How similar are P450s and what can their differences teach us?

Cytochromes P450 form a very large superfamily of proteins which metabolize substrates from steroids to fatty acids to drugs and are found in organisms from protists to mammals. P450s all appear to take on a similar structural fold, yet frequently having less than 20% sequence identity and having vastly different substrates. Within the structural fold there appears to be a highly conserved core, as determined from the comparison of the structures of the six crystallized, soluble P450s. There are also variable regions which by and large appear to be associated with substrate recognition, substrate binding, and redox partner binding. Molecular dynamics simulations of motion in P450cam and P450BM-3 indicate that substrate binding and product release require substantial motion around the "substrate access channel." Additionally, at the 11th International Conference on Cytochrome P450 Biochemistry, Biophysics, and Molecular Biology and briefly here, the first structure of a microsomal eukaryotic P450 will be presented and compared to the already determined structures by Drs. Johnson and McRee. Finally, with a better understanding of the structure/function relationship of P450s, one will be better able to modify P450s to metabolize the substrates of choice or produce needed valuable chemicals.     

Plant cytochrome P450s: nomenclature and involvement in natural product biosynthesis

Cytochrome P450s constitute the largest family of enzymatic proteins in plants acting on various endogenous and xenobiotic molecules. They are monooxygenases that insert one oxygen atom into inert hydrophobic molecules to make them more reactive and hydro-soluble. Besides for physiological functions, the extremely versatile cytochrome P450 biocatalysts are highly demanded in the fields of biotechnology, medicine, and phytoremediation. The nature of reactions catalyzed by P450s is irreversible, which makes these enzymes attractions in the evolution of plant metabolic pathways. P450s are prime targets in metabolic engineering approaches for improving plant defense against insects and pathogens and for production of secondary metabolites such as the anti-neoplastic drugs taxol or indole alkaloids. The emerging examples of P450 involvement in natural product synthesis in traditional medicinal plant species are becoming increasingly interesting, as they provide new alternatives to modern medicines. In view of the divergent roles of P450s, we review their classification and nomenclature, functions and evolution, role in biosynthesis of secondary metabolites, and use as tools in pharmacology.     

豌豆蚜基因组P450基因家族的分析

细胞色素P450在各种内源和外源物质代谢中起着非常重要的作用。利用豌豆蚜Acyrthosiphon pisum基因组mRNA和氨基酸数据库研究P450基因功能的进化规律。通过生物信息学方法对豌豆蚜全基因组P450进行分析, 结果显示: 在豌豆蚜基因组中发现69个P450基因, 它们分别属于13个P450家族和18个亚家族, 是一个典型的多基因家族。进一步将这些基因与豌豆蚜ESTs数据库进行了比对分析, 其中39个候选基因有EST证据, 证明了这些P450基因在转录水平的真实性。以氨基酸相似度大于60%为标准对豌豆蚜基因组中P450基因进行分组, 69个P450基因中, 除18个基因序列因差异太大, 不能被归入任何一组, 其余51个可归入10个组, 其中8个组(包含47条序列)适合于正选择和基因转换分析。正选择和基因转换分析结果表明: 仅有1个组(含9个基因)显著受到正选择压力作用, 正选择概率大于95%的氨基酸位点分别是20T和27N, 20T位于底物识别位点SRS1, 27N位于D. helix; 有3个组(包含8个基因）显示显著的基因转换事件。 参与基因转换的基因均为CYP4家族成员, 分别是CYP4C, CYP4G和CYP4V亚家族。 参与基因转换的成员之间的蛋白相似度较高（70％~95％）, 且XM_001944991与XM_001951794同存在于SCAFFOLD12542上, XM_001945510与XM_001944057同存在于SCAFFOLD7010上。这可能暗示豌豆蚜P450基因通过基因复制, 然后通过基因转换使P450获得新的功能, 以适应多变的生存环境。此外, 鉴定出20个不同的基序, 其中有5条基序在90％以上的基因中出现。      

