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.

Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases.

Engineering functional thermostable proteins using ancestral sequence reconstruction.

Natural proteins are often only slightly more stable in the native state than the denatured state, and an increase in environmental temperature can easily shift the balance toward unfolding. Therefore, the engineering of proteins to improve protein stability is an area of intensive research. Thermostable proteins are required to withstand industrial process conditions, for increased shelf-life of protein therapeutics, for developing robust 'biobricks' for synthetic biology applications, and for research purposes (e.

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.

Ancestral sequence reconstruction of the CYP711 family reveals functional divergence in strigolactone biosynthetic enzymes associated with gene duplication events in monocot grasses.

The strigolactone (SL) class of phytohormones shows broad chemical diversity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the diversification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences.

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors.

Determinants of thermostability in the cytochrome P450 fold.

Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The diversity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions.