Search

The pathogenic fungus Candida albicans contains genes encoding five fatty acid hydroxylases belonging to the CYP52 family in its genome. Our previous study reported that CYP52A21 demonstrated typical omega-hydroxylation of lauric acid (Kim D, Cryle MJ, De Voss JJ, Ortiz de Montellano PR (2007) Arch Biochem Biophys 464, 213-220). Functional characterization of CYP52 fatty acid hydroxylases was studied, and their selectivity for hydroxylation was analyzed. Genes for four other CYP52 members (CYP52A22, CYP52A23, CYP52A24, and CYP52C3) from C. albicans were cloned, and their recombinant enzymes were expressed in Escherichia coli. CO-binding spectral analyses showed that the functional P450 holoenzyme was obtained only in CYP52A23, while no holoenzyme peak was observed in the other three CYP52 enzymes. Spectral change of the type II binding was observed in purified CYP52A23 when titrated with fatty acids but none was observed with alkanes. The gas chromatography-mass spectrometry (GC-MS) analysis revealed that CYP52A23 predominantly exhibited omega-hydroxylation activity during the oxidation reaction of fatty acids. Interestingly, it was found that CYP52A23 preferred longer-chain fatty acids (stearic acid and arachidic acid) for its catalytic activities while CYP52A21 preferred mid-chain fatty acids (lauric acid and mystic acid). To analyze the selectivity of fatty acids, hybrid mutagenesis of genes encoding CYP52A21 and CYP52A23 by overlap extension polymerase chain reaction was conducted. Two hybrid mutants containing the N-terminal fragments of CYP52A21 and C-terminal fragments of CYP52A23 displayed higher catalytic activity in palmitic acid and arachidic acid. These results suggested that the C-terminal part of CYP52A23 may be responsible for its preference to longer-chain fatty acids.

This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8C9 to the corresponding alkene was also detected.

CYP4B1 is an enigmatic mammalian cytochrome P450 monooxygenase acting at the interface between xenobiotic and endobiotic metabolism. A prominent CYP4B1 substrate is the furan pro-toxin 4-ipomeanol (IPO). Our recent investigation on metabolism of IPO related compounds that maintain the furan functionality of IPO while replacing its alcohol group with alkyl chains of varying structure and length revealed that, in addition to cytotoxic reactive metabolite formation (resulting from furan activation) non-cytotoxic ω-hydroxylation at the alkyl chain can also occur. We hypothesized that substrate reorientations may happen in the active site of CYP4B1. These findings prompted us to re-investigate oxidation of unsaturated fatty acids and fatty alcohols with C9–C16 carbon chain length by CYP4B1. Strikingly, we found that besides the previously reported ω- and ω-1-hydroxylations, CYP4B1 is also capable of α-, β-, γ-, and δ-fatty acid hydroxylation. In contrast, fatty alcohols of the same chain length are exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking results for the corresponding CYP4B1-substrate complexes revealed that fatty acids can adopt U-shaped bonding conformations, such that carbon atoms in both arms may approach the heme-iron. Quantum chemical estimates of activation energies of the hydrogen radical abstraction by the reactive compound 1 as well as electron densities of the substrate orbitals led to the conclusion that fatty acid and fatty alcohol oxidations by CYP4B1 are kinetically controlled reactions.

Hydroxylation activity at the 6β-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6β-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6β-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for progesterone 6β-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6β-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6β-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6β-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.

We have reported the interaction of two flavonoids namely quercetin (Q) and morin (M) with double stranded poly(A).poly(U) (herein after A.U) and triple stranded poly(U).poly(A)*poly(U) (herein after U.A*U, dot represents the Watson-Crick and asterisk represents Hoogsteen base pairing respectively) in this article. It has been observed that relative positions of hydroxyl groups on the B-ring of the flavonoids affect the stabilization of RNA. The double strand as well as the triple strand of RNA-polymers become more stabilized in presence of Q, however both the duplex and triplex remain unaffected in presence of M. The presence of catechol moiety on the B-ring of Q is supposed to be responsible for the stabilization. Moreover, after exploiting a series of biophysical experiments, it has been found that, triple helical RNA becomes more stabilized over its parent duplex in presence of Q. Fluorescence quenching, viscosity measurement and helix melting results establish the fact that Q binds with both forms of RNA through the mode of intercalation while M does not bind at all to either forms of RNA.