Plant Cytochrome P450 Monooxygenases

Plant systems utilize a diverse array of cytochrome P450 monooxygenases (P450s) in their biosynthetic and detoxification pathways. The classic forms of these enzymes are heme-dependent mixed function oxidases that utilize NADPH or NADH and molecular oxygen to produce functionalized organic products. The nonclassical forms are monooxygenases that either do not utilize flavoproteins for dioxygen activation or fail to incorporate molecular oxygen into their final product. Biosynthetic P450s play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products. Other catabolic P450s metabolize toxic herbicides and insecticides into nontoxic products or, conversely, activate nontoxic substances into toxic products. Biochemical and molecular characterizations on a number of plant P450s have indicated that the relationships between these heme proteins and their substrates are at least as complex as those that exist in mammalian systems. Examples now exist of plant P450s that metabolize: a narrow range of substrates to yield different products, a single substrate to yield different products, multiple substrates to yield the same product, or a single substrate sequentially to yield discrete intermediates in the biosynthesis of a single product. Extensive divergence of catalytic site as well as noncatalytic site residues accounts for the high degree of primary structure variation in the P450 gene superfamily and the diverse array of substrates synthesized and/or detoxified by these proteins. Classic P450s still retain a highly conserved F-G-R-C-G motif in their catalytic site and conserved amino acids in their oxygen binding pocket; nonclassical P450s diverge at several of these positions. A broad range of cloning and transient expression strategies are suitable for plant P450 studies and these have allowed for the isolation and characterization of a number of P450 cDNAs and genes. Because many of these sequences have been cloned only recently, much remains to be learned about the substrate specificities of P450 reactions in plants and the mechanisms by which their genes are regulated.     

Computational solvent mapping reveals the importance of local conformational changes for broad substrate specificity in mammalian cytochromes P450

Computational solvent mapping moves small organic molecules as probes around a protein surface, finds favorable binding positions, clusters the conformations, and ranks the clusters on the basis of their average free energy. Prior mapping studies of enzymes, crystallized in either substrate-free or substrate-bound form, have shown that the largest number of solvent probe clusters invariably overlaps in the active site. We have applied this method to five cytochromes P450. As expected, the mapping of two bacterial P450s, P450 cam (CYP101) and P450 BM-3 (CYP102), identified the substrate-binding sites in both ligand-bound and ligand-free P450 structures. However, the mapping finds the active site only in the ligand-bound structures of the three mammalian P450s, 2C5, 2C9, and 2B4. Thus, despite the large cavities seen in the unbound structures of these enzymes, the features required for binding small molecules are formed only in the process of substrate binding. The ability of adjusting their binding sites to substrates that differ in size, shape, and polarity is likely to be responsible for the broad substrate specificity of these mammalian P450s. Similar behavior was seen at "hot spots" of protein-protein interfaces that can also bind small molecules in grooves created by induced fit. In addition, the binding of S-warfarin to P450 2C9 creates a high-affinity site for a second ligand, which may help to explain the prevalence of drug-drug interactions involving this and other mammalian P450s.     

Gluconeogenesis and the peroxisome

The interaction between hydroperoxides, cytochrome P450 and 8-anilino-1-naphthalenesulfonic acid (ANS) has been investigated. The addition of ANS to the cytochrome P450 solution did not effect the P450 Soret absorption peak or the reduced CO difference spectrum, suggesting that ANS may not bind to P450 heme directly. H2O2 or CuOOH alone did not effect ANS fluorescence and absorption spectra indicating that no detectable reaction occurs between hydroperoxide and ANS in the absence of P450. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, the addition of either H2O2 or CuOOH, however, leads to a decrease in ANS absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the ANS elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of max of ANS. Moreover , a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was detected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the reconstituted system of cytochrome P450 and NADPH-P450 reductase is metabolized by the reconstituted system of P450 and hydroperoxide, suggesting the activities of these two systems may not be completely comparable. (Mol Cell Biochem 167: 159-168, 1997)     

Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b₅. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA). No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6-, 16-, and 21-hydroxyprogesterone. Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450. Based on the V_max/K_m values for P450 3A9 using either 21-hydroxprogesterone or 6-hydroxyprogesterone as a substrate, 4-pregnen-6, 21-diol-3, 20-dione may be formed either by 6-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.     

