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 purely chemical approaches, introducing delays into the drug pipeline. An alternative is to use the cytochrome P450 enzymes (P450s) that produce the metabolites in vivo, taking advantage of the enzyme's inherently chiral active site to achieve regio- and stereoselectivity. Importantly, biotransformations are more sustainable: they proceed under mild conditions and avoid environmentally damaging solvents and transition metal catalysts. Recombinant enzymes avoid the need to use animal liver microsomes. However, native enzymes must be stabilized to work for extended periods or at elevated temperatures, 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 obtaining metabolites not produced by human or rodent liver microsomes. Importantly, no alteration in assay conditions from standard protocols for microsomal incubations was necessary. Thus, such bacterial membranes 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: In drug development it is often chemically challenging, to synthesize authentic metabolites of drug candidates for structural identification and evaluation of activity and safety. Biosynthesis using microsomes or recombinant human enzymes is confounded by the instability of the enzymes. Here we show that thermostable ancestral cytochrome P450 enzymes derived from P450 families responsible for human drug metabolism offer advantages over the native human forms in being more robust and over microbial enzymes in faithfully reflecting human drug metabolism.

Use of engineered cytochromes P450 for accelerating drug discovery and development.

Numerous steps in drug development, including the generation of authentic metabolites and late-stage functionalization of candidates, necessitate the modification of often complex molecules, such as natural products. While it can be challenging to make the required regio- and stereoselective alterations to a molecule using purely chemical catalysis, enzymes can introduce changes to complex molecules with a high degree of stereo- and regioselectivity. Cytochrome P450 enzymes are biocatalysts of unequalled versatility, capable of regio- and stereoselective functionalization of unactivated CH bonds by monooxygenation. Collectively they catalyze over 60 different biotransformations on structurally and functionally diverse organic molecules, including natural products, drugs, steroids, organic acids and other lipophilic molecules. This catalytic versatility and substrate range makes them likely candidates for application as potential biocatalysts for industrial chemistry. However, several aspects of the P450 catalytic cycle and other characteristics have limited their implementation to date in industry, including: their lability at elevated temperature, in the presence of solvents, and over lengthy incubation times; the typically low efficiency with which they metabolize non-natural substrates; and their lack of specificity for a single metabolic pathway. Protein engineering by rational design or directed evolution provides a way to engineer P450s for industrial use. Here we review the progress made to date toward engineering the properties of P450s, especially eukaryotic forms, for industrial application, and including the recent expansion of their catalytic repertoire to include non-natural reactions.

SeqScrub: a web tool for automatic cleaning and annotation of FASTA file headers for bioinformatic applications.

Data consistency is necessary for effective bioinformatic analysis. SeqScrub is a web tool that parses and maintains consistent information about protein and DNA sequences in FASTA file format, checks if records are current, and adds taxonomic information by matching identifiers against entries in authoritative biological sequence databases. SeqScrub provides a powerful, yet simple workflow for managing, enriching and exchanging data, which is crucial to establish a record of provenance for sequences found from broad and varied searches; for example, using BLAST on continually updated genome sequence sets. Headers standardized using SeqScrub can be parsed by a majority of bioinformatic tools, stay uniformly named between collaborators and contain informative labels to aid management of reproducible, scientific data. SeqScrub is available at http://bioinf.scmb.uq.edu.au/seqscrub.