Stress-responsive dihydroxy flavonoids exhibit capability to inhibit the accretion of reactive oxygen species (ROS). The formation of these dihydroxy flavonols is catalyzed by flavonoid hydroxylases which are among the rate limiting enzymes of flavonoid biosynthesis pathway. Although flavonoid hydroxylases have been identified in several plant species but their role in abiotic stress is not explicitly documented. In the present study we report identification of all the flavonoid biosynthesis pathway genes of Crocus sativus and their expression profiling. We also report functional characterization of flavonoid 3' hydroxylase (CsF3'H) and attempt to explore its physiological role in vitro and in planta. The results indicated that CsF3'H is 1608 bp long encoding 535 amino acids. Docking and enzyme kinetic studies revealed that CsF3'H catalyzes hydroxylation of naringenin and dihydrokaempferol to eriodictoyl and dihydroquercetin respectively, but exhibits higher affinity for naringenin. Further, CsF3'H showed comparatively higher expression in floral tissues particularly stigma and its expression was significantly enhanced in response to UV-B, dehydration and salinity stress indicative of its role in stress. The expression of CsF3'H was associated with concomitant accumulation of eriodictoyl and dihydroquercetin. Transient overexpression of CsF3'H in Nicotiana benthamiana leads to the accumulation of substantial amounts of eriodictoyl and dihydroquercetin. Further, it was observed that transient expression of CsF3'H conferred tolerance to UV-B and dehydration stress as was evident from higher chlorophyll and soluble sugar and lower MDA contents. Taken together, these results suggest that CsF3'H confers tolerance to UV-B and dehydration in planta through synthesis of dihydroflavonols.

Cytochromes P450 are enzymes that catalyse the oxidation of a wide variety of compounds that range from small volatile compounds, such as monoterpenes to larger compounds like steroids. These enzymes can be modified to selectively oxidise substrates of interest, thereby making them attractive for applications in the biotechnology industry. In this study, we screened a small library of terpenes and terpenoid compounds against P450cin and two P450cin mutants, N242A and N242T, that have previously been shown to affect selectivity. Initial screening indicated that P450cin could catalyse the oxidation of most of the monoterpenes tested; however, sesquiterpenes were not substrates for this enzyme or the N242A mutant. Additionally, both P450cin mutants were found to be able to oxidise other bicyclic monoterpenes. For example, the oxidation of (R)- and (S)-camphor by N242T favoured the production of 5-endo-hydroxycamphor (65–77% of the total products, dependent on the enantiomer), which was similar to that previously observed for (R)-camphor with N242A (73%). Selectivity was also observed for both (R)- and (S)-limonene where N242A predominantly produced the cis-limonene 1,2-epoxide (80% of the products following (R)-limonene oxidation) as compared to P450cin (23% of the total products with (R)-limonene). Of the three enzymes screened, only P450cin was observed to catalyse the oxidation of the aromatic terpene p-cymene. All six possible hydroxylation products were generated from an in vivo expression system catalysing the oxidation of p-cymene and were assigned based on 1H NMR and GC-MS fragmentation patterns. Overall, these results have provided the foundation for pursuing new P450cin mutants that can selectively oxidise various monoterpenes for biocatalytic applications.

Flavin-dependent monooxygenases catalyze a wide variety of redox reactions in important biological processes and are responsible for the synthesis of highly complex natural products. Although much has been learned about FMO chemistry in the last ~80 years of research, several aspects of the reactions catalyzed by these enzymes remain unknown. In this review, we summarize recent advancements in the flavin-dependent monooxygenase field including aspects of flavin dynamics, formation and stabilization of reactive species, and the hydroxylation mechanism. Novel catalysis of flavin-dependent N-oxidases involving consecutive oxidations of amines to generate oximes or nitrones is presented and the biological relevance of the products is discussed. In addition, the activity of some FMOs have been shown to be essential for the virulence of several human pathogens. We also discuss the biomedical relevance of FMOs in antibiotic resistance and the efforts to identify inhibitors against some members of this important and growing family enzymes.

The ubiquitous flavoenzymes commonly catalyze redox chemistry such as the monooxygenation of organic substrates and are both widely utilized in nature (e.g., in primary and secondary metabolism) and of significant industrial interest. In this work, we highlight the structural and mechanistic characteristics of the distinct types of flavoprotein monooxygenases (FPMOs). We thereby illustrate the chemical basis of FPMO catalysis, which enables reactions such as (aromatic) hydroxylation, epoxidation, (de)halogenation, heteroatom oxygenation, Baeyer-Villiger oxidation, α-hydroxylation of ketones, or non-oxidative carbon-hetero bond cleavage. This seemingly unmatched versatility in oxygenation chemistry results from extensive fine-tuning and regiospecific functionalization of the flavin cofactor that is tightly controlled by the surrounding protein matrix. Accordingly, FPMOs steer the formation of covalent flavin-oxygen adducts for oxygen transfer in the form of the classical flavin-C4a-(hydro)peroxide or the recently discovered N5-functionalized flavins (i.e. the flavin-N5-oxide and the flavin-N5-peroxide), while in rare cases covalent oxygen adduct formation may be foregone entirely. Finally, we speculate about hitherto undiscovered flavin-mediated oxygenation reactions and compare FPMOs to cytochrome P450 monooxygenases, before addressing open questions and challenges for the future investigation of FPMOs.

l -lysine ( l -Lys) N6-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l -Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)+ or l -Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l -Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l -Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2′-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l -Lys binding.

