Mutation Of Cytochrome P450-BM3 Variant M13 And The Application Of M13 Mutants In Hydroxylation Modifications Of Natural Products

Cytochromes P450(P450s)are members of a superfamily of mono-oxygenases that catalyze multiple oxidation reactions.They have been widely studied because they can insert oxygen between inert carbon-hydrogen bonds.Also,P450 proteins catalyze the oxidation,dealkylation and dehalogenation reactions of heteroatoms such as N,S and so on.Despite the catalytic capability of diverse reactions,P450s have lower catalytic activity and stability.Complex reductive systems and reductases present challenges for the receptor-to-ligand recognition and research about electron transfer between donors and receptors.CYP102A1(P450-BM3)derived from Bacillus megaterium is a soluble and self-sufficient fusion of a cytochrome P450 and a diflavin-containing reductase.Compared with other P450s,it has unique advantages such as higher hydroxylation ability,higher plasticity for engineering,and better soluble expression etc.The extensive applications of P450 BM3,however,are limited due to its specific hydroxylation towards several mid-to long-chain fatty acids.Protein engineering strategies including rational design and random mutagenesis are used to modify the structure of BM3 to broaden substrate spectrum and increase the activity.Here,our work focuses on the catalytic study of the BM3 variant M13,including the dihydroxylation ability about steroids and the promiscuity of the substrates.In addition,the catalytic ability of genistein is improved by rational engineering of Ml 3.The main findings are as follows:There are few reports about dihydoxylation reactions catalyzed by BM3,especially for steroidal compounds.In this work,seven kinds of steroid compounds such as testosterone,androstenedione and progesterone were tested.It indicated that some steroid compounds could be hydroxylated to yield multiple products.The result of mass spectrometry showed that two dihydroxylated products were produced in the reactions with androstenedione and progesterone.All these results showed that M13 had strong hydroxylation ability and could form dihydroxylated products in the reactions.It suggested that M13 had catalytic potential in hydroxylations of drug-like small molecules.1.Probing substrate promiscuity of M13In this study,molecular docking was used for the research of substrate promiscuity of M13.We established a computer-assisted method for rapid study of substrate promiscuity,which is timeless,effortless and cost-saving.The experiment results showed that the substrates can be hydroxylated to form new product only when the conformation meets certain requirements in distance and energy.Here we proposed a standard which act as a rule the reactant must follow.Different types of substrates were analyzed by docking according to this standard.This algorithm was further verified by in-vitro experiments.In addition,it was found that M13 not only catalyze the monohydroxylation of some medicinal molecules such as quinine,cinchonine,bufalin and hydrocortisone,but also catalyze the dihydroxylation of androstenedione and progesterone.M13 was thus regarded as a promising catalytic tool for hydroxylations of natural products.2.Improving hydroxylation efficiency towards genistein through engineered M13In this study,rational approaches were firstly employed to probe M13 structure.A single residue of M13,namely isoleucine at position 86(186),was selected for site-saturation mutagenesis.A mutant M13I86C was thus generated and the hydroxylation efficiency towards genistein catalyzed with M13I86C increased by 4 times than that of M13.Next virtual docking of M13I86C with genistein was performed.A mutation M13P18W/I86C was generated using PCR-based mutation approaches.Compared to M13I86C,the hydroxylation efficiency of M13P18W/I86C towards genistein increased 1.6 times.In-vitro experiments showed that the hydroxylation efficiency towards genistein catalyzed by double mutant variant M13P18W/I86C improved 6-7 times compared to the M13.In addition,the key residue combinations(F390/P392 and F390/K391)in genistein hydroxylation were identified by combinatorial alanine scanning experiments.