Crystal structures of cytochrome P450nor and its mutants (Ser286-->Val, Thr) in the ferric resting state at cryogenic temperature: a comparative analysis with monooxygenase cytochrome P450s

Cytochrome P450nor (P450nor) is a heme enzyme isolated from the denitrifying fungus Fusarium oxysporum and catalyzes the NO reduction to N2O. Crystal structures of the wild type and two Ser286 mutants (Ser286-->Val, Ser286-->Thr) of P450nor have been determined for the ferric resting forms at a 1.7 A resolution at cryogenic temperature (100 K). We carried out three comparative analyses: (1) between the structures of P450nor at room temperature and cryogenic temperature, (2) between the structures of P450nor and four monooxygenase P450s, and (3) between the structures of the WT and the Ser286 mutant enzymes of P450nor. Comparison of the charge distribution on the protein surface suggests that proton and electron flow to the heme site is quite different in P450nor than in monooxygenase P450s. On the basis of the mutant structures, it was found that a special hydrogen-bonding network, Wat99-Ser286-Wat39-Asp393-solvent, acts as a proton delivery pathway in NO reduction by P450nor. In addition, the positively charged cluster located beneath the B'-helix is suggested as possible NADH binding site in P450nor, from which the direct two-electron transfer to the heme site allows to generate the characteristic intermediate in the NO reduction. These structural characteristics were not observed in structures of monooxygenase P450s, implying that these are factors determining the unique NO reduction activity of P450nor.     

Fungal denitrification and nitric oxide reductase cytochrome P450nor

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N(2)O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N(2)O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N(2) or N(2)O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.     

Role of the conserved threonine 309 in mechanism of oxidation by cytochrome P450 2D6

Based on sequence alignments and homology modeling, threonine 309 in cytochrome P450 2D6 (CYP2D6) is proposed to be the conserved I-helix threonine, which is supposed to be involved in dioxygen activation by CYPs. The T309V mutant of CYP2D6 displayed a strong shift from O-dealkylation to N-dealkylation reactions in oxidation of dextromethorphan and 3,4-methylenedioxymethylamphetamine. This may be explained by an elevated ratio of hydroperoxo-iron to oxenoid-iron of the oxygenating species. In consistence, using cumene hydroperoxide, which directly forms the oxenoid-iron, the T309V mutant again selectively catalyzed the O-dealkylation reactions. The changed ratio of oxygenating species can also explain the decreased activity and changed regioselectivity that were observed in 7-methoxy-4-(aminomethyl)-coumarin and bufuralol oxidation, respectively, by the T309V mutant. Interestingly, the T309V mutant always showed a significantly increased, up to 75-fold, higher activity compared to that of the wild-type when using cumene hydroperoxide. These results indicate that T309 in CYP2D6 is involved in maintaining the balance of multiple oxygenating species and thus influences substrate and regioselectivity.     

Role of Glu318 and Thr319 in the catalytic function of cytochrome P450d (P4501A2): effects of mutations on the methanol hydroxylation.

Polar amino acids in the (putative) distal site are well conserved in P450s. For example, Glu318 for P450d is well conserved as either Glu or Asp for P450s, and Thr319 for P450d is also conserved for P450s. We have studied how mutations at Glu318 and Thr319 of P450d influence the catalytic activity toward methanol associated with the activation of O2. Catalytic activities of Glu318Asp, Glu318Ala, and Thr319Ala mutants toward methanol were 60, 25, and 38%, respectively, compared with that of the wild type. O2 consumption and NADPH oxidation rates of each mutants varied corresponding to the catalytic activities. However, surprisingly, efficiency (16-40%) of incorporated O to the substrate vs. consumed O2 for the Glu318Ala and Thr319Ala mutants were higher than that (9%) of the wild type. In addition, H2O2, which is produced from uncoupling for the wild-type P450d, was not observed for reaction of the Glu318Ala and Thr319Ala mutants. It seemed that consumed O2 was partially reduced to 2 mol of H2O by 4-electron transfer from NADPH for the wild-type and Thr319Ala mutant. However, for the two Glu318 mutants, it appeared that the consumed O2 was not reduced in the same way. It was thus suggested that the conserved Glu318 and Thr319 of P450d are not essential for the activation of O2 in the methanol oxidation. Role of the water molecule or the methanol molecule in the catalytic function was implied.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

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。     

Mutation probability of cytochrome P450 based on a genetic algorithm and support vector machine

The support vector machine (SVM), an effective statistical learning method, has been widely used in mutation prediction. Two factors, i.e., feature selection and parameter setting, have shown great influence on the efficiency and accuracy of SVM classification. In this study, according to the principles of a genetic algorithm (GA) and SVM, we developed a GA-SVM program and applied it to human cytochrome P450s (CYP450s), which are important monooxygenases in phase I drug metabolism. The program optimizes features and parameters simultaneously, and hence fewer features are used and the overall prediction accuracy is improved. We focus on the mutation of non-synonymous single nucleotide polymorphisms (nsSNPs) in protein sequences that appear to exhibit significant influences on drug metabolism. The final predictive model has a quite satisfactory performance, with the prediction accuracy of 61% and cross-validation accuracy of 73%. The results indicate that the GA-SVM program is a powerful tool in optimizing mutation predictive models of nsSNPs of human CYP450s.     

