Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

西花蓟马细胞色素P450基因cDNA片段克隆与mRNA表达分析

细胞色素P450介导的解毒作用增强是昆虫对杀虫剂产生抗性的重要机制。本文通过RT-PCR克隆了西花蓟马CYP4家族5个细胞色素P450基因c DNA片段。多重序列比对发现,这5个基因在氨基酸水平的一致性在40%-76%之间。采用实时荧光定量PCR对5个CYP4基因的mRNA表达水平的分析发现,阿维菌素抗性品系CYP4-1、CYP4-2和CYP4-5的表达水平分别是敏感品系的3.50、4.00和2.48倍,表明西花蓟马对阿维菌素的抗性可能与这3个CYP4基因的过量表达相关。     

细胞色素P450酶与微生物药物创制北大核心CSCD

细胞色素P450酶广泛存在于动植物和微生物体内,具有底物结构多样性和催化反应类型多样性,在天然产物生物合成中扮演重要作用。P450酶可在温和条件下高选择性地催化结构复杂有机化合物中惰性C-H键的氧化反应,具备化学催化剂难以比拟的优势,因此在微生物制药领域具有广阔的应用空间。本文综述了参与天然产物生物合成的P450酶近年来的研究进展;P450酶的酶工程改造、生物转化实践及其在微生物药物创制方面的应用现状;探讨了P450酶的工业应用瓶颈及其解决途径;并对P450酶未来的应用前景进行了展望。     

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.     

Resonance Raman scattering of cytochrome P450 BM3 and effect of imidazole inhibitors

Resonance Raman scattering from cytochrome P450 BM3 is obtained with a Raman microprobe using 406-nm excitation with an accumulation time of a few seconds. The small sample size and rapid measurement time make the routine characterization of P450 systems by resonance Raman spectroscopy easier. Addition of imidazole and imidazole derivatives as inhibitors causes the appearance of additional peaks due to vinyl modes, increases the relative intensity of symmetric modes that would be A(1g) in D(4h) symmetry, and causes a large drop in the intensity of nu(11). This information indicates that the ligation of imidazoles to the heme iron causes the alignment of the vinyl modes with the plane of the heme ring and reduces the out of plane distortion of the ring. The effect of both inhibitors is similar but there is a subtle difference in the extent of the reduction in the intensity of nu(11), which suggests that steric effects within the pocket are having some effect.     

In vitro modulation of the P450 activities of hamster and human lung slices

The respiratory tract is a portal of entry for many environmental chemicals. The respiratory tract plays an important role in the detoxification or metabolic activation of these chemicals, e.g., via cytochrome P450 enzymes. Alterations in the capabilities of these enzymes to metabolize inhaled compounds can, therefore, affect the toxicity of the chemicals. The pulmonary cytochrome P450 activity has been studied in many species, but relatively little is known about this activity in the human lung tissue. In this limited study, we have investigated the possibility of modulating in vitro the P450 activity in lung slices from hamsters and humans. The alkoxyresorufin-O-dealkylase activity was measured in the S9 fraction of lung slices incubated for 24 h with 106 mol/L 20-methylcholanthrene (3MC) or -naphthoflavone (N). The ethoxyresorufin-O-deethylase (EROD) activity was increased by 3MC and N in lung slices of both species. The benzyloxyresorufin-O-deethylase (BROD) activity was decreased after incubation with 3MC but increased with N. These data show that in vitro modulation in lung slices is feasible, although technical improvement is still needed, particularly in relation to the viability of the slices.     

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.     

原核生物细胞色素P450酶系及其应用

细胞色素P450酶系广泛分布于各种生物中,它们通常由一组基因超家族编码并含有血红素,能够催化一系列化学反应,具有多种生物学功能。特别是原核生物P450酶在催化内源性和外源性化合物的反应中具有重要的工业生产应用价值,成为近年来P450酶系研究的热点。本文对近年来原核生物P450酶系的重组表达和生物催化领域的研究进展进行综述。     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.     

Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis

To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity). The recombinant reductase was characterized and compared to other reductases by heterologous expression in insect cells and was shown to support reconstituted taxoid 10beta-hydroxylase activity with an efficiency comparable to that of other plant-derived reductases. Coexpression in yeast of the reductase along with T. cuspidata taxoid 10beta-hydroxylase, which catalyzes an early step of taxoid biosynthesis, demonstrated significant enhancement of hydroxylase activity compared to that supported by the endogenous yeast reductase alone. Functional transgenic coupling of the Taxus reductase with a homologous cytochrome P450 taxoid hydroxylase represents an important initial step in reconstructing Taxol biosynthesis in a microbial host.     

亚致死浓度氰氟虫腙对小菜蛾三种解毒酶及P450 mRNA表达量的影响

【目的】为探讨亚致死浓度氰氟虫腙对小菜蛾Plutella xylostella(L.)3种解毒酶和细胞色素P450基因表达量的影响。【方法】采用叶片浸渍法,测定用氰氟虫腙LC10和LC25处理72 h后小菜蛾羧酸酯酶、谷胱甘肽S-转移酶和多功能氧化酶3种解毒酶的活性;应用实时荧光定量PCR(Real-time PCR)方法测定了其在24、48和72 h时对P450基因(Px CYP4、Px CYP6和Px CYP9家族)表达的影响。【结果】结果表明,氰氟虫腙LC10和LC25处理对羧酸酯酶活性影响不显著;对谷胱甘肽S-转移酶活性存在显著抑制作用(P<0.05);LC25处理后多功能氧化酶活性显著增加(P<0.05),为对照的1.39倍。同时,LC25氰氟虫腙处理组小菜蛾P450基因Px CYP4M19、Px CYP6BF1V2、Px CYP6CN1和Px CYP6CV2 m RNA的相对表达量分别为对照组的2.33~8.5倍、1.32~3.48倍、1.18~2.53倍、2.02~7.22倍。【结论】结果显示,氰氟虫腙可能通过诱导这4种P450基因m RNA的上调表达而增强了小菜蛾多功能氧化酶酶活性。     

细胞色素P450在植物与昆虫相互关系中的作用

细胞色素P4 5 0在植物与昆虫相互关系中发挥重要的作用 ,植物可以利用P4 5 0来合成有毒物质以防御昆虫的取食 ,而昆虫则利用P4 5 0对植物毒素进行代谢解毒 ,昆虫以植物代谢中间物为原料合成自身活性物质的过程也有P4 5 0的参与。通过长期的协同进化 ,植物与昆虫的相互作用不仅表现在P4 5 0底物特异性方面 ,也反映在P4 5 0的表达调控上。     

Molecular analysis of some camel cytochrome P450 enzymes reveals lower evolution and drug-binding properties

Camels bear unique genotypes and phenotypes for adaptation of their harsh environment. They have unique visual systems, sniffing, water metabolism, and heat-control mechanisms that are different from other creatures. The recent announcement for the complete sequence of camel genome will allow for the discovery of many secrets of camel life. In this context, the genetic bases of camel drug-metabolizing enzymes are still unknown. Furthermore, the genomic content of camel that rendered it highly susceptible to some drugs (as monensin and salinomycin) and became easily intoxicated needs to be investigated. The objectives of this work are the annotation of camel genome and retrieval of camel for cytochrome P450 (CYP) 1A1, 2C, and 3A enzymes. This is followed by comprehensive phylogenetic, evolution, molecular modeling, and docking studies. In comparison with the human enzymes, camel CYPs showed lower evolution rate, especially CYP1A1. Furthermore, the binding of monensin, salinomycin, alfa-naphthoflavone, felodepine, and ritonavir was weaker in camel enzymes. Interestingly, rerank score indicated instable binding of monensin and salinomycin with camel CYP1A1 as well as salinomycin with camel CYP2C. The results of this work suggest that camels are more susceptible to toxicity with compounds undergoing metabolic oxidation. This conclusion was based on lower evolution rate and lower binding potency of camels compared with the human enzymes.     

