A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity

Abstract

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...

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

“…Of 96 clones screened on solid media, one showed elevated indigo production compared to the parental forms. Similarly, from the clones obtained upon CYP1A (shuffled from CYP1A1 and CYP1A2) different activity profiles were seen with higher specific activity on individual compounds (e.g., clone 22; 9 times the CYP1A1 specific activity toward luciferin 6′-chloroethyl ether); novel activities (e.g., clone 35; activity toward 6′-deoxyluciferin and p-nitrophenol); and broadening of substrate range observed in particular clones (e.g., clone 9; activity toward both selective substrates luciferin 6′-methyl ether and luciferin 6′-chloroethyl ether as well as toward 6′-deoxyluciferin and p-nitrophenol) [148]. …”

Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity

“…P450s belonging to the same evolutionary branch share significant nucleotide sequence homology, yet often have unique functional properties, making them excellent starting material for DNA shuffling. Libraries of P450 mutants from the P450 3A and P450 1A subfamilies capable of accepting substrates not metabolized by the parental enzymes [61,62] and P450 3A-derived mutants with altered product specificity [62] have been created. Libraries of P450 mutants from the P450 3A and P450 1A subfamilies capable of accepting substrates not metabolized by the parental enzymes [61,62] and P450 3A-derived mutants with altered product specificity [62] have been created.…”

“…SCHEMA estimates the disruption to tertiary structure when segments of a protein are inherited from multiple parental sequences, and thus can be used to identify optimal recombination sites for constructing semirational libraries of shuffled proteins. A further development of the SCHEMA algorithm enhanced the structural integrity and functional diversity of a library created by recombination of human P450 1A1 and P450 1A2 [42] compared with similar libraries made by DNA family shuffling [59,61]. Of the resulting mutants, 47 % encoded folded intact haemoprotein and >73 % of the folded proteins had detectable peroxygenase activity.…”

Biooxidation with Cytochrome P450 Monooxygenases

“…They observed different activity profiles toward various luciferin derivatives among active clones, including improved specific activity, novel activities, and broadening of substrate range [147,148].…”