A dynamic understanding of cytochrome P450 structure and function through solution NMR

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Nitric oxide reductase (P450nor) from Fusarium oxysporum

In the present review we wanted to highlight the characteristic features of cytochtome P450 NADH-NO reductase (P450nor) from Fusarium oxysporum which belongs to the heme-thiolate protein family. This enzyme catalyzes the reduction of two NO molecules to N2O. The discovery, isolation, identification and crystallography are described in detail. Special emphasis was focused on the mechanism of NO reduction and possible electronic configurations of the 444 nm intermediate were discussed. Among heme-thiolate proteins nitric oxide reductase (P450nor) is unique since it catalyzes the conversion to dinitrogen oxide as a reductive process. However, it joins the typical physical characteristics of other P450 proteins including the ferric NO complex which can be considered as the enzyme-substrate complex of the enzyme. At a closer look some of its properties like a tilted structure and a shorter Fe-N distance indicate properties for a facilitated hydride transfer from NADH. The resulting intermediate forms the product in a subsequent reaction with the NO radical. For this rate-limiting step at physiological NO levels electron transfer is postulated as a common feature with other heme-thiolate mechanisms. P450nor seems to have an important role in protecting the fungus from NO inhibition of mitochondria especially when dioxygen becomes limiting.     

The role of Thr268 and Phe393 in cytochrome P450 BM3

In flavocytochrome P450 BM3 there are several active site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate heme reduction potential through interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (DeltaE(m) [WT+arachidonate (120 microM)]=+138 mV). Substitution of Thr268 by an alanine or asparagine residue causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (DeltaE(m) [T268A+arachidonate (120 microM)]=+73 mV, DeltaE(m) [T268N+arachidonate (120 microM)]=+9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady-state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. In addition, substitutions of the threonine lead to an increase in the production of hydrogen peroxide in preference to hydroxylated product. These results suggest an important role for this active site threonine in substrate recognition and in maintaining an efficiently functioning enzyme. However, the dependence of the rate constants for oxy-ferrous decay on reduction potential raises some questions as to the importance of Thr268 in iron-oxo stabilisation.     

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.     

High-resolution crystal structure of cytochrome P450cam

The crystal structure of Pseudomonas putida cytochrome P450cam with its substrate, camphor, bound has been refined to R = 0.19 at a normal resolution of 1.63 A. While the 1.63 A model confirms our initial analysis based on the 2.6 A model, the higher resolution structure has revealed important new details. These include a more precise assignment of sequence to secondary structure, the identification of three cis-proline residues, and a more detailed picture of substrate-protein interactions. In addition, 204 ordered solvent molecules have been found, one of which appears to be a cation. The cation stabilizes an unfavorable polypeptide conformation involved in forming part of the active site pocket, suggesting that the cation may be the metal ion binding site associated with the well-known ability of metal ions to enhance formation of the enzyme-substrate complex. Another unusual polypeptide conformation forms the proposed oxygen-binding pocket. A localized distortion and widening of the distal helix provides a pocket for molecular oxygen. An intricate system of side-chain to backbone hydrogen bonds aids in stabilizing the required local disruption in helical geometry. Sequence homologies strongly suggest a common oxygen-binding pocket in all P450 species. Further sequence comparisons between P450 species indicate common three-dimensional structures with changes focused in a region of the molecule postulated to be associated with the control of substrate specificity.     

Crystal structure of cytochrome P450 MoxA from Nonomuraea recticatena (CYP105)

Cytochrome P450 MoxA (P450moxA) from a rare actinomycete Nonomuraea recticatena belongs to the CYP105 family and exhibits remarkably broad substrate specificity. Here, we demonstrate that P450moxA acts on several luciferin derivatives, which were originally identified as substrates of the human microsomal P450s. We also describe the crystal structure of P450moxA in substrate-free form. Structural comparison with various bacterial and human microsomal P450s reveals that the P450moxA structure is most closely related to that of the fungal nitric oxide reductase P450nor (CYP55A1). Final refined model of P450moxA comprises almost all the residues, including the "BC-loop" and "FG-loop" regions pivotal for substrate recognition, and the current structure thus defines a well-ordered substrate-binding pocket. Clear electron density map reveals that the MES molecule is bound to the substrate-binding site, and the sixth coordination position of the heme iron is not occupied by a water molecule, probably due to the presence of MES molecule in the vicinity of the heme. The unexpected binding of the MES molecule might reflect the ability of P450moxA to accommodate a broad range of structurally diverse compounds.     

