Phanerochaete chrysosporium NADPH-cytochrome P450 reductase kinetic mechanism

The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) gene and >123 cytochrome P450 (CYP) genes. How a single CPR can drive many CYPs is an important area of study. We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the cytochrome P450 catalytic system. Native CPR and a NH(2)-terminally truncated derivative lacking 23 amino acids have been overexpressed in Escherichia coli and purified to electrophoretic homogeneity. Steady-state kinetics of cytochrome c reductase activity revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reflecting differences in CPR kinetic mechanisms even within a single kingdom of life. Removal of the N-terminal anchor of P. chrysosporium CPR did not alter the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modification for structural studies.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Purification, cDNA cloning and functional expression of NADPH-cytochrome P450 reductase from Centaurium erythraea cell cultures

Solubilised NADPH-cytochrome P450 reductase (CPR) was purified from the microsomal fraction of centaury ( Centaurium erythraea ) cell cultures by Q-anion exchange chromatography and affinity chromatography on adenosine 2',5'-diphosphate agarose. SDS-PAGE demonstrated the presence of three CPR isoforms with molecular masses of 77, 79 and 81 kDa. The 79- and 81-kDa isoforms were identified as glycoproteins when blotted following SDS-PAGE and subjected to a sugar detection procedure. A homology-based approach led to the isolation of a CPR cDNA encoding the 77-kDa isoform. The enzyme was a class I CPR, possessing a short N-terminus upstream of the membrane anchor. The amino acid sequence contained a putative N -glycosylation site, indicating that the two major isoforms of 77 and 79 kDa are related through attachment of an oligosaccharide chain. This glycosylation process was also found upon heterologous expression in yeast. When co-expressed in yeast together with centaury coniferyl alcohol 5-hydroxylase, CPR efficiently supported the activity of the P450 enzyme. The genome of C. erythraea was found to contain a second CPR gene. RT-PCR experiments using gene-specific primers revealed differential regulation of the two CPR genes. While CPR 2 mRNA was strongly induced by the addition of methyl jasmonate to the cell cultures, the CPR 1 expression level did not change after this elicitation.     

NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability

NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain. The triple mutant was purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD. Deficiency in FAD content was overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site Ping-Pong mechanism with similar kinetic constants arguing against any global structural changes. Analysis of the single mutants indicates that the proline to alanine substitution has no impact, but that both leucine to phenylalanine substitutions are essential for FMN binding and maximum stability of the enzyme.     

Hydroxylation of quinocetone and carbadox is mediated by CYP1As in the chicken (Gallus gallus)

Quinoxaline derivatives (quinoxalines) comprise a class of drugs that have been widely used as animal antimicrobial agents and feed additives. Although the metabolism of quinoxaline drugs has been mostly studied using chicken liver microsomes, the biochemical mechanism of biotransformation of these chemicals in the chicken has yet to be characterized. In this study, using bacteria produced enzymes, we demonstrated that both CYP1A4 and CYP1A5 participate in the oxidative metabolism of quinoxalines. For CYP1A5, three hydroxylated metabolites of quinocetone were generated. In addition, CYP1A5 is able to hydroxylate carbadox. For CYP1A4, only one hydroxylated product of quinocetone on the phenyl ring was identified. Neither CYP1A5 nor CYP1A4 showed hydroxylation activity towards mequindox and cyadox. Our results suggest that CYP1A4 and CYP1A5 have different and somewhat overlapping substrate specificity in quinoxaline metabolism, and CYP1A5 represents a crucial enzyme in hydroxylation of both quinocetone and carbadox.     

Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO₂) the recalcitrant fused-ring high molecular weight (?4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30 years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.     

The NADPH-cytochrome P450 reductase family in Trypanosoma cruzi is involved in the sterol biosynthesis pathway

Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP⁺/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations.     

