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We investigated the effects of CYP2A6 genotypes on nicotine metabolism, focused from nicotine to cotinine and its additional 3'-hydroxylating resulted in trans-3'-hydroxycotinine formation. In the subjects genotyped by PCR-RFLP method, one cigarette smoking experiment was performed and urine samples were collected for 24 h. In all subjects who smoked, we detected nicotine, cotinine and trans-3'-hydroxycotinine in urine by GC-MS analysis. In whole deletion of CYP2A6, urinary excretion amounts of cotinine and trans-3'-hydroxycotinine were significantly smaller than those in the wild-type of CYP2A6*1. A lack of CYP2A6 reduces the formation of cotinine and trans-3'-hydroxycotinine, but not entirely reduces the trans-3'-hydroxycotinine formation. Unknown cotinine 3'-hydroxylating activity except CYP2A6 are suspected in humans.

Directed evolution (DE) inspired by natural evolution (NE) has been achieving tremendous successes in protein/enzyme engineering. However, the conventional ‘one-protein-for-one-task’ DE cannot match the ‘multi-proteins-for-multi-tasks’ NE in terms of screening throughput and efficiency, thus often failing to meet the fast-growing demands for biocatalysts with desired properties. In this study, we design a novel ‘multi-enzyme-for-multi-substrate’ (MEMS) DE model and establish the proof-of-concept by running a NE-mimicking and higher-throughput screening on the basis of ‘two-P450s-against-seven-substrates’ (2P×7S) in one pot. With the significantly improved throughput and hit-rate, we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes (P450 BM3 and P450cam) in laboratory. Further structural analysis of the two functionally convergent P450 variants provide important insights into how distinct active-sites can reach a common catalytic goal.