Effect of Conformational Dynamics on Substrate Recognition and Specificity as Probed by the Introduction of a de Novo Disulfide Bond into Cytochrome P450 2B1

The conformational dynamics of cytochrome P450 2B1 (CYP2B1) were investigated through the introduction of a disulfide bond to link the I- and K-helices by generation of a double Cys variant, Y309C/S360C. The consequences of the disulfide bonding were examined both experimentally and in silico by molecular dynamics simulations. Under high hydrostatic pressures, the partial inactivation volume for the Y309C/S360C variant was determined to be −21 cm³mol⁻¹, which is more than twice as much as those of the wild type (WT) and single Cys variants (Y309C, S360C). This result indicates that the engineered disulfide bond has substantially reduced the protein plasticity of the Y309C/S360C variant. Under steady-state turnover conditions, the S360C variant catalyzed the N-demethylation of benzphetamine and O-deethylation of 7-ethoxy-trifluoromethylcoumarin as the WT did, whereas the Y309C variant retained only 39% of the N-demethylation activity and 66% of the O-deethylation activity compared with the WT. Interestingly, the Y309C/S360C variant restored the N-demethylation activity to the same level as that of the WT but decreased the O-deethylation activity to only 19% of the WT. Furthermore, the Y309C/S360C variant showed increased substrate specificity for testosterone over androstenedione. Molecular dynamics simulations revealed that the engineered disulfide bond altered substrate access channels. Taken together, these results suggest that protein dynamics play an important role in regulating substrate entry and recognition.Liver microsomal cytochromes P450 (CYP or P450)² metabolize a large number of clinically used drugs that have diverse steric and functional moieties. Despite low sequence homology among CYPs from different families, all P450s invariably contain a heme cofactor that is coordinated to a thiolate and catalyze the oxidative metabolism, mostly through hydroxylation, of substrates. However, production of reactive intermediates by P450s is often associated with drug toxicity and carcinogenesis, and inhibition or induction of a specific P450 isoform may lead to adverse drug-drug interactions (1). From a clinical and pharmacological perspective, it is important to understand the structure, function, and dynamics of P450s.Structural studies of P450s by x-ray crystallography in the past decade have provided us with a wealth of information regarding the structural organization, critical active site residues, and proton delivery pathways of P450s (2–4). In particular, these structural analyses have consistently shown that certain regions of the P450 structures such as the F/G and B/B′-C loops are extremely flexible and can undergo large conformational changes to accommodate substrates of various sizes, although the overall folding pattern of all P450s is conserved. For instance, an open conformation was observed in the ligand-free CYP2B4 crystal structure, whereas a closed conformation was reported for the CPI-bound CYP2B4 (3, 5). The open-to-closed conformational change involves large motions of the F- and G-helices and the F/G and B/B′-C loops. Based on comparisons of the crystal structures of CYP2B4 bound with inhibitors of different sizes, Zhao et al. (6) identified five plastic regions in P450s, including the B/B′-C loop (PR2) and F/G loop (PR4). Binding of ketoconazole or erythromycin to CYP3A4 led to a large increase in the active site volume (>80% increase) because of conformational changes primarily in the PR4, but interestingly the F- and G-helices moved in the opposite direction (7). These authors proposed that the extreme flexibility of CYP3A4 accounts for its promiscuity, as CYP3A4 metabolizes nearly ∼50% of all clinically used drugs. The complexity of the conformational flexibility and dynamics are also revealed in an MD simulation study of CYP3A4, 2C9 and 2A6 (8). Importantly, this molecular dynamics (MD) simulation study shows that the three-dimensional structure of P450s is more flexible in solution than was observed in the crystal structure.Despite intensive studies of the crystal structures of microsomal P450s, insights into the conformational dynamics of P450s in solution, particularly in relation to their functional importance, are lacking. A laser flash photolysis study of CO rebinding to CYP2E1 in solution revealed that the binding of substrates such as ethanol, pyrazole, and acetaminophen restricts the conformational flexibility of CYP2E1, as the kinetics for the rebinding of CO to ligand-bound CYP2E1 are significantly slower than those for the ligand-free CYP2E1 (9). A solution thermodynamics study of CYP2B4 supports the notion that CYP2B4 is remarkably flexible, as the entropy substantially decreases upon inhibitor binding resulting from reduction of the hydrophobic surface (10). In this study, a de novo disulfide bond is engineered into CYP2B1 and the consequences resulting from the disulfide bonding are examined both experimentally and in silico using MD simulations. To discern the effect of the de novo disulfide bond apart from the Cys mutagenesis, both the single and double Cys variants were characterized in detail. To our knowledge, this is the first report that investigates the consequences of limiting conformational dynamics in a P450 by incorporating a disulfide bond. Our results demonstrate that protein dynamics play an important role in regulating substrate entry/product egress channels and substrate recognition and provide insights that will be valuable for rational drug design and protein engineering.     

Do mammalian cytochrome P450s show multiple ligand access pathways and ligand channelling?

Understanding substrate binding and product release in cytochrome P450 (CYP) enzymes is important for explaining their key role in drug metabolism, toxicity, xenobiotic degradation and biosynthesis. Here, molecular simulations of substrate and product exit from the buried active site of a mammalian P450, the microsomal CYP2C5, identified a dominant exit channel, termed pathway (pw) 2c. Previous simulations with soluble bacterial P450s showed a different dominant egress channel, pw2a. Combining these, we propose two mechanisms in CYP2C5: (i) a one-way route by which lipophilic substrates access the enzyme from the membrane by pw2a and hydroxylated products egress along pw2c; and (ii) a two-way route for access and egress, along pw2c, for soluble compounds. The proposed differences in substrate access and product egress routes between membrane-bound mammalian P450s and soluble bacterial P450s highlight the adaptability of the P450 fold to the requirements of differing cellular locations and substrate specificity profiles.     

Cooperativity in cytochrome P450 3A4: linkages in substrate binding, spin state, uncoupling, and product formation

Understanding the detailed metabolic mechanisms of membrane-associated cytochromes P450 is often hampered by heterogeneity, ill-defined oligomeric state of the enzyme, and variation in the stoichiometry of the functional P450.reductase complexes in various reconstituted systems. Here, we describe the detailed characterization of a functionally homogeneous 1:1 complex of cytochrome P450 3A4 (CYP3A4) and cytochrome P450 reductase solubilized via self-assembly in a nanoscale phospholipid bilayer. CYP3A4 in this complex showed a nearly complete conversion from the low- to high-spin state when saturated with testosterone (TS) and no noticeable modulation due to the presence of cytochrome P450 reductase. Global analysis of equilibrium substrate binding and steady-state NADPH consumption kinetics provided precise resolution of the fractional contributions to turnover of CYP3A4 intermediates with one, two, or three TS molecules bound. The first binding event accelerates NADPH consumption but does not result in significant product formation due to essentially complete uncoupling. Binding of the second substrate molecule is critically important for catalysis, as the product formation rate reaches a maximum value with two TS molecules bound, whereas the third binding event significantly improves the coupling efficiency of redox equivalent usage with no further increase in product formation rate. The resolution of the fractional contributions of binding intermediates of CYP3A4 into experimentally observed overall spin shift and the rates of steady-state NADPH oxidation and product formation provide new detailed insight into the mechanisms of cooperativity and allosteric regulation in this human cytochrome P450.     

Process intensification for cytochrome P450 BM3-catalyzed oxy-functionalization of dodecanoic acid

Selective oxy-functionalization of nonactivated C-H bonds is a long-standing “dream reaction” of organic synthesis for which chemical methodology is not well developed. Mono-oxygenase enzymes are promising catalysts for such oxy-functionalization to establish. Limitation on their applicability arises from low reaction output. Here, we showed an integrated approach of process engineering to the intensification of the cytochrome P450 BM3-catalyzed hydroxylation of dodecanoic acid (C12:0). Using P450 BM3 together with glucose dehydrogenase for regeneration of nicotinamide adenine dinucleotide phosphate (NADPH), we compared soluble and co-immobilized enzymes in O₂-gassed and pH-controlled conversions at high final substrate concentrations (≥40mM). We identified the main engineering parameters of process output (i.e., O₂ supply; mixing correlated with immobilized enzyme stability; foam control correlated with product isolation; substrate solubilization) and succeeded in disentangling their complex interrelationship for systematic process optimization. Running the reaction at O₂-limited conditions at up to 500-ml scale (10% dimethyl sulfoxide; silicone antifoam), we developed a substrate feeding strategy based on O₂ feedback control. Thus, we achieved high reaction rates of 1.86g·L⁻¹·hr⁻¹ and near complete conversion (≥90%) of 80mM (16g/L) C12:0 with good selectivity (≤5% overoxidation). We showed that “uncoupled reaction” of the P450 BM3 (~95% utilization of NADPH and O₂ not leading to hydroxylation) with the C12:0 hydroxylated product limited the process efficiency at high product concentration. Hydroxylated product (~7g; ≥92% purity) was recovered from 500ml reaction in 82% yield using ethyl-acetate extraction. Collectively, these results demonstrate key engineering parameters for the biocatalytic oxy-functionalization and show their integration into a coherent strategy for process intensification.     

