Monooxygenation Reactions Research Articles

Metabolism of 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 by CYP24A1 and biological activity of its 24R-hydroxylated metabolite

Our previous study revealed that the 2α-substituted vitamin D analog 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exhibited a higher osteocalcin promoter transactivation activity in human osteosarcoma cells and a greater effect on bone mineral density in a rat model of osteoporosis than 1α,25(OH)2D3 without increasing blood calcium concentration. Thus, we hypothesized that AH-1 could be a promising therapeutic agent for osteoporosis without any serious side effects. In this study, we compared the CYP24A1-dependent metabolism of AH-1 with that of 1α,25(OH)2D3. The resistance to CYP24A1-dependent metabolism could be an important property of vitamin D analogs in prolonging their biological effects. A kinetic analysis was performed using a membrane fraction prepared from recombinant E. coli expressing human CYP24A1. The kcat/Km (μM−1 min−1) value for AH-1 was 31% of that for 1α,25(OH)2D3, suggesting that AH-1 is not as resistant to CYP24A1-dependent metabolism as the other C2-substituted vitamin D analogs such as eldecalcitol [2β-hydroxypropoxy-1α,25(OH)2D3]. The major metabolite of AH-1 was the 24R-hydroxylated metabolite, which had high vitamin D receptor (VDR) binding affinity and high HL-60 cell differentiation activity similar to AH-1 itself. In contrast, 1α,25(OH)2D3 was metabolized by multistep monooxygenation reactions, which led to the loss of affinity for VDR. Thus, the greater therapeutic effects of AH-1 than those of 1α,25(OH)2D3 in in vivo studies using osteoporosis rat models may be due to 24R-hydroxy-AH-1 whose VDR affinity was 91% of that of AH-1.

Molecular and in silico cloning, identification, and preharvest period expression analysis of a putative cytochrome P450 monooxygenase gene from Camellia sinensis (L.) Kuntze (tea).

Cytochrome P450 monooxygenases are one of the largest heme-containing protein groups, and the majority of them catalyze hydroxylation reactions dependent on nicotinamide adenine dinucleotide phosphate and oxygen. Cytochrome P450 (CYP) enzymes function in a wide range of monooxygenation reactions essential in primary and secondary metabolism in plants. Camellia sinensis (L.) Kuntze is a commercially and economically valuable plant due to its medicinally important secondary metabolites and as a beloved beverage. Cytochrome P450 monooxygenases play a significant role in the biosynthesis of a variety of secondary metabolites in tea. Although the biosynthesis of secondary metabolites has been investigated in detail, there have been limited studies conducted on identifying the genetic mechanisms of CYP-catalyzed secondary metabolic pathways in the C. sinensis (tea) plant. In our study, we characterized a putative C. sinensis (L.) Kuntze cytochrome P450 monooxygenase gene (Csp450), which has 1759 bp full-length cDNA with 49 bp of 5' and 183 bp of 3' untranslated regions. eTh CDS of the gene is 1527 bp and 508 amino acids in length. BLAST results of the deduced amino acid sequence revealed a high similarity with the CYP704C1-like superfamily. Preharvest period gene expression analysis from May, July, and September did not show any difference.

Competition for electrons between pyridine and quinoline during their simultaneous biodegradation.

Biodegradation of pyridine and quinoline is initiated with mono-oxygenation reactions that require an intracellular electron donor. Simultaneous biodegradation of both substrates should set up competition for the intracellular electron donor that may inhibit one or more of the mono-oxygenation steps. An internal circulation baffled biofilm reactor (ICBBR) was used to evaluate the impacts of competition during pyridine and quinoline biodegradation. Compared with independent biodegradation, pyridine and quinoline removal rates were slowed when biodegraded simultaneously, although the pyridine removal rate decreased more than for quinoline. The first mono-oxygenation of quinoline (to 2-hydroxyquinoline) always was faster than the first mono-oxygenation of pyridine (to 2-hydroxypyridine), and the difference was accentuated with pyridine and quinoline which were biodegraded simultaneously due to the competition for intracellular electron donor. Competition also existed between the second mono-oxygenations, and the removal rate of 2-hydroxypyridine was faster than the rate for 2-hydroxyquinoline, even though the rate was faster for quinoline than pyridine. Adding an exogenous electron donor accelerated all mono-oxygenations in proportion to the amount of donor added, but the increments were greater for quinoline due to its higher affinity for intracellular electron donors than pyridine. When actual coking wastewater was used as the background matrix, removals of pyridine and quinoline exhibited the same competitive trends.

Subcellular Targeting of Nitric Oxide Synthases Mediated by Their N-Terminal Motifs.