烟草甲细胞色素P450还原酶基因的分子特征与表达模式分析

烟草甲Lasioderma serricorne是一种重要的仓储害虫,长期化学防治导致烟草甲已对多种传统熏蒸剂产生抗性,但其对新型熏蒸剂甲酸乙酯仍处于敏感水平。因此明确其体内细胞色素P450还原酶（cytochrome P450 reductase, CPR）对甲酸乙酯的代谢解毒功能,对开展该药剂的抗性监测及延缓抗性的发生发展具有重要意义。本研究旨在克隆烟草甲LsCPR基因,分析其分子特征和表达特性,为进一步明确其在烟草甲对甲酸乙酯解毒代谢过程中的作用奠定基础。利用RT-PCR技术克隆烟草甲LsCPR基因的开放阅读框（open reading frame, ORF）;利用生物信息学软件分析LsCPR编码蛋白的结构、特征和系统进化关系。采用实时定量PCR技术检测LsCPR在烟草甲不同发育阶段（低龄幼虫、高龄幼虫、蛹、低龄成虫、高龄成虫）、幼虫不同组织（表皮、肠道、脂肪体和马氏管）以及甲酸乙酯熏蒸胁迫后的表达模式。烟草甲LsCPR基因的ORF为2 046 bp（GenBank登录号：MZ423209）,编码681个氨基酸,具有典型的昆虫CPR基因FMN区域、NADPH区域和FAD等保守结构域。系统发育分析表明,烟草甲LsCPR与鞘翅目昆虫聚为一支,且与赤拟谷盗Tribolium castaneum CPR亲缘关系最近。LsCPR在烟草甲不同发育阶段均有表达,在高龄幼虫期的表达水平较高;在幼虫体内的表达部位主要在肠道,其次为脂肪体和马氏管,而在表皮的表达水平最低。LC10、LC30和LC50 3种浓度的甲酸乙酯处理24 h后,烟草甲LsCPR表达量随着胁迫浓度升高而上调且显著高于对照;甲酸乙酯LC50处理烟草甲不同时间（3、6、12、24和48 h）后,LsCPR基因上调表达,24 h时达到表达高峰。推测LsCPR是参与烟草甲代谢甲酸乙酯的候选基因。     

Characterization of two NADPH: Cytochrome P450 reductases from cotton (Gossypium hirsutum)

Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton (Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including biosynthesis of secondary metabolites.     

High-level heterologous expression of fungal cytochrome P450s in Escherichia coli

A thorough understanding of the sequence–structure–function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using large-scale screening, we confirmed that at least 27 P450s could be expressed with/without simple sequence deletion at the 5′ end of cDNAs, which encode the N-terminal hydrophobic domain of the enzyme. Moreover, we identified N-terminal amino acid sequences that can potentially be used to construct chimeric P450s, which could dramatically improve their expression levels even when the expression of the wild-type sequence was unpromising. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.     

Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers

Heterologous expression of CYP73A5, an Arabidopsis cytochrome P450 monooxygenase, in baculovirus-infected insect cells yields correctly configured P450 detectable by reduced CO spectral analysis in microsomes and cell lysates. Co-expression of a housefly NADPH P450 reductase substantially increases the ability of this P450 to hydroxylate trans-cinnamic acid, its natural phenylpropanoid substrate. For development of high-throughput P450 substrate profiling procedures, membrane proteins derived from cells overexpressing CYP73A5 and/or NADPH P450 reductase were incorporated into soluble His(6)-tagged nanoscale lipid bilayers (Nanodiscs) using a simple self-assembly process. Biochemical characterizations of nickel affinity-purified and size-fractionated Nanodiscs indicate that CYP73A5 protein assembled into Nanodiscs in the absence of NADPH P450 reductase maintains its ability to bind its t-cinnamic acid substrate. CYP73A5 protein co-assembled with P450 reductase into Nanodiscs hydroxylates t-cinnamic acid using reduced pyridine nucleotide as an electron source. These data indicate that baculovirus-expressed P450s assembled in Nanodiscs can be used to define the chemical binding profiles and enzymatic activities of these monooxygenases.     