The 1.5-? Structure of XplA-heme,an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

XplA is a cytochrome P450 of unique structural organization, consisting of a heme- domain that is C-terminally fused to its native flavodoxin redox partner. XplA, along with flavodoxin reductase XplB, has been shown to catalyze the breakdown of the nitramine explosive and pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition explosive) by reductive denitration. The structure of the heme domain of XplA (XplA-heme) has been solved in two crystal forms: as a dimer in space group P2₁ to a resolution of 1.9 Å and as a monomer in space group P2₁2₁2 to a resolution of 1.5 Å, with the ligand imidazole bound at the heme iron. Although it shares the overall fold of cytochromes P450 of known structure, XplA-heme is unusual in that the kinked I-helix that traverses the distal face of the heme is broken by Met-394 and Ala-395 in place of the well conserved Asp/Glu plus Thr/Ser, important in oxidative P450s for the scission of the dioxygen bond prior to substrate oxygenation. The heme environment of XplA-heme is hydrophobic, featuring a cluster of three methionines above the heme, including Met-394. Imidazole was observed bound to the heme iron and is in close proximity to the side chain of Gln-438, which is situated over the distal face of the heme. Imidazole is also hydrogen-bonded to a water molecule that sits in place of the threonine side-chain hydroxyl exemplified by Thr-252 in Cyt-P450cam. Both Gln-438 → Ala and Ala-395 → Thr mutants of XplA-heme displayed markedly reduced activity compared with the wild type for royal demolition explosive degradation when combined with surrogate electron donors.Royal demolition explosive (RDX)² or cyclotrimethylenetrinitramine 1 (see Fig. 1) is a widely used explosive compound with both military and civil applications. The extensive global usage of RDX has resulted in concerns over environmental contamination, because it is both recalcitrant to degradation, leading to contamination in soil and ground water, and a potent convulsant and possible carcinogen. The bioremediation of RDX has thus been the focus of increasing research in recent years, with a number of bacterial strains reported to catalyze its degradation (1–3). Among these, Bruce and co-workers, using a selective enrichment technique, isolated Rhodococcus rhodochrous strain 11Y from an RDX-contaminated site, which was able to grow on RDX as the sole nitrogen source (4). The products of biotransformation of RDX by this bacterium were shown to be nitrite and formaldehyde. A gene cluster in strain 11Y essential for RDX degradation was identified and shown to contain a novel cytochrome P450, termed XplA, and a redox partner, XplB, which were shown together to be capable of catalyzing the biotransformation of RDX in vitro (5). Near identical xplA and xplB genes have now been identified in strains within the Actinomycetales isolated from geographically distinct sites (6). Interestingly, these genes have been found to be plasmid encoded providing compelling evidence for recent lateral gene transfer. In the interests of developing an efficient technology for the targeted phytoremediation of RDX, the XplA-XplB system was expressed in transgenic strains of Arabidopsis thaliana and shown to successfully remediate RDX from contaminated soil (5, 7).Open in a separate windowFIGURE 1.Biocatalyzed routes proposed for the degradation of the nitramine exposive RDX 1 by cytochrome P450 XplA from Rhodococcus rhodochrous 11Y. Pathway A corresponds to the route proposed under anaerobic conditions to give the cleavage product metabolite methylene dinitramine 6; pathway B is that postulated to occur under aerobic conditions to give cleavage product 4-nitro-2,4-diazabutanal 7.RDX has an unusual chemical structure, featuring three nitramine (N-NO₂) bonds on a saturated six-membered ring, a functionality that has few precedents among natural products (8). The products isolated from XplA-XplB-catalyzed degradation of RDX in vitro were shown to be different under aerobic and anaerobic conditions after