From a catalytic point of view, the three mammalian nitric oxide synthases (NOSs) function in an almost identical way. The N-terminal oxygenase domain catalyzes the conversion of l-arginine to l-citrulline plus ·NO in two sequential oxidation steps. Once l-arginine binds to the active site positioned above the heme moiety, two consecutive monooxygenation reactions take place. In the first step, l-arginine is hydroxylated to make Nω-hydroxy-l-arginine in a process that requires 1 molecule of NADPH and 1 molecule of O2 per mol of l-arginine reacted. In the second step, Nω-hydroxy-l-arginine, never leaving the active site, is oxidized to ·NO plus l-citrulline and 1 molecule of O2 and 0.5 molecules of NADPH are consumed. Since nitric oxide is an important signaling molecule that participates in a number of biological processes, including neurotransmission, vasodilation, and immune response, synthesis and release of ·NO in vivo must be exquisitely regulated both in time and in space. Hence, NOSs have evolved introducing in their amino acid sequences subcellular targeting motifs, most of them located at their N-termini. Deletion studies performed on recombinant, purified NOSs have revealed that part of the N-terminus of all three NOS can be eliminated with the resulting mutant enzymes still being catalytically active. Likewise, NOS isoforms lacking part of their N-terminus when transfected in cells render mislocalized, active proteins. In this review we will comment on the current knowledge of these subcellular targeting signals present in nNOS, iNOS, and eNOS.

Divergent mechanisms of iron-containing enzymes for hydrocarbon biosynthesis.

Increasing levels of energy consumption, dwindling resources, and environmental considerations have served as compelling motivations to explore renewable alternatives to petroleum-based fuels, including enzymatic routes for hydrocarbon synthesis. Phylogenetically diverse species have long been recognized to produce hydrocarbons, but many of the enzymes responsible have been identified within the past decade. The enzymatic conversion of Cn chain length fatty aldehydes (or acids) to Cn-1 hydrocarbons, alkanes or alkenes, involves a C-C scission reaction. Surprisingly, the enzymes involved in hydrocarbon synthesis utilize non-heme mononuclear iron, dinuclear iron, and thiolate-ligated heme cofactors that are most often associated with monooxygenation reactions. In this review, we examine the mechanisms of several enzymes involved in hydrocarbon biosynthesis, with specific emphasis on the structural and electronic changes that enable this functional switch.

Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies.

Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.

RETRACTED ARTICLE: Role of UV photolysis in accelerating the biodegradation of 2,4,6-TCP.

2,4,6-TCP, a kind of chlorinated aromatic and aliphatic compound, is difficult to be biodegraded by ordinary microorganisms. UV photolysis and biodegradation of 2,4,6-TCP by Bacillus amyloliquefaciens intimate coupling is a potential means to accelerate its biotransformation. The initial steps of 2,4,6-TCP biodegradation involve mono-oxygenation reactions that have molecular oxygen and an intracellular electron carrier as cosubstrates. It was demonstrated that B. amyloliquefaciens has the 2,4,6-TCP monooxygenase gene tcpA which could encode 2,4,6-TCP monooxygenase (TCP-MO). TCP-MO would catalytically decompose 2,4,6-TCP into 2,6-DCHQ. We employed an internal loop photolytic biofilm reactor for 2,4,6-TCP degradation. Sequentially coupled photolysis and biodegradation experimental results suggested that 2,4,6-TCP removal rate in P+B (TCP(UV)+phenol) protocol was higher by 77 and 103% when compared to B (TCP+phenol) and B (TCP-only) protocols respectively. The corresponding loss rate coefficient (k) values were 0.069, 0.039, 0.034mg/L·min-1 respectively. This is because UV photolysis converted 2,4,6-TCP into its intermediates: 2,4-dichlorophenol (2,4-DCP), 4-monochlorophenol (4-MCP), phenol, 2,6-dichloro-p-hydroquinone (2,6-DCHQ), with all displaying less inhibition to bacterial action. In addition, phenol was the crucial UV-photolysis product from 2,4,6-TCP, its catabolic oxidation generating internal electron carriers that may accelerate the initial steps of 2,4,6-TCP biodegradation. Intimately coupled photolysis and biodegradation experimental results suggested that 2,4,6-TCP removal rate in P&B (TCP+phenol) protocol was higher by 166 and 681% when compared to P&B (TCP-only) and P+B protocols respectively. The corresponding loss rate coefficient (k) values were 0.539, 0.203, 0.069mg/L·min-1 respectively. It provided sufficient evidence to demonstrate that intimately coupled photolysis and biodegradation accelerated 2,4,6-TCP removal much faster than sequentially coupled photolysis and biodegradation. In addition, oxidation of phenol was the mechanism by which intimately coupled photolysis and biodegradation accelerated rapid 2,4,6-TCP removal producing electron equivalents that stimulated the initial mono-oxygenation reactions for 2,4,6-TCP biodegradation. It is important to note that 2,6-DCHQ (produced from UV-photolysis products or initial mono-oxygenation reactions) would be catalