Metabolic imidacloprid resistance in the brown planthopper,Nilaparvata lugens,relies on multiple P450 enzymes

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance.     

Quantitative structure-activity relationships (QSARs) for inhibitors and substrates of CYP2B enzymes: importance of compound lipophilicity in explanation of potency differences

The results of quantitative structure-activity relationship (QSAR) studies on inhibitors and substrates of cytochrome P450 2B (CYP2B) subfamily enzymes are reported. It was found that lipophilicity (in the form of log P) is the most important property for explaining the variations in inhibitory activity, and there are similarities between QSARs for both substrates and inhibitors for CYP2B6 (human), and also between those of other CYP2B enzymes, such as CYP2B1 (rat) and CYP2B4 (rabbit). Both linear and quadratic lipophilicity relationships are evidenced in human and other mammalian species, and the particular type of expression found is probably due to the nature of the compounds under investigation, as it is usually the homologous series which tend to show quadratic relationships in log P. The findings from QSAR studies can be rationalized by molecular modelling of the active site interactions with both P450 crystal structures and homology models of CYP2B subfamily enzymes.     

Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes

A detailed analysis of substrate selectivity within the cytochrome P450 2 (CYP2) family is reported. From a consideration of specific interactions between drug substrates for human CYP2 family enzymes and the putative active sites of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, it is likely that the number and disposition of hydrogen bond donor/acceptors and aromatic rings within the various P450 substrate molecules determines their enzyme selectivity and binding affinity, together with directing their preferred routes of metabolism by the CYP2 enzymes concerned. Although many aliphatic residues are present in most P450 active sites, it would appear that their main contribution centers around hydrophobic interactions and desolvation processes accompanying substrate binding. Molecular modeling studies based on the recent CYP2C5 crystal structure appear to show close agreement with site-directed mutagenesis experiments and with information on substrate metabolism and selectivity within the CYP2 family.     

X‐ray crystal structure of cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae strain B2

The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8‐cineole. Here we report the crystallization and X‐ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450‐fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well‐ordered substrate‐binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8‐cineole‐hydroxylating P450 enzymes. Proteins 2017; 85:945–950. © 2016 Wiley Periodicals, Inc.     

The activity of human CYP2D6 in low water organic solvents

P450 enzymes are of high interest for synthetic applications due to their ability to catalyze hydroxylation reactions at inactivated C-H bonds. The low solubility of many substrates in buffer, however, is limiting the applications of P450s. Our recent demonstration that the P450 enzymes CYP2D6 and CYP3A4 can function very well in biphasic solvent systems is one step towards overcoming this drawback, but is not practical when substrates or products are unstable in water, or with water-soluble products. An alternative strategy, which also facilitates enzyme recycling, is to directly resuspend lyophilized enzyme into nearly anhydrous organic solvents. Interestingly, we report here that CYP2D6 colyophilized with trehalose and suspended in n-decane shows higher activity than in aqueous buffer. This study demonstrates the unexpected high tolerance of CYP2D6 to some low water organic solvents and provides an alternative strategy to facilitate the use of this enzyme in synthesis.     

细胞色素P450介导的果蝇抗药性研究进展

细胞色素P450 (cytochrome P450, CYP450)超基因家族是由一些数量多而功能复杂的血红蛋白酶基因所组成,该代谢酶系作为一种几乎地球上所有需氧生物都存在的重要生存策略,可以调控多种内源物质及外源化合物的代谢,参与了众多重要的生命过程,代谢解毒作用是该酶系重要功能之一。细胞色素P450的代谢解毒作用受药物影响,机体通过改变基因表达量,实现增强代谢解毒,加快机体对于有害物质的代谢,从而使得机体对有害环境产生一定的适应性,进而使得机体产生耐药性或抗药性。本研究说明果蝇细胞色素P450介导的杀虫剂类药物代谢机制及代谢抗性的特点等方面的研究,对明确果蝇的抗药性机制研究具有参考意义。     

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo.     