The first step in the degradation of p-hydroxyphenylacetic acid (HPA) is catalyzed by the two-component enzyme p-hydroxyphenylacetate 3-hydroxylase (HPAH). The two components of Acinetobacter baumannii HPAH are known as C1 and C2, respectively. C1 is a flavin reductase that uses NADH to generate reduced flavin mononucleotide (FMNH-), which is used by C2 in the hydroxylation of HPA. Interestingly, although HPA is not directly involved in the reaction catalyzed by C1, the presence of HPA dramatically increases the FMN reduction rate. Amino acid sequence analysis revealed that C1 contains two domains: an N-terminal flavin reductase domain, and a C-terminal MarR domain. Although MarR proteins typically function as transcription regulators, the MarR domain of C1 was found to play an auto-inhibitory role. Here, we report a crystal structure of C1 and small-angle X-ray scattering (SAXS) studies that revealed that C1 undergoes a substantial conformational change in the presence of HPA, concomitant with the increase in the rate of flavin reduction. Amino acid residues that are important for HPA binding and regulation of C1 activity were identified by site-directed mutagenesis. Amino acid sequence similarity analysis revealed several as yet uncharacterized flavin reductases with N- or C-terminal fusions.

Cylindrospermopsin, a cytotoxin from cyanobacteria, is biosynthesized by a complex pathway, which involves CyrI, an iron and 2-oxoglutarate dependent hydroxylase that transforms 7-deoxy-cylindrospermopsin into cylindrospermopsin and its epimer, 7-epi-cylindrospermopsin, in the last step. The activity of CyrI from Oscillatoria sp. PCC 7926 depends on Fe(II) (Km = 2.1 μM), and 2-oxoglutarate (Km = 3.2 μM), and is strongly inhibited by 7-deoxy-cylindrospermopsin at concentration higher than 1 μM. Using tryptophan fluorescence, we measured the binding to CyrI of Fe(II) (KD = 0.02 μM) and 2-oxoglutarate (KD = 53 μM and KD = 1.1 μM in the absence or presence of 10 μM Fe(II), respectively). The Oscillatoria sp. PCC 6506 CyrI mutants H157A, D159A, H247A, and R257A were all inactive, and impaired in the binding of Fe(II) or 2-oxoglutarate, confirming the identity of the iron ligands and the role of R257 in the binding of 2-oxoglutarate. We constructed several chimeric enzymes using the Oscillatoria sp. PCC 7926 CyrI protein (stereoselective) and that from Oscillatoria sp. PCC 6506 (not stereoselective) to help understanding the structural factors that influence the stereoselectivity of the hydroxylation. Our data suggest that a predicted α-helix in CyrI (positions 87–108) seems to modulate the stereoselectivity of the reaction.

p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to yield 3,4-dihydroxyphenylacetate (DHPA). In this study, we investigated whether variants of the oxygenase component (C2) could catalyze hydroxylation of 4-hydroxyphenylethylamines to synthesize catecholamine derivatives. Single turnover product analysis showed that the R263D variant can catalyze hydroxylation of tyramine to form dopamine with the highest yield (57%). The enzyme was also found to have dual substrate charge specificity because it can also maintain reasonable hydroxylation efficiency of HPA (86%). This property is different from the R263E variant, which can hydroxylate HPA (73%) but not tyramine. The R263A variant can hydroxylate HPA (72%) and tyramine to a small extent (7%). Stopped-flow experiments indicated that tyramine and HPA prefer binding to R263D after C4a-hydroperoxy-FMN formation, while tyramine cannot bind to the wild-type or R263E enzymes. Data also indicate that the hydroxylation rate constant is the rate-limiting step. The R263D variant was used as a starting enzyme for further mutation to obtain other variants for the synthesis of additional catecholamine drugs. The R263D/Y398D double mutant enzyme showed interesting results in that it was able to catalyze the hydroxylation of octopamine to form norepinephrine. However, the enzyme still lacked stereo-selectivity in its reaction.

A self-sufficient CYP102 family encoding gene (Krac_9955) has been identified from the bacterium Ktedonobacter racemifer DSM44963 which belongs to the Chloroflexi phylum. The characterisation of the substrate range of this enzyme was hampered by low levels of production using E. coli. The yield and purity of the Krac9555 enzyme was improved using a codon optimised gene, the introduction of a tag and modification of the purification protocol. The heme domain was isolated and in vitro analysis of substrate binding and turnover was performed. Krac9955 was found to preferentially bind alkyl- and alkyloxy-benzoic acids (≥95% high spin, Kd < 3 μM) over saturated and unsaturated fatty acids. Unusually for a self-sufficient CYP102 family member Krac9955 showed low levels of NAD(P)H oxidation activity for all the substrates tested though product formation was observed for many. For nearly all substrates the preferred site of hydroxylation of Krac9955 was eight carbons away from the carboxylate group with certain reactions proceeding at ≥ 90% selectivity. Krac9955 differs from CYP102A1 (P450Bm3), and is the first self-sufficient member of the CYP102 family of P450 enzymes which is not optimised for fast fatty acid hydroxylation close to the ω-terminus.

The light-driven hybrid P450 enzyme approach utilizing the photochemical properties of a covalently attached Ru(II)-diimine photosensitizer was extended to the archaeal Sulfolobus acidocaldarius CYP119 enzyme leading to high photocatalytic activity in the hydroxylation of the chromogenic substrate, 11-nitrophenoxyundecanoic acid. The determined kcat was greater than those reported with various natural redox partners. In addition, the sacrificial electron donor, diethyldithiocarbamate, used in the photocatalytic reaction is shown to play a dual role. It acts as an efficient quencher of the Ru(II) excited state leading to a highly reducing species necessary to inject electrons into the heme. It is also known for its antioxidant properties and is shown herein to be a useful probe to determine coupling efficiency in the light-driven hybrid enzymes.