An ultrastructural study of the cysts in chicken ultimobranchial glands,with special reference to C-cells

Summary The ultimobranchial glands of the chicken were examined by electron microscopy and immunocytochemistry using a calcitonin antiserum. Electron microscopy confirmed the presence of C-cells, containing numerous secretory granules storing calcitonin, in the luminal lining of cyst-like structures found in these glands. These cells were furnished with prominent microvillar projections at their luminal surface, and the cytoplasm of the apical region was filled with fibril material. Furthermore, the cells contained prominent junctional complexes and desmosomes at their apico-lateral surfaces. In these C-cells, secretory granules were concentrated near the lumen and some were attached to the apical cell membrane. The luminal content of the cysts had a colloid-like and flocculent appearance, and was frequently seen attached to the cytoplasmic projections or apical cell membrane of the C-cells. Since the cysts progressively increase in volume and number with age, it is suggested that they may partly play a role in the storage of excess or unneeded hormonal products.     

Candida albicans NADPH-P450 reductase: Expression, purification, and characterization of recombinant protein

Candida albicans is responsible for serious fungal infections in humans. Analysis of its genome identified NCP1 gene coding for a putative NADPH-P450 reductase (NPR) enzyme. This enzyme appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence. In this study, we report the characterization of the functional features of NADPH-P450 reductase from C. albicans. The recombinant C. albicans NPR protein harboring a 6×(His)-tag was expressed heterologously in Escherichia coli, and was purified. Purified C. albicans NPR has an absorption maximum at 453 nm, indicating the feature of an oxidized flavin cofactor, which was decreased by the addition of NADPH. It also evidenced NADPH-dependent cytochrome c or nitroblue tetrazolium reducing activity. This purified reductase protein was successfully able to substitute for purified mammalian NPR in the reconstitution of the human P450 1A2-catalyzed O-deethylation of 7-ethoxyresorufin. These results indicate that purified C. albicans NPR is an orthologous reductase protein that supports cytochrome P450 or heme oxygenase enzymes in C. albicans.     

Functional expression and structural characterization of ORF cDNA encoding chitinase of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)

Chitin is an important component of the exoskeleton and peritrophic matrix in insects. Its bio-degradation is initiated by the endo-splitting chitinase. We cloned an ORF cDNA encoding chitinase from the last instar larva of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), into E. coli to confirm its functionality. Its amino acid sequence was compared with previously described lepidopteran chitinases. S. exigua chitinase expression enhanced cell growth approx. 1.5 fold in transformed E. coli than in the wild strain in a 1% colloidal chitin-containing medium with insufficient regular nutrients. Compared with the wild strain, the two intracellular chitin degradation derivatives, glucosamine and N-acetylglucosamine, increased approx. 5.8 and 1.5 fold, respectively, and extracellular chitinase activity in the transformed strain was about 1.6 fold higher. The ORF of S. exigua chitinase-encoding cDNA including stop codon was composed of 1674 bp nucleotides and the calculated molecular weight of the deduced 557 amino acid residues was about 62.6 kDa. The ORF consisted of an N-terminal leading signal peptide (AA 1-20), a catalytic domain (AA 21-392), a linker region (AA 393-493), and a C-terminal chitin-binding domain (AA 494-557) showing a typical molting fluid chitinase structure. Phylogenetic analysis determined that all 5 noctuid chitinases were grouped together, while two bombycid enzymes and one tortricid enzyme mapped together in one lineage. In the noctuid group, the sub-lineages reflected their taxonomic relationships at the Genus level.     

Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus

CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized. We report here the cloning and expression of CYP102A5, a new member of this family that is very closely related to CYP102A4 from Bacillus anthracis. Characterization of the substrate specificity of CYP102A5 shows that it, like the other CYP102s, will metabolize saturated and unsaturated fatty acids as well as N-acylamino acids. CYP102A5 catalyzes very fast substrate oxidation, showing one of the highest turnover rates for any P450 monooxygenase studied so far. It does so with more specificity than other CYP102s, yielding primarily ω-1 and ω-2 hydroxylated products. Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in CYP102A5 than in CYP102A1, providing a likely explanation for the increased monooxygenation rate. The availability of this new, very fast fusion P450 will provide a great tool for comparative structure-function studies between CYP102A5 and the other characterized CYP102s.