Department of Life Sciences and Systems Biology, University of Torino via Accademia Albertina 13, 10123, Torino, Italy.
Chemistry. 2012 Mar 19;18(12):3582-8. doi: 10.1002/chem.201102470. Epub 2012 Feb 15.
The soluble, catalytically self-sufficient cytochrome P450 BM3 from Bacillus megaterium is a good candidate as biocatalyst for the synthesis of drug metabolites. To this end, error-prone polymerase chain reaction (PCR) was used to generate a library of P450 BM3 mutants with novel activities toward drugs. The double mutant Asp251Gly/Gln307His (A2) with activities towards diclofenac, ibuprofen and tolbutamide was identified by screening with the alkali method. This is based on the detection of NADPH oxidation during enzymatic turnover on whole Escherichia coli cells heterologously expressing the P450 BM3 mutants in the presence of the target substrates. The three drugs screened are marker substrates of human liver cytochromes P450 belonging to the 2C subfamily. Interestingly the mutations Asp251Gly/Gln307His are located on the protein surface and they are not directly involved in substrate binding and turnover. Dissociation constants and K(M) values of mutant A2 for diclofenac, ibuprofen and tolbutamide are in the micromolar range. Catalysis leads to hydroxylations in specific positions, producing 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide, respectively.
来自巨大芽孢杆菌的可溶性、催化自足细胞色素 P450 BM3 是合成药物代谢物的生物催化剂的良好候选物。为此，易错聚合酶链反应 (PCR) 用于生成针对药物具有新活性的 P450 BM3 突变体文库。通过碱法筛选，鉴定出对双氯芬酸、布洛芬和甲苯磺丁脲具有活性的双突变体 Asp251Gly/Gln307His (A2)。这是基于在存在靶底物的情况下，在异源表达 P450 BM3 突变体的整个大肠杆菌细胞的酶周转过程中检测 NADPH 氧化。筛选出的三种药物是属于 2C 亚家族的人肝细胞色素 P450 的标记底物。有趣的是，突变体 A2 的 Asp251Gly/Gln307His 突变位于蛋白质表面，它们不直接参与底物结合和周转。突变体 A2 对双氯芬酸、布洛芬和甲苯磺丁脲的解离常数和 K(M) 值均在微摩尔范围内。催化导致在特定位置的羟化，分别产生 4'-羟基双氯芬酸、2-羟基布洛芬和 4-羟基甲苯磺丁脲。