Differential effect of copper (II) on the cytochrome P450 enzymes and NADPH-cytochrome P450 reductase: inhibition of cytochrome P450-catalyzed reactions by copper (II) ion

Inhibitory effects of Cu(2+) on the cytochrome P450 (P450)-catalyzed reactions of liver microsomes and reconstituted systems containing purified P450 and NADPH-P450 reductase (NPR) were seen. However, Zn(2+), Mg(2+), Mn(2+), Ca(2+), and Co(2+) had no apparent effects on the activities of microsomal P450s. Cu(2+) inhibited the reactions catalyzed by purified P450s 1A2 and 3A4 with IC(50) values of 5.7 and 8.4 microM, respectively. Cu(2+) also inhibited reduction of cytochrome c by NPR (IC(50) value of 5.8 microM). Copper caused a decrease in semiquinone levels of NPR, although it did not disturb the rate of formation of semiquinone. P450 reactions supported by an oxygen surrogate, tert-butyl hydroperoxide, instead of NPR and NADPH, were inhibited by the presence of Cu(2+). The results indicate that Cu(2+) inhibits the P450-catalyzed reactions by affecting both P450s and NPR. It was also found that the inhibition of catalytic activities of P450s by Cu(2+) involves overall conformational changes of P450s and NPR, investigated by CD and intrinsic fluorescence spectroscopy. These results suggest that the inhibitory effect of Cu(2+) on the P450-catalyzed reactions may come from the inability of an efficient electron transfer from NPR to P450 and also the dysfunctional conformation of NPR and P450.     

Formation of a novel reversible cytochrome P450 spectral intermediate: role of threonine 303 in P450 2E1 inactivation

tert-Butyl acetylene (tBA) is a mechanism-based inactivator of cytochromes P450 2E1 and 2E1 T303A; however, the inactivation of the T303A mutant could be reversed by overnight dialysis. The inactivation of P450 2E1 T303A, but not the wild-type 2E1 enzyme, by tBA resulted in the formation of a novel reversible acetylene-iron spectral intermediate with an absorption maximum at 485 nm. The formation of this intermediate required oxygen and could be monitored spectrally with time. Although the alternate oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of wild-type P450 2E1 by tBA in a reductase- and NADPH-free system, only tBHP supported the inactivation of the 2E1 T303A mutant. The losses in enzymatic activity occurred concomitantly with losses in the native P450 heme, which were accompanied by the formation of tBA-adducted heme products. The inactivations supported by tBHP and CHP were completely irreversible with overnight dialysis. Spectral binding constants (K(s)) for the binding of tBA to the 2E1 P450s together with models of the enzymes with the acetylenic inactivator bound in the active site suggest that the T303A mutation results in increased hydrophobic interactions between tBA and nearby P450 residues, leading to a higher binding affinity for the acetylene compound in the mutant enzyme. Together, these data support a role for the highly conserved T303 residue in proton delivery to the active site of P450 2E1 and in the inactivation of the 2E1 P450s by small acetylenic compounds.     

Differential effects of flavonoids on testosterone-metabolizing cytochrome P450s

Flavonoids are widely distributed phytochemicals, whose modulation of cytochrome P450 mediated carcinogen metabolism is well established. Less well studied is their effect on P450 dependent metabolism of endogenous substrates. To address this question we evaluated a series of twelve flavonoids and hematoxylin for their effect on P450-mediated steroid hydroxylation by rat liver microsomes. Site-specific 7α-, 6β- and 2α-hydroxylation of testosterone by P450s 2A1, 3A2 and 2C11, respectively, was measured. Highly selective patterns of inhibition or activation of these P450s were observed. 3,6-dichloro-2′-isopropyloxy-4′-methylflavone was the most potent inhibitor of P450 2C11 while cyanidin chloride most potently inhibited P450s 2A1 and 3A2. The flavonoid analogue hematoxylin was unique in that it activated 2C11 (by 2.5 fold) yet inhibited both 2A1 and 3A2 (by 60%). These results indicate that consumption of dietary flavonoids may likewise alter the metabolite profile of steroids and other physiological P450 substrates.     

Crystallization,preliminary diffraction and electron paramagnetic resonance studies of a single crystal of cytochrome P450nor

Cytochrome P450nor (P450nor) is a heme-containing nitric oxide reductase from the denitrifying fungus, Fusarium oxysporum. This enzyme catalyzes the reduction of NO to N₂O. In the present study, we report results from preliminary crystallographic and electron paramagnetic resonance (EPR) analysis of a single crystal of P450nor. The crystal was grown in 100 mM MES buffer at pH 5.6 using PEG 4000 as a precipitant. It belongs to the orthorhombic system with cell dimensions of a=54.99 Å, b=82.66 Å, c=87.21 Å, and the space group is P2₁2₁2₁. The crystal diffracts synchrotron radiation at higher than 2.0 Å resolution, and therefore it is suitable for X-ray crystal structure analysis at atomic resolution. Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the heme iron. The structure solution is currently underway by means of MIR and MAD techniques. EPR analysis determined the orientation of the heme within the P450nor crystal.