P450 enzymes from the bacterium Novosphingobium aromaticivorans

Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.     

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.     

Alkane activation by P450 oxygenases

The hydroxylation of alkane molecules, especially at terminal positions, is a challenging reaction. Enzymes that catalyze this reaction could be used to produce high-value compounds from aliphatic and alkyl-substituted substrates. However, until a few years ago, all known alkane hydroxylating enzymes were membrane-bound, and difficult to use. Recently, three bacterial P450 enzymes of the (soluble) CYP101 and CYP102 families were engineered to hydroxylate alkanes, but even after extensive efforts hydroxylation was mainly at sub-terminal positions. More recently, a new soluble P450 family (CYP153) was identified and characterized, which activates the terminal position of alkanes and alkyl-substituted compounds with very high regio-selectivity. The use of CYP153s in biotechnological applications is now being explored.     

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle: EVIDENCE FOR PHYSICAL COMPLEX FORMATION*

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.     

Alkane activation by P450 oxygenases

The hydroxylation of alkane molecules, especially at terminal positions, is a challenging reaction. Enzymes that catalyze this reaction could be used to produce high-value compounds from aliphatic and alkyl-substituted substrates. However, until a few years ago, all known alkane hydroxylating enzymes were membrane-bound, and difficult to use. Recently, three bacterial P450 enzymes of the (soluble) CYP101 and CYP102 families were engineered to hydroxylate alkanes, but even after extensive efforts hydroxylation was mainly at sub-terminal positions. More recently, a new soluble P450 family (CYP153) was identified and characterized, which activates the terminal position of alkanes and alkyl-substituted compounds with very high regio-selectivity. The use of CYP153s in biotechnological applications is now being explored.     

Cytochrome P450 enzymes from the metabolically diverse bacterium Rhodopseudomonas palustris

Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to approximately 2.0A have been obtained.     

Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58–67 and 100–109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth.     

Identification of the cytochrome P450 enzymes responsible for the omega-hydroxylation of phytanic acid

Patients suffering from Refsum disease have a defect in the alpha-oxidation pathway which results in the accumulation of phytanic acid in plasma and tissues. Our previous studies have shown that phytanic acid is also a substrate for the omega-oxidation pathway. With the use of specific inhibitors we now show that members of the cytochrome P450 (CYP450) family 4 class are responsible for phytanic acid omega-hydroxylation. Incubations with microsomes containing human recombinant CYP450s (Supersomes) revealed that multiple CYP450 enzymes of the family 4 class are able to omega-hydroxylate phytanic acid with the following order of efficiency: CYP4F3A>CYP4F3B>CYP4F2>CYP4A11.     

人类细胞色素P450与药物氧化代谢遗传多态性分子机制的研究现状

近年,在表型及基因型上均发现存在药物氧化代谢多态性,特别是对于人类细胞色素Ｐ４５０氧化酶与药氧化代谢遗传多态性的关系进行了深入的研究。有关ＣＹＰ２Ｄ６、ＣＹＰ２Ｃ１９等的突变已大多被鉴定;ＣＹＰ１Ａ１、ＣＹＰ１Ａ２等在表型存在多态性而确切的遗传机制尚不清楚。     

Analysis of cytochrome P450 genes in silkworm genome (Bombyx mori)

Cytochrome P450 monooxygenases (P450) are im-portant metabolic enzymes involved in the metabolism not only of a wide range of endogenous compounds such as fatty acids, steroids, hormones or vitamins, but also of exogenous substrates such as drugs, chemicals including environmental pollutants, such carcinogens as polycyclic aromatic hydrocarbons, and pesticides[1]. P450s are found virtually in all aerobic organisms, including organisms as diverse as in insects, plants, mammals, birds and bacter…     

Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli

Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b₅ (Cyt-b₅) was further coexpressed with CPR, indicating that Cyt-b₅ supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b₅ but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an “alternative” electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways.