Sequence diversity in the monooxygenases involved in oxime production in plant defense and signaling: a conservative revision in the nomenclature of the highly complex CYP79 family.

Cytochrome P450 monooxygenases of the CYP79 family catalyze conversion of specific amino acids into oximes feeding into a variety of metabolic plant pathways. Here we present an extensive phylogenetic tree of the CYP79 family built on carefully curated sequences collected across the entire plant kingdom. Based on a monophyletic origin of the P450s, a set of evolutionarily distinct branches was identified.

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.

Strigolactones and Shoot Branching: What Is the Real Hormone and How Does It Work?

There have been substantial advances in our understanding of many aspects of strigolactone regulation of branching since the discovery of strigolactones as phytohormones. These include further insights into the network of phytohormones and other signals that regulate branching, as well as deep insights into strigolactone biosynthesis, metabolism, transport, perception and downstream signaling. In this review, we provide an update on recent advances in our understanding of how the strigolactone pathway co-ordinately and dynamically regulates bud outgrowth and pose some important outstanding questions that are yet to be resolved.

Solar-powered P450 catalysis: Engineering electron transfer pathways from photosynthesis to P450s.

With the increasing focus on green chemistry, biocatalysis is becoming more widely used in the pharmaceutical and other chemical industries for sustainable production of high value and structurally complex chemicals. Cytochrome P450 monooxygenases (P450s) are attractive biocatalysts for industrial application due to their ability to transform a huge range of substrates in a stereo- and regiospecific manner. However, despite their appeal, the industrial application of P450s is limited by their dependence on costly reduced nicotinamide adenine dinucleotide phosphate (NADPH) and one or more auxiliary redox partner proteins.

Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts.

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules.

Opportunities for Accelerating Drug Discovery and Development by Using Engineered Drug-Metabolizing Enzymes.

The study of drug metabolism is fundamental to drug discovery and development (DDD) since by mediating the clearance of most drugs, metabolic enzymes influence their bioavailability and duration of action. Biotransformation can also produce pharmacologically active or toxic products, which complicates the evaluation of the therapeutic benefit versus liability of potential drugs but also provides opportunities to explore the chemical space around a lead. The structures and relative abundance of metabolites are determined by the substrate and reaction specificity of biotransformation enzymes and their catalytic efficiency.

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.

Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP).

Analyzing the natural evolution of proteins by ancestral sequence reconstruction (ASR) can provide valuable information about the changes in sequence and structure that drive the development of novel protein functions. However, ASR has also been used as a protein engineering tool, as it often generates thermostable proteins which can serve as robust and evolvable templates for enzyme engineering. Importantly, ASR has the potential to provide an insight into the history of insertions and deletions that have occurred in the evolution of a protein family.

Resurrection and characterization of ancestral CYP11A1 enzymes.

Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties.

Oxygen Surrogate Systems for Supporting Human Drug-Metabolizing Cytochrome P450 Enzymes.

Oxygen surrogates (OSs) have been used to support cytochrome P450 (P450) enzymes for diverse purposes in drug metabolism research, including reaction phenotyping, mechanistic and inhibition studies, studies of redox partner interactions, and to avoid the need for NADPH or a redox partner. They also have been used in engineering P450s for more cost-effective, NADPH-independent biocatalysis. However, despite their broad application, little is known of the preference of individual P450s for different OSs or the substrate dependence of OS-supported activity.

Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability.

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with values of ∼30-40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites.

Rational evolution of the cofactor-binding site of cytochrome P450 reductase yields variants with increased activity towards specific cytochrome P450 enzymes.

NADPH-cytochrome P450 reductase (CPR) is the natural redox partner of microsomal cytochrome P450 enzymes. CPR shows a stringent preference for NADPH over the less expensive cofactor, NADH, economically limiting its use as a biocatalyst. The complexity of cofactor-linked CPR protein dynamics and the incomplete understanding of the interaction of CPR with both cofactors and electron acceptors present challenges for the successful rational engineering of a CPR with enhanced activity with NADH.

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.

The GMC superfamily of oxidoreductases revisited: analysis and evolution of fungal GMC oxidoreductases.

Background: The glucose-methanol-choline (GMC) superfamily is a large and functionally diverse family of oxidoreductases that share a common structural fold. Fungal members of this superfamily that are characterised and relevant for lignocellulose degradation include aryl-alcohol oxidoreductase, alcohol oxidase, cellobiose dehydrogenase, glucose oxidase, glucose dehydrogenase, pyranose dehydrogenase, and pyranose oxidase, which together form family AA3 of the auxiliary activities in the CAZy database of carbohydrate-active enzymes. Overall, little is known about the extant sequence space of these GMC oxidoreductases and their phylogenetic relations.

Habitat associations of bats in a working rangeland landscape.

Land-use change has resulted in rangeland loss and degradation globally. These changes include conversion of native grasslands for row-crop agriculture as well as degradation of remaining rangeland due to fragmentation and changing disturbance regimes. Understanding how these and other factors influence wildlife use of rangelands is important for conservation and management of wildlife populations.

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides.

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides.

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.

Exploring the past and the future of protein evolution with ancestral sequence reconstruction: the 'retro' approach to protein engineering.

A central goal in molecular evolution is to understand the ways in which genes and proteins evolve in response to changing environments. In the absence of intact DNA from fossils, ancestral sequence reconstruction (ASR) can be used to infer the evolutionary precursors of extant proteins. To date, ancestral proteins belonging to eubacteria, archaea, yeast and vertebrates have been inferred that have been hypothesized to date from between several million to over 3 billion years ago.