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Enhanced bacterial expression of several mammalian cytochrome P450s by codon optimization and chaperone coexpression.

ABSTRACT: To elucidate the effects of codon optimization and chaperone coexpression on the heterologous expression of mammalian cytochrome P450s (P450) in Escherichia coli, the expression of P450s 2B1, 2S1, 2U1, 2W1, and 27C1 were investigated. With codon optimization for N-terminus or the entire gene, the expression levels of P450 27C1, 2U1 and 2W1 increased 22-fold, 3.6-fold and 2.1-fold, respectively, while those for P450s 2B1 and 2S1 remained unchanged. With coexpression of E. coli molecular chaperones GroEL/ES, the expression level increased up to 14-fold for P450 27C1, and 3- to 5-fold for P450s 2B1, 2S1, and 2W1. Simultaneous application of these two techniques resulted in synergetic effects.

SUBMITTER: Wu ZL

PROVIDER: S-EPMC4188451 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Enhanced bacterial expression of several mammalian cytochrome P450s by codon optimization and chaperone coexpression.

Wu Zhong-Liu ZL Qiao Jing J Zhang Zhi-Gang ZG Guengerich F Peter FP Liu Yan Y Pei Xiao-Qiong XQ

Biotechnology letters 20090626 10

To elucidate the effects of codon optimization and chaperone coexpression on the heterologous expression of mammalian cytochrome P450s (P450) in Escherichia coli, the expression of P450s 2B1, 2S1, 2U1, 2W1, and 27C1 were investigated. With codon optimization for N-terminus or the entire gene, the expression levels of P450 27C1, 2U1 and 2W1 increased 22-fold, 3.6-fold and 2.1-fold, respectively, while those for P450s 2B1 and 2S1 remained unchanged. With coexpression of E. coli molecular chaperone ...[more]

PMID: 19557307

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Project description:The superfamily of cytochrome P450s forms a large class of heme monooxygenases with more than 13,000 enzymes represented in organisms from all biological kingdoms. Despite impressive variability in sizes, sequences, location, and function, all cytochrome P450s from various organisms have very similar tertiary structures within the same fold. Here we show that systematic comparison of all available X-ray structures of cytochrome P450s reveals the presence of two distinct structural classes of cytochrome P450s. For all membrane bound enzymes, except the CYP51 family, the beta-domain and the A-propionate heme side chain are shifted towards the proximal side of the heme plane, which may result in an increase of the volume of the substrate binding pocket and an opening of a potential channel for the substrate access and/or product escape directly into the membrane. This structural feature is also observed in several soluble cytochrome P450s, such as CYP108, CYP151, and CYP158A2, which catalyze transformations of bulky substrates. Alternatively, both beta-domains and the A-propionate side chains in the soluble isozymes extend towards the distal site of the heme. This difference between the structures of soluble and membrane bound cytochrome P450s can be rationalized through the presence of several amino acid inserts in the latter class which are involved in direct interactions with the membrane, namely the F'- and G'-helices. Molecular dynamics using the most abundant human cytochrome P450, CYP3A4, incorporated into a model POPC bilayer reveals the facile conservation of a substrate access channel, directed into the membrane between the B-C loop and the beta domain, and the closure of the peripheral substrate access channel directed through the B-C loop. This is in contrast to the case when the same simulation is run in buffer, where no such channel closing occurs. Taken together, these results reveal a key structural difference between membrane bound and soluble cytochrome P450s with important functional implications induced by the lipid bilayer.

Project description:BackgroundHoney bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored.Methodology/principal findingsWe used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey.Conclusions/significanceThese results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey.

Dataset Information

Enhanced bacterial expression of several mammalian cytochrome P450s by codon optimization and chaperone coexpression.

Publications

Enhanced bacterial expression of several mammalian cytochrome P450s by codon optimization and chaperone coexpression.

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