Exploration of Natural and Artificial Sequence Spaces: Towards a Functional Remodeling of Membrane-bound Cytochrome P450

Abécassis, Valérie; Urban, Philippe; Aggerbeck, Lawrence P.; Truan, Gilles; Pompon, Denis

doi:10.1080/102424203100012150

Cited by 7 publications

(2 citation statements)

References 22 publications

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“…In contrast to mutants derived by saturation mutagenesis, shuffled P450 1A mutants did not necessarily align with either P450 1A1 or 1A2 parent in a correlation analysis with methoxy- and ethoxyresorufins. With a third, structurally dissimilar substrate, mutants showing similar phenotypes with respect to the alkoxyresorufins could be further distinguished , . Other recent studies have confirmed the ability of DNA shuffling to generate libraries of functional diversity with a relatively high proportion of properly folded P450s , .…”

Section: Engineering Activitymentioning

confidence: 86%

“…The Pompon group published the first results on DNA shuffling of mammalian P450s (111). In a mosaic library derived from P450 1A1 and P450 1A2, 12% of clones showed activity toward naphthalene, a common substrate of both P450 1A1 and P450 1A2 (111), and were catalytically diverse with respect to five different substrates (112,113). In contrast to mutants derived by saturation mutagenesis, shuffled P450 1A mutants did not necessarily align with either P450 1A1 or 1A2 parent in a correlation analysis with methoxy-and ethoxyresorufins.…”

Section: Engineering Activitymentioning

confidence: 99%

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Engineering Cytochrome P450 Enzymes

Gillam

2007

Chem. Res. Toxicol.

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The last 20 years have seen the widespread and routine application of methods in molecular biology such as molecular cloning, recombinant protein expression, and the polymerase chain reaction. This has had implications not only for the study of toxicological mechanisms but also for the exploitation of enzymes involved in xenobiotic clearance. The engineering of P450s has been performed with several purposes. The first and most fundamental has been to enable successful recombinant expression in host systems such as bacteria. This in turn has led to efforts to solubilize the proteins as a prerequisite to crystallization and structure determination. Lagging behind has been the engineering of enzyme activity, hampered in part by our still-meager comprehension of fundamental structure-function relationships in P450s. However, the emerging technique of directed evolution holds promise in delivering both engineered enzymes for use in biocatalysis and incidental improvements in our understanding of sequence-structure and sequence-function relationships, provided that data mining can extract the fundamental correlations underpinning the data. From the very first studies on recombinant P450s, efforts were directed toward constructing fusions between P450s and redox partners in the hope of generating more efficient enzymes. While this aim has been allowed to lie fallow for some time, this area merits further investigation as does the development of surface-displayed P450 systems for biocatalytic and biosensor applications. The final application of engineered P450s will require other aspects of their biology to be addressed, such as tolerance to heat, solvents, and high substrate and product concentrations. The most important application of these enzymes in toxicology in the near future is likely to be the biocatalytic generation of drug metabolites for the pharmaceutical industry. Further tailoring will be necessary for specific toxicological applications, such as in bioremediation.

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Section: Engineering Activitymentioning

confidence: 86%

Section: Engineering Activitymentioning

confidence: 99%

Engineering Cytochrome P450 Enzymes

Gillam

2007

Chem. Res. Toxicol.

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Engineering and assaying of cytochrome P450 biocatalysts

et al. 2008

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Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.

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Plant cytochrome P450-mediated herbicide metabolism

2006

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In the last two decades it has become apparent that enzymes of the P450 monooxygenase (P450) superfamily are responsible for the Phase I metabolism of numerous herbicides representing several classes of organic compounds. The majority of experimental evidence for P450 involvement in herbicide metabolism has been derived from in vitro studies in which the catalytic activity of plant microsomes towards herbicidal substrates was measured in the presence of various P450 inhibitors and activators. While the studies with microsomes elicited much appreciation for the pivotal roles of plant P450s in herbicide metabolism, detailed characterization of these enzymes only became possible after the isolation of genes encoding specific isoforms responsible for herbicide conversion. Several lines of evidence suggest that the development of herbicide resistance in weeds by enhanced detoxification is frequently associated with elevated levels of P450 activity. Enhanced detoxification-based herbicide resistance is particularly difficult to control, because it can involve resistance to multiple, chemically unrelated classes of herbicides. Continued research efforts are aimed at elucidating the role of P450s in the metabolic fates of herbicides in plants and the development of herbicide resistance in weeds. Recent advances made in the isolation and genetic manipulation of P450 enzymes have created new opportunities for their application in engineering herbicide tolerance and bioremediation.

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Exploration of Natural and Artificial Sequence Spaces: Towards a Functional Remodeling of Membrane-bound Cytochrome P450

Cited by 7 publications

References 22 publications

Engineering Cytochrome P450 Enzymes

Engineering Cytochrome P450 Enzymes

Engineering and assaying of cytochrome P450 biocatalysts

Plant cytochrome P450-mediated herbicide metabolism

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