were screened and undergone a rather systematic investigation after purified by Ni-NTA affinity chromatography.First, according to present knowledge about structure and function of cytochrome P450 BM-3, a mutation library holding amino acid substitutions in D168 was constructed after optimizing mutagenesis parameters with E. coli BL21.Second, the random mutagenesis of the monooxygenase domain of the P450 BM-3 (F87V/A74G/L188Q/E435T) mutant was performed by error-prone PCR. Mn²⁺ concentration as a gene mutagenesis factor in vitro was optimized and six different Mn²⁺ levels were used to perform error-prone PCR. Because of the vector, pET28a (+), containing a copy of glucose dehydrogenase, mutants co-express P450 BM3 and glucose dehydrogenase simultaneously which is good for the regeneration for NADPH indispensable to indole hydroxylation. Two Variants, D168R and D168W, were distinguished by their catalytic production composition of indole hydroxylation in 96-well microtiter plate. The main part in the red production of D168W was confirmed as indirubin which came from the change of the indole hydroxylation position.Third, Mutant D168R and D168W were investigated further about their enzymatic activity properties. The mutant D168W shown lower hydroxylation activity toward p-nitrophenoxydecanoic acid than the parent did. In order to determine the kinetic parameters of the mutant enzymes to indole, a modified method taking advantage of the full-wave scanning function of Ultrospec3300 pro was employed to monitor the reaction progress by real-time and the relationships between NADPH consumption and product formation were clearly demonstrated.After all, results indicated that the mutant enzyme D168R not only exhibited higher affinity and activity to indole but also increased electron coupling efficiency than the parent. Indirubin (90%) was formed as main product in indole hydroxylation production of variant D168R should have to do with the changed catalytic regioselectivity of D168R, which favoring the formation of indirubin.