Cytochrome P450 (CYP) 147F1 from Streptomyces peucetius is a new CYP subfamily of that has been identified as ω-fatty acid hydroxylase. We describe the identification of CYP147F1 as a fatty acid hydroxylase by screening for the substrate using a substrate binding assay. Screening of substrates resulted in the identification of fatty acid groups of compounds as potential hits for CYP147F1 substrates. Fatty acids from C10:0 to C18:0 all showed type I shift spectra indicating their potential as substrates. Among several fatty acids tested, lauric acid, myrsitic acid, and palmitic acid were used to characterize CYP147F1. CYP147F1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida as surrogate electron transfer partners. Kinetic parameters, including the dissociation constant, Km, NADH consumption assay, production formation rate, and coupling efficiency for CYP147F1 were also determined.

The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.

Flavin-dependent monooxygenase (FMO) from Methylophaga sp. strain SK1 catalyzes the NADPH- and oxygen-dependent hydroxylation of a number of xenobiotics. Reduction of the flavin cofactor by NADPH is required for activation of molecular oxygen. The role of a conserved tryptophan at position 47 was probed by site-directed mutagenesis. FMOW47A resulted in an insoluble inactive protein; in contrast, FMOW47F was soluble and active. The spectrum of the flavin in the mutant enzyme was redshifted, indicating a change in the flavin environment. The kcat values for NADPH, trimethylamine, and methimazole, decreased 5-8-fold. Primary kinetic isotope effect values were higher, indicating that hydride transfer is more rate-limiting in the mutant enzyme. This is supported by a decrease in the rate constant for flavin reduction and in the solvent kinetic isotope effect values. Results from molecular dynamics simulations show reduced flexibility in active site residues and, in particular, the nicotinamide moiety of NADP+ in FMOW47F. This was supported by thermal denaturation experiments. Together, the data suggests that W47 plays a role in maintaining the overall protein flexibility that is required for conformational changes important in hydride transfer.

l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼ 10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10-50 μM) and increases kcat (10-25 s(-1)) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼ 10-fold higher than that of the other forms (3-5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues.

(15)N heavy isotope effects are especially useful when detail is sought pertaining to the reaction mechanism for the cleavage of a C-N bond. Their potential in assisting to describe the mechanism of N-demethylation of tertiary amines by the action of cytochrome P450 monooxygenase has been investigated. As a working model for the first step, oxidation of the N-methyl group to N-methoxyl, tropine and a cytochrome P450 monooxygenase reaction centre composed of a truncated heme with sulfhydryl as the axial ligand were used. It is apparent that this first step of the reaction proceeds via a hydrogen atom transfer mechanism. Transition states for this step are described for both the high spin ((4)TS(H)) and low spin ((2)TS(H)) pathways in both gas and solvation states. Hence, overall normal secondary (15)N KIE could be calculated for the reaction path modeled in the low spin state, and inverse for the reaction modeled in the high spin state. This partial reaction has been identified as the probable rate limiting step. The model for the second step, fission of the C-N bond, consisted of N-methoxylnortropine and two molecules of water. A transition state described for this step, TS(CN), gives a strongly inverse overall theoretical (15)N KIE.

Manganese-oxo porphyrins have been well studied as biomimetic models of cytochromes P450 and are known to be able to catalyze substrate hydroxylation reactions. Recent experimental studies [J.Y. Lee, Y.-M. Lee, H. Kotani, W. Nam, S. Fukuzumi, Chem. Commun. (2009) 704] showed that Mn(V)-oxo porphyrins react rapidly with 10-methyl-9,10-dihydroacridine (AcrH(2)) via a proton-coupled-electron-transfer followed by an electron transfer. In this work, we present a computational study on the reactivity patterns of Mn(V)-oxo and Mn(IV)-oxo with respect to AcrH(2). This study shows that although both oxidants are capable of hydroxylating AcrH(2), the Mn(V)-oxo species is the more active oxidant. We have generalized these observations with thermodynamic cycles that explain the reaction mechanisms and electron transfer processes. For the Mn(V)-oxo mechanism the reactions proceed with a fast spin state crossing from the ground state singlet to the triplet spin state prior to a hydrogen atom transfer followed by another electron transfer. The present results are fully consistent with previous studies on iron-oxo porphyrins and manganese-oxo porphyrins and shows that the interplay of low lying singlet and triplet spin state surfaces influences the reaction mechanisms and kinetics.

Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC–MS. Regioselective hydroxylation of C12–C20 saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, ketohydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H218O2 established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates., This study was funded by the PEROXICATS (KBBE-2010-4-265397) and BIORENEW (NMP2-CT-2006-026456) EU-projects, and the ELLE (AGL2008-00709) and RAPERO (BIO2008-01533) Spanish MICINN projects co-financed by FEDER funds.