真菌细胞色素P450在大肠杆菌中的表达

【背景】真菌细胞色素P450蛋白在大肠杆菌中表达水平低甚至不表达,近期研究发现通过对该类蛋白氨基端(N端)氨基酸序列的修饰可优化其表达水平。【目的】在大肠杆菌系统中表达预测功能为P450酶的焦曲霉094102菌株的Au8002蛋白,为真菌P450蛋白在大肠杆菌表达系统中的N端氨基酸序列修饰策略提供有效依据。【方法】对野生型P450蛋白Au8002的氨基酸序列进行分析,对其N端序列进行了3种序列修饰,并在诱导蛋白表达时添加P450生物合成前体5-氨基乙酰丙酸(5-ALA),研究N端氨基酸序列修饰策略及前体添加对真菌P450在大肠杆菌中蛋白表达的影响。【结果】SDS-PAGE和Westernblot检测结果显示,对目的蛋白进行的3种氨基酸序列修饰均使Au8002蛋白获得了表达,前体5-ALA的添加提高了目的蛋白表达量。其中对目的蛋白进行N端全长截短时可部分增加其可溶性,同时也验证了其特征性的CO结合能力。【结论】对预测为P450酶的菌株094102蛋白Au8002氨基端(N端)氨基酸序列的修饰有效解决了其在大肠杆菌内不表达的难题,实现了其可溶性表达;另一方面P450生物合成前体5-ALA的添加也能有效提高该类蛋白的表达水平,上述策略对改善其它该类蛋白在大肠杆菌内的表达水平具有借鉴意义。     

Adult specific expression and induction of cytochrome P450tpr in house flies

Cytochrome P450_tpr is a xenobiotic metabolizing P450 that is found in house flies (Musca domestica). To better understand the regulation of cytochrome P450_tpr, the effects of 21 potential monooxygenase inducers were examined for their ability to induce total cytochromes P450 and cytochrome P450_tpr levels in adult flies. Six compounds caused induction of total cytochromes P450 per mg protein in adult susceptible (CS) house flies: ethanol (1.6-fold), phenobarbital in food (1.5-fold) or water (1.5-fold), naphthalene (1.3-fold), DDT (1.3-fold), xanthotoxin (1.4-fold), and α-pinene (1.2-fold). Six compounds were found to be inducers of cytochrome P450_tpr: piperonyl butoxide in food (1.9-fold), phenobarbital in food (1.4-fold) and water (3.4-fold), clofibrate (1.3-fold), xanthotoxin (1.3-fold), methohexital (1.3-fold), and isosafrole (1.3-fold). Comparison of our results with house fly P450 6A1 indicates that there are specific inducers for each of these individual P450s as well as compounds that induce both P450s. Total P450s were inducible by PB in CS house fly larvae, but not in LPR larvae. Immunoblotting revealed no detectable P450_tpr in control or PB-treated larvae in either strain. Thus, although total P450s are inducible in the susceptible strain larvae, P450_tpr does not appear to be normally present or inducible with PB in larvae of either strain. Northern blots of phenobarbital (in water) treated CS flies indicated that there was a 4.2-fold increase in the P450_tpr (i.e., CYP6D1) mRNA levels over the untreated flies. In the multiresistant LPR strain there was no apparent induction of CYP6D1 mRNA by phenobarbital. Following phenobarbital induction, the level of CYP6D1 mRNA in the CS strain was about half of the level in the LPR strain. © 1996 Wiley-Liss, Inc.     

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.     

An inhibition study of beauvericin on human and rat cytochrome P450 enzymes and its pharmacokinetics in rats

Beauvericin is a secondary metabolite natural product from microorganisms and has been shown to have a new potential antifungal activity. In this study, the metabolism and inhibition of beauvericin in human liver microsomes (HLM) and rat liver microsomes (RLM) were investigated. The apparent K_m and V_max of beauvericin in HLM were determined by substrate depletion approach and its inhibitory effects on cytochromes P450 (CYP) activities were evaluated using probe substrates, with IC₅₀ and the (K_i) values were 1.2 μM (0.5 μM) and 1.3 μM (1.9 μM), respectively for CYP3A4/5 (midazolam) and CYP2C19 (mephenytoin). Similarly, beauvericin was also a potent inhibitor for CYP3A1/2 (IC₅₀: 1.3 μM) in RLM. Furthermore, the pharmacokinetics of beauvericin in the rat were studied after p.o administration alone and co-administration with ketoconazole, which indicated a pharmacodynamic function may play a role in the synergistic effect on antifungal activity.