initial denitration to imine intermediate 2 (Fig. 1) (5). Under anaerobic conditions A, 2 would undergo hydration to give intermediate 4. Ring cleavage would yield the isolable metabolite methylene dinitramine 6. The aerobic process B was thought to proceed via successive denitration of two nitramine groups, to give the di-imine 3, which would then be twice hydrated to give the unstable intermediate 5, which would undergo spontaneous ring cleavage to give the isolable product 4-nitro-2,4-diazabutanal 7, formaldehyde, and two equivalents of nitrite.Although the reductive denitration of, for example, glycerol trinitrate (9) and other nitroheterocylic drugs (10), has previously been described for mammalian cytochromes P450, the best known reactions catalyzed by this family of enzymes include a wide range of oxidative chemical reactions, including hydroxylation, heteroatom dealkylation, and C–C bond cleavage reactions most commonly using molecular oxygen and involving cleavage of the O–O bond after dioxygen is bound by the heme-iron (11). A sequence alignment of some cyt-P450 for which the structures have been determined (Fig. 2) shows that the well conserved residues Glu/Asp-Thr/Ser that are thought to be a requirement for oxidative chemistry (12, 13) and to aid cleavage of the scissile O–O bond are not conserved in XplA, being replaced by methionine-394 and alanine-395. In the interests of illuminating the molecular mechanism and substrate specificity in XplA, we were thus interested in obtaining a structure of the heme domain of the enzyme. In this report, the structure of XplA-heme domain in two crystal forms is presented: the first with two molecules in the asymmetric unit, derived from the attempted crystallization of full-length XplA and the other derived from the subcloned, isolated XplA-heme domain, with one molecule in the asymmetric unit and the heme iron ligated to the substrate analogue imidazole. The results reveal a possible access channel for ligand transport and, in combination with mutational studies, demonstrate a highly unusual active site environment for substrate binding, suggesting substrate binding or catalytic roles for Gln-438 and Ala-395.Open in a separate windowFIGURE 2.Amino acid sequence alignment for portions of selected CYP heme domains for which the structures have been solved. P450_pikC (from Streptomyces venezuelae; Uniprot accession no. {"type":"entrez-protein","attrs":{"text":"O87605","term_id":"75539043","term_text":"O87605"}}O87605); P450_cin (from Citrobacter braakii; {"type":"entrez-protein","attrs":{"text":"Q8VQ86","term_id":"75529099","term_text":"Q8VQ86"}}Q8VQ86); P450_eryF (Saccharopolyspora erythrea; {"type":"entrez-protein","attrs":{"text":"Q00441","term_id":"729202","term_text":"Q00441"}}Q00441); P450_moxa (Nonomuraea recticatena; {"type":"entrez-protein","attrs":{"text":"Q2L6S8","term_id":"123293472","term_text":"Q2L6S8"}}Q2L6S8); P450_staP (Streptomyces sp. TP-A0274; {"type":"entrez-protein","attrs":{"text":"Q83WG3","term_id":"81321418","term_text":"Q83WG3"}}Q83WG3); P450_BioI (B. subtilis; {"type":"entrez-protein","attrs":{"text":"P53554","term_id":"1705469","term_text":"P53554"}}P53554); P450_epok (Sorangium cellulosum; {"type":"entrez-protein","attrs":{"text":"Q9KIZ4","term_id":"41688519","term_text":"Q9KIZ4"}}Q9KIZ4); P450_terp (Pseudomonas sp.; {"type":"entrez-protein","attrs":{"text":"P33006","term_id":"416837","term_text":"P33006"}}P33006); P450_xpla (Rhodococcus sp. 11Y; {"type":"entrez-protein","attrs":{"text":"Q8GPH7","term_id":"81322300","term_text":"Q8GPH7"}}Q8GPH7); P450_cam (Pseudomonas putida; {"type":"entrez-protein","attrs":{"text":"P00183","term_id":"117297","term_text":"P00183"}}P00183); and P450_BM3 (Bacillus megaterium; {"type":"entrez-protein","attrs":{"text":"P14779","term_id":"117298","term_text":"P14779"}}P14779). Each catalyzes oxidative chemistry and most apart from P450_cin contain the (E/D)(S/T) dyad (shown in red) demonstrated to be important in the scission of the dioxygen bond as a prerequisite of substrate mono-oxygenation. XplA possesses Met-394 and Ala-395 (shown in green).     

Cytochrome P450 monooxygenases: perspectives for synthetic application

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.     

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。     

RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius

Background

NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides.

Methodology/Principal Findings

The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs.

Conclusions/Significance

These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.     

Structures of prostacyclin synthase and its complexes with substrate analog and inhibitor reveal a ligand-specific heme conformation change

Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H(2). PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H(2), leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels.     

Pinpointing P450s associated with pyrethroid metabolism in the dengue vector, Aedes aegypti: developing new tools to combat insecticide resistance

Background

Pyrethroids are increasingly used to block the transmission of diseases spread by Aedes aegypti such as dengue and yellow fever. However, insecticide resistance poses a serious threat, thus there is an urgent need to identify the genes and proteins associated with pyrethroid resistance in order to produce effective counter measures. In Ae. aegypti, overexpression of P450s such as the CYP9J32 gene have been linked with pyrethroid resistance. Our aim was to confirm the role of CYP9J32 and other P450s in insecticide metabolism in order to identify potential diagnostic resistance markers.

Methodology/Principal Findings

We have expressed CYP9J32 in Escherichia coli and show that the enzyme can metabolize the pyrethroids permethrin and deltamethrin. In addition, three other Ae. aegypti P450s (CYP9J24, CYP9J26, CYP9J28) were found capable of pyrethroid metabolism, albeit with lower activity. Both Ae. aegypti and Anopheles gambiae P450s (CYP''s 6M2, 6Z2, 6P3) were screened against fluorogenic and luminescent substrates to identify potential diagnostic probes for P450 activity. Luciferin-PPXE was preferentially metabolised by the three major pyrethroid metabolisers (CYP9J32, CYP6M2 and CYP6P3), identifying a potential diagnostic substrate for these P450s.

Conclusions/Significance

P450s have been identified with the potential to confer pyrethroid resistance in Ae.aegypti. It is recommended that over expression of these enzymes should be monitored as indicators of resistance where pyrethroids are used.     

On the identity and reactivity patterns of the “second oxidant” of the T252A mutant of cytochrome P450cam in the oxidation of 5-methylenenylcamphor

Density functional calculations show that in the absence of Compound I, the primary oxidant species of P450, the precursor species, Compound 0 (FeOOH), can effect double bond activation of 5-methylenylcamphor (1). The mechanism is initiated by homolytic cleavage of the O–O bond and formation of an OH radical bound to the Compound II species by hydrogen bonding interactions. Subsequently, the so-formed OH radical can either activate the double bond of 1 or attack the meso position of the heme en route to heme degradation. The calculations show that double bond activation is preferred over attack on the heme. Past the double bond activation, the intermediate can either lead to epoxidation or to a glycol formation. The glycol formation is predicted to be preferred, although in the P450_cam pocket the competition may be closer. Therefore, in the absence of Compound I, Compound 0 will be capable of epoxidizing double bonds. Previous studies [E. Derat, D. Kumar, H. Hirao, S. Shaik, J. Am. Chem. Soc. 128 (2006) 473–484] showed that in the case of a substrate that can undergo only C–H activation, the bound OH prefers heme degradation over hydrogen abstraction. Since the epoxidation barrier for Compound I is much smaller than that of Compound 0 (12.8 vs. 18.9 kcal/mol), when Compound I is present in the cycle, Compound 0 will be silent. As such, our mechanism explains lucidly why T252A P450_cam can epoxidize olefins like 5-methylenylcamphor but is ineffective in camphor hydroxylation [S. Jin, T.M. Makris, T. A. Bryson, S.G. Sligar, J.H. Dawson, J. Am. Chem. Soc. 125 (2003) 3406–3407]. Our calculations show that the glycol formation is a marker reaction of Compound 0 with 5-methylenylcamphor. If this product can be found in T252A P450_cam or in similar mutants of other P450 isozymes, this will constitute a more definitive proof for the action of Cpd 0 in P450 enzymes.