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:10.1080/1024242031000121502

Cited by 3 publications

(3 citation statements)

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“…Regardless of the area of application, however, the feasibility of utilizing P450s to confer herbicide resistance or decontaminate polluted areas will greatly depend on our abilities to provide P450 enzymes with high catalytic activity towards the desired substrate(s). Several approaches have been successful in enhancing P450 activity including rational protein design (Bell et al 2003;Carmichael and Wong 2001), directed enzyme evolution (Abecassis et al 2000;Abecassis et al 2003) and the optimization of the redox environment in plants by simultaneously overexpressing a NADPH:P450 reductase (Siminszky et al 2003); therefore, P450s specifically tailored for high efficiency herbicide metabolism may soon become available.…”

Section: Practical Applicationsmentioning

confidence: 99%

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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Section: Practical Applicationsmentioning

confidence: 99%

Plant cytochrome P450-mediated herbicide metabolism

2006

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“…assay for cleavage product identification was used to identify bacterial proteases [47]; again the analysis of a fingerprint was instrumental to this approach. Finally, functional analysis of cytochrome P450 variants with multiple substrates has shown that their activity profile (or fingerprint) is linked with variations in their amino acid sequence [48 ].…”

Section: Figurementioning

confidence: 98%

Enzyme assays for high-throughput screening

Goddard

Reymond

2004

Current Opinion in Biotechnology

242

123

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“…A CYP1A library was also created by DNA family shuffling using DNase I-mediated fragmentation of human CYP1A1 and CYP1A2 and screened for naphthalene hydroxylation, yielding approximately 20% of active clones (Abécassis et al, 2000). Further characterization of such libraries revealed mutants with significantly altered profiles compared with those of CYP1A1 and CYP1A2 (Abécassis et al, 2003;Taly et al, 2007).…”

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confidence: 99%

A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity

Johnston

Huang²,

Voss³

et al. 2007

Drug Metab Dispos

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ABSTRACT:The cytochrome P450 enzymes (P450s) that mediate mammalian xenobiotic metabolism are highly versatile monooxygenases, which show wide and overlapping substrate ranges but generally poor catalytic rates. Re-engineering of these P450s may enable the development of useful biocatalysts for industrial applications. In the current study, restriction enzyme-mediated DNA family shuffling was used to create a library from human CYP1A1 and CYP1A2. Among sequenced clones (four randomly selected and eight functional clones), 5.9 ؎ 2.3 crossovers and 1.5 ؎ 1.5 spontaneous mutations (mean ؎ S.D.) were detected per mutant. A high level of structural integrity as well as diverse functionality were found, with 53% of clones expressed at significant levels (>50 nM P450 hemoprotein) and 23% of clones showing activity on one or more of the following compounds: luciferin 6-chloroethyl ether (luciferin-CEE), luciferin 6-methyl ether (luciferin-ME), 6-deoxyluciferin (luciferin-H), the ethylene glycol ester of luciferin 6-methyl ether, 7-ethoxyresorufin, and p-nitrophenol (PNP). Different activity profiles were seen with higher specific activity on individual compounds (e.g., clone 22; 9 times the CYP1A1 specific activity toward luciferin-CEE), novel activities (e.g., clone 35; activity toward luciferin-H and PNP), and broadening of substrate range observed in particular clones (e.g., clone 9; activity toward both selective substrates luciferin-ME and luciferin-CEE as well as toward luciferin-H and PNP). In summary, forms were found with distinct and novel activity profiles, despite the relatively small number of mutants examined. In addition, the whole-cell metabolic assays described here provide simple, high-throughput methods useful for screening larger libraries.Enzymes are exquisitely specific biocatalysts, able to modify even complex organic chemicals with a high degree of stereo-and regioselectivity by virtue of their complex, stereoselective, three-dimensional structures. Directed or artificial evolution is being used to engineer enzymes with novel catalytic and physicochemical properties that can be used as biocatalysts for industrial applications. The development of enzyme libraries relies upon the ability to navigate a practically infinite sequence space of possible enzymes while identifying novel and desired characteristics in library clones. DNA family shuffling is a powerful technique that allows sparse sampling of a large region of the sequence space, whereas techniques based on random mutagenesis either explore more limited sequence space close to the parental starting point or are prohibitively massive in scope (Crameri et al., 1998).Cytochrome P450 (P450) enzymes are ideal starting points for directed evolution. The need for organisms to clear a diversity of xenobiotics has given several mammalian P450s the capability to metabolize a wide range of foreign chemicals via monooxygenation, with considerable regiospecificity on even large and complex molecules (Gillam, 2005). Despite this diversity of activity, th...

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

Cited by 3 publications

References 0 publications

Plant cytochrome P450-mediated herbicide metabolism

Plant cytochrome P450-mediated herbicide metabolism

Enzyme assays for high-throughput screening

A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity

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