Biocatalysis using thermostable cytochrome P450 enzymes in bacterial membranes - comparison of metabolic pathways with human liver microsomes and recombinant human enzymes.

Detailed structural characterization of small molecule metabolites is desirable during all stages of drug development, and often relies on the synthesis of metabolite standards. However, introducing structural changes into already complex, highly functionalized small molecules both regio- and stereo-selectively can be challenging using approaches that rely purely on synthetic organic chemistry, introducing delays and bottlenecks into the drug development pipeline. An alternative is to use enzymes such as the cytochromes P450 (P450s) that produce the metabolites in vivo, taking advantage of the inherent chirality of the enzyme's active site to achieve regio- and stereoselectivity. Importantly, such biotransformations typically proceed under milder conditions and avoid the need for environmentally damaging solvents and transition metal catalysts . However native enzymes must be stabilized to work under process reaction conditions, and stabilizing mutations can alter catalytic activity. Here we assessed a set of novel, thermostable P450s in bacterial membranes, a format analogous to liver microsomes, for their ability to metabolize drugs through various pathways, and compared them to human liver microsomes. Collectively, the thermostable P450s could replicate the metabolic pathways seen with human liver microsomes, including bioactivation to protein-reactive intermediates. Novel metabolites were found suggesting the possibility of anticipating metabolites seen in preclinical species but not humans. Importantly, no alteration in assay conditions from standard protocols for microsomal incubations was necessary. Thus, such 'bacterial microsomes' represent an analogous metabolite generation system to liver microsomes in terms of metabolites produced and ease of use, but which provides access to more diversity of metabolite structures. Significance Statement Drug-metabolizing P450 enzymes catalyze useful chemistry for drug development but are unsuited for use under process conditions due to their poor stability and low solvent tolerance. Here we show that thermostable ancestral P450s can catalyze the same types of chemistry and are useful substitute biocatalysts for metabolite generation and late-stage functionalization. Ancestral enzymes in bacterial membrane fractions ('bacterial microsomes') can be used in the same way as liver microsomes or recombinant human P450s, without significant modification of standard operating procedures.