Tetrahydrofuran monooxygenase (Thm) catalyzes the NADH-and oxygen-dependent hydroxylation of tetrahydrofuran to 2-hydroxytetrahydrofuran. Thm is composed of a hydroxylase enzyme, a regulatory subunit, and an oxidoreductase named ThmD. ThmD was expressed in Escherichia coli as a fusion to maltose-binding protein (MBP) and isolated to homogeneity after removal of the MBP. Purified ThmD contains covalently bound FAD, [2Fe-2S] center, and was shown to use ferricyanide, cytochrome c, 2,6-dichloroindophenol, and to a lesser extent, oxygen as surrogate electron acceptors. ThmD displays 160-fold preference for NADH over NADPH and functions as a monomer. The flavin-binding domain of ThmD (ThmD-FD) was purified and characterized. ThmD-FD displayed similar activity as the full-length ThmD and showed a unique flavin spectrum with a major peak at 463nm and a small peak at 396 nm. Computational modeling and mutagenesis analyses suggest a novel three-dimensional fold or covalent flavin attachment in ThmD.

In the petals of Dahlia variabilis, hydroxylation of chalcones at position 3 can be detected, except the well-known flavonoid 3'-hydroxylation. Although the reaction is well characterized at the enzymatic level, it remained unclear whether it is catalyzed by a flavonoid 3'-hydroxylase (F3'H, EC1.14.13.21, CYP75B) with broad substrate specificity. Two novel allelic variants of F3'H were cloned from D. variabilis, which differ only in three amino acids within their 508 residues. The corresponding recombinant enzymes show significant differences in their chalcone 3-hydroxylase (CH3H) activity. A substitution of alanine at position 425 with valine enables CH3H activity, whereas the reciprocal substitution leads to a loss of CH3H activity. Interaction of the valine at position 425 with not yet identified structural properties seems to be decisive for chalcone acceptance. This is the first identification of an F3'H which is able to catalyze chalcone 3-hydroxylation to a physiologically relevant extent from any plant species.

CYP4F22 and CYP4F8 are expressed in epidermis, and mutations of CYP4F22 are associated with lamellar ichthyosis. Epoxyalcohols (HEETs) and epoxides (EETs) of 20:4n-6 appear to be important for the water permeability barrier of skin. Our aim was to study the MS/MS spectra and fragmentation of these compounds and to determine whether they were oxidized by CYP4F22 or CYP4F8 expressed in yeast. HEETs were prepared from 15-hydroperoxyeicosatetraenoic acid (15-HPETE), 12-HPETE, and their [(2)H(8)]labeled isotopomers, and separated by normal phase-HPLC with MS/MS analysis. CYP4F22 oxygenated 20:4n-6 at C-18, whereas metabolites of HEETs could not be identified. CYP4F8 formed omega3 hydroxy metabolites of HEETs derived from 12R-HPETE with 11,12-epoxy-10-hydroxy configuration, but not HEETs derived from 15S-HPETE. 8,9-EET and 11,12-EET were also subject to omega3 hydroxylation by CYP4F8. We conclude that CYP4F8 and CYP4F22 oxidize 20:4n-6 and that CYP4F8 selectively oxidizes 8,9-EET, 11,12-EET, and 10,11R,12R-HEET at the omega3 position.

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) and 5-pyridoxic acid oxygenase are flavoenzymes catalyzing an aromatic hydroxylation and a ring-cleavage reaction. Both enzymes are involved in biodegradation of vitamin B6 in bacteria. Oxygen-tracer experiments have shown that the enzymes are monooxygnases since only one atom of molecular oxygen is incorporated into the products. Kinetics of MHPCO has shown that the enzyme is similar to single-component flavoprotein hydroxylases in that the binding of MHPC is required prior to the flavin reduction by NADH, and C4a-hydroperoxy-FAD and C4a-hydroxy-FAD are found as intermediates. Investigation on the protonation status of the substrate upon binding to the enzyme has shown that only the tri-ionic form of MHPC is bound at the MHPCO active site. Using a series of FAD analogues with substituents at the 8-position of the isoalloxazine ring, the oxygenation of MHPC by the C4a-hydroperoxy-FAD was shown to occur via an electrophilic aromatic substitution mechanism. Recently, the X-ray structures of MHPCO and a complex of MHPC-MHPCO at 2.1A have been reported and show the presence of nine water molecules in the enzyme active site. Based on structural data, a few residues, Tyr82, Tyr223, Arg181, were suggested to be important for catalysis of MHPCO.

Flavoprotein monooxygenases reduce flavins, speed their reaction with oxygen, and stabilize a C4a-oxygen adduct long enough to use this reactive species to transfer an oxygen atom to a substrate. The flavin-oxygen adduct can be the C4a-peroxide anion, in which case it reacts as a nucleophile. The protonated adduct - the C4a-hydroperoxide - reacts as an electrophile. The elimination of H(2)O(2) competes with substrate oxygenation. This side-reaction is suppressed, preventing the waste of NAD(P)H and the production of toxic H(2)O(2). Several strategies have been uncovered that prevent the deleterious side-reaction while still allowing substrate hydroxylation.

There are 75 full length cytochrome P450 (CYP) genes known in the genome of the nematode Caenorhabditis elegans. The individual biological functions of the vast majority are mostly as yet unknown. Here the impact of cytochrome P450 isoforms on the metabolism of PCB52, an ortho-substituted, non-coplanar 2,2',5,5'-tetrachlorbiphenyl, as a model PCB of these worldwide distributed pollutants is investigated. Organic extracts, isolated from treated worms and analyzed by GC/MS, contained two obvious PCB52-derived products which have been identified as C3-, C4- and/or C6-hydroxy-PCB52. Moreover, these hydroxylase reactions strictly required the functional expression of the NADPH-dependent cytochrome P450 reductase (CPR) encoding emb-8 gene, which was recently shown to be essential also for several other cytochrome P450-dependent enzymatic reactions. Multiple and subsequent single RNAi-gene silencing experiments, as well as the use of cyp-mutant strains, identified members of the CYP-14A subfamily and CYP-34A6 as the major isoforms contributing to PCB52 metabolism in C. elegans. In the gene-silenced worms and mutants, the reduction in formation of hydroxylated products ranged from 55% to 78%. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent PCB metabolites and may thus facilitate future studies on biotransformation.

The last few steps in the biosynthesis of the anticancer drug Taxol in yew (Taxus) species are thought to involve the attachment of beta-phenylalanine to the C13-O-position of the advanced taxane diterpenoid intermediate baccatin III to yield N-debenzoyl-2'-deoxytaxol, followed by hydroxylation on the side chain at the C2'-position to afford N-debenzoyltaxol, and finally N-benzoylation to complete the pathway. A cDNA encoding the N-benzoyl transferase that catalyzes the terminal step of the reaction sequence was previously isolated from a family of transferase clones (derived from an induced Taxus cell cDNA library) by functional characterization of the corresponding recombinant enzyme using the available surrogate substrate N-debenzoyl-2'-deoxytaxol [K. Walker, R. Long, R. Croteau, Proc. Nat. Acad. Sci. USA 99 (2002) 9166-9171]. Semi-synthetic N-debenzoyltaxol was prepared by coupling of 7-triethylsilybaccatin III and (2R,3S)-beta-phenylisoserine protected as the N-Boc N,O-isopropylidene derivative by means of carbodiimide activation and formic acid deprotections. The selectivity of the recombinant N-transferase for N-debenzoyltaxol was evaluated, and the enzyme was shown to prefer, by a catalytic efficiency factor of two, N-debenzoyltaxol over N-debenzoyl-2'-deoxytaxol as the taxoid co-substrate in the benzoyl transfer reaction, consistent with the assembly sequence involving 2'-hydroxylation prior to N-benzoylation. Selectivity for the acyl/aroyl-CoA co-substrate was also examined, and the enzyme was shown to prefer benzoyl-CoA. Transfer from tigloyl-CoA to N-debenzoyltaxol to afford cephalomannine (Taxol B) was not observed, nor was transfer observed from hexanoyl-CoA or butanoyl-CoA to yield Taxol C or Taxol D, respectively. These results support the proposed sequence of reactions for C13-O-side chain assembly in Taxol biosynthesis, and suggest that other N-transferases are responsible for the formation of related, late pathway, N-acylated taxoids.

Cytochrome P450 (CYP) enzymes involved in mammalian xenobiotic metabolism are attractive targets for the engineering of biocatalysts since they have broad and overlapping substrate and reaction substrate specificities. In this report, a library of chimeric mutants was prepared from CYP2C8, CYP2C9, CYP2C18 and CYP2C19 by DNA family shuffling. Twelve randomly selected clones were fully sequenced and showed 9 +/- 2 crossovers and 1.5 +/- 0.5 spontaneous mutations per similar to 1.5 kbp open reading frame. CYP hemoprotein expression was observed in 50% (microaerobic culture) to 54% (aerobic culture) of clones. The functional diversity of the library was assessed using three luminogenic substrates, diclofenac and indole as probe substrates. A random sample of 26 clones revealed two clones with activity towards luciferin ME, one towards luciferin H and five towards diclofenac 4'-hydroxylation. One mutant showed activity towards all three substrates. Of 96 clones screened on solid media, one showed elevated indigo production compared to the parental forms. Turnover rates for luciferin ME and H metabolism by CYP2C9 and mutants were at least one order of magnitude higher in experiments with membranes compared to whole cells, consistent with impaired product egress from cells. Apparent K-m values were increased in whole cell incubations with luciferin H suggesting impaired access of the substrate to the active site of the enzymes in whole cells. Finally screening with a panel of CYP2C ligands using CYP2C9 or active mutants revealed different patterns of inhibition and heteroactivation of metabolism of luciferin analogs. (C) 2007 Elsevier Inc. All rights reserved.

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k(cat)/K(m) for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K(m) (0.72 microM vs. 18 microM). I209A also demonstrated enhanced selectivity for testosterone 16beta-hydroxylation over 16alpha-hydroxylation. In contrast, V183L showed a 4-fold increased k(cat) for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k(cat)/K(m) for testosterone 16alpha-hydroxylation. V183L also displayed a 1.7-fold higher k(cat)/K(m) than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a approximately 4-fold decrease in K(m). Introduction of the V183L mutation into full-length P450 2B11 did not enhance the k(cat)/K(m). Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs.

The SidA ornithine N5-monooxygenase from Aspergillus fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP(+) and ornithine shows that the nicontinamide riboside group of NADP is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites.

Streptomyces peucetius ATCC27952 contains the cytochrome P450 monoxygenase DoxA that is responsible for the hydroxylation of daunorubicin into doxorubicin. Although S. peucetius ATCC27952 contains several potential redox partners, the most suitable endogenous electron-transport system is still unclear; therefore, we conducted a study of potential redox partners using Accelrys Discovery Studio 3.5. Recombinant DoxA along with its redox partners from S. peucetius FDX1, FDR2, and FDX3, and the putidaredoxin and putidaredoxin reductase from Pseudomonas putida that are essential equivalents of the class I type of bacterial electron-transport system were over-expressed and purified. The successful development of an efficient redox system was achieved by an in vitro enzymatic catalysis reaction with DoxA. The optimal pH for the activation of the heme was 7.6 and the optimal temperature was 30 °C. Our findings suggest a two-fold increase of DoxA activity via the NADH → FDR2 → FDX1 → DoxA pathway for the hydroxylation of the daunorubicin, and indicate that the usage of a native redox partner may increase daunorubicin-derived doxorubicin production due to the inclusion of DoxA.

Recombinant CYP4F8 and CYP4F12 metabolize prostaglandin H2 (PGH2) analogs by omega2- and omega3-hydroxylation and arachidonic acid (20:4n-6) by omega3-hydroxylation. CYP4F8 was found to catalyze epoxidation of docosahexaenoic acid (22:6n-3) and docosapentaenoic acid (22:5n-3) and omega3-hydroxylation of 22:5n-6. CYP4F12 oxidized 22:6n-3 and 22:5n-3 in the same way, but 22:5n-6 was a poor substrate. The products were identified by liquid chromatography-mass spectrometry. The missense mutation 374AT of CYP4F8 (Tyr125Phe in substrate recognition site-1 (SRS-1)) occurs in low frequency. This variant oxidized two PGH2 analogs, U-51605 and U-44069, in analogy with CYP4F8, but 20:4n-6 and 22:5n-6 were not oxidized. CYP4F enzymes with omega-hydroxylase activity contain a heme-binding Glu residue, whereas CYP4F8 (and CYP4F12) with omega2- and omega 3-hydroxylase activities has a Gly residue in this position of SRS-4. The mutant CYP4F8 Gly328Glu oxidized U-51605 and U-44069 as recombinant CYP4F8, but the hydroxylation of arachidonic acid was shifted from C-18 to C-19. Single amino acid substitutions in SRS-1 and SRS-4 of CYP4F8 may thus influence oxygenation of certain substrates. We conclude that CYP4F8 and CYP4F12 catalyze epoxidation of 22:6n-3 and 22:5n-3, and CYP4F8 omega3-hydroxylation of 22:5n-6.

Unlike monooxygenases, molybdenum-containing hydroxylases catalyze the hydroxylation of carbon centers using oxygen derived ultimately from water, rather than O(2), as the source of the oxygen atom incorporated into the product, and do not require an external source of reducing equivalents. The mechanism by which this interesting chemistry takes place has been the subject of investigation for some time, and in the last several years the chemical course of the reaction has become increasingly well understood. The present minireview summarizes recent mechanistic and structure/function studies of members of this large and growing family of enzymes.

The effect of plasticizers phthalate esters (PEs) on health is a controversial subject. PEs are likely to be estrogenic, but the results on the potency obtained by many investigators are still inconsistent and the endocrine disrupting mechanism remains to be clarified. Here, we show that PEs acquire unequivocal binding affinities for human estrogen receptors (ERs) through ring hydroxylation that is possible in the environment and through metabolism. Unexpectedly, the acquired affinities of hydroxylated PEs (PEs-OH) were enhanced by elongation and branching of the ester alkyl chains. PEs-OH with alkyl chains more than six carbons may grope for a new binding site, which is inaccessible to PEs-OH with short chains. The strongest ER-binding affinity among the tested PEs-OH was close to that of diethylstilbestrol, the most potent synthetic ER-binder. Ring hydroxylation would be a new clue to the clarification of the endocrine disruption mechanism of PEs.

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

Human cytochrome P450 1A2 catalyzes important reactions in xenobiotic metabolism, including the N-hydroxylation of carcinogenic aromatic amines. In 2001, Chevalier et al. [Hum. Mutat. 17 (2001) 355] reported four new P450 1A2 sequence variants in the human population. We have now expressed these variants in Escherichia coli and measured protein expression (optical spectroscopy of holoenzyme and immunoblotting) and bioactivation of IQ (2-amino-3-methylimidazo[4,5- f ]quinoline) and MeIQ (2-amino-2,4-dimethylimidazo[4,5- f ]quinoline) in the lacZ reversion mutagenicity test. Enzyme kinetic analyses were performed for N -hydroxylation of five heterocyclic amine substrates and for O-deethylation of phenacetin. The most drastic effect was that of the R431W substitution: no holoenzyme was detectable. This residue is located in the “meander” peptide region and earlier site-directed mutagenesis studies demonstrated that it is critical for maintenance of protein tertiary structure. The other three variants had subtly different catalytic activities compared to the wild-type enzyme.

A number of enzymes from the cytochrome P450 family show atypical (non-Michaelis-Menten) kinetic behavior resulting from substrate activation, inhibition, partial inhibition, biphasic saturation, or autoactivation. Herein, we provide a technique that can identify multiple substrate occupancy in the same active site of a P450 as a result of an altered kinetic profile. Using an isotope effect on product ratios confirms that the enzyme-substrate (ES) complex responsible for omega hydroxylation of palmitic acid (palmitate) is in rapid equilibrium with the ES complex that leads to omega-1 hydroxylation of palmitate. Co-incubation of a second substrate, lauric acid (laurate), results in a change in the ratio of omega to omega-1 hydroxylated palmitate. Furthermore, an isotope effect on palmitate is observed when deuterated laurate is co-incubated with non-deuterated palmitate. These results are only consistent with both substrates being in the same active site simultaneously. This mode of binding explains how the F87A mutant of P450BM3 is able to produce the omega alcohol, a product that arises from the high-energy primary radical.

3-Hydroxykynurenine is a tryptophan metabolite with an o-aminophenol structure. It is both a tyrosinase activator and a substrate, reducing the lag phase, stimulating the monophenolase activity, and being oxidized to xanthommatin. In the early stage of monophenol hydroxylation, catechol accumulation takes place, whereas 3-hydroxykynurenine is substantially unchanged and no significant amounts of the o-quinone are produced. These results suggest an activating action of 3-hydroxykynurenine toward o-hydroxylation of monophenols. 3-Hydroxykynurenine could therefore well act as a physiological device to control phenolics metabolism to catechols and quinonoids.

Biosynthesis of nitric oxide (NO) is catalyzed by NO synthase (NOS) through a two-step oxidation of L-arginine (Arg) with formation of an intermediate, GN-hydroxy-L-Arg (NHA). In this study we have employed mutagenesis to investigate how residues Y357 and R365 which interact primarily with the substrate Arg and (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)B) modulate these two steps of the NOS reaction. Mutant Y357F preserved most wild-type heme characteristics and NADPH oxidation ability. However, mutation of this residue markedly increased the dissociation constants for both Arg and NHA by 20-fold and decreased the NO synthesis from Arg by 85% compared to that of wild type. Mutation of Y357 had less effect on the rate of NO generated from NHA. Mutant R365L purified in the presence of Arg had a normal heme environment and retained 9 and 55% of the wild-type NO formation rate from Arg and NHA, respectively. When Arg was removed from buffer, R365L instantly became a low-spin state (Soret peak at 418 nm) with the resultant loss of H(4)B and instability of the heme-CO complex. The low-spin R365L exhibited an NADPH oxidation rate higher than that of wild type. Its Arg-driven NO formation was decreased to near the limit of detection, whereas the rate of NHA-driven NO synthesis was one third that of wild type. This NHA-driven NO formation completely relied on H(4)B and was not sensitive to superoxide dismutase or catalase but was inhibited by imidazole. The wild-type eNOS required 14 microM NHA and 0.39 microM H(4)B to reach the half-maximal NHA-driven NO formation rate (EC(50)), while R365L needed 59 microM NHA and 0.73 microM H(4)B to achieve EC(50). The differential effect of mutation on Arg and NHA oxidation suggests that distinct heme-based active oxidants are responsible for each step of NO synthesis.

Metabolism of polychlorinated dibenzo-p-dioxins by CYP1A subfamily was examined by using the recombinant yeast microsomes. In substrate specificity and reaction specificity, considerable species differences between rats and humans were observed in both CYP1A1- and CYP1A2-dependent metabolism of dioxins. Among four CYPs, rat CYP1A1 showed the highest activity toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. To reveal the mechanism of dioxin metabolism, we examined rat CYP1A1-dependent metabolism of 2-chloro-dibenzo-p-dioxin. In addition to hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and cleavage of an ether linkage of the dioxin ring were observed. In particular, the cleavage of an ether linkage of the dioxin ring appeared most important for the detoxication of dioxins. Based on these results, the metabolic pathways of 2-chloro-dibenzo-p-dioxin by rat CYP1A1 were proposed. The metabolic pathways contain most of the metabolites observed in vivo using experimental animals, suggesting that P450 monooxygenase systems including CYP1A1 are greatly responsible for dioxin metabolism in vivo.

Two notable features of the thermophilic CYP119, an Arg154–Glu212 salt bridge between the F–G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K s , K m , and k cat values. The salt bridge mutants, like wild-type CYP119, melt at 91±1°C, whereas mutation of individual residues in the extended aromatic cluster lowers the T m by 10–15 °C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154–Glu212 salt bridge, contributes to the thermal stability of CYP119.

Resonance Raman spectroscopy at 2.5 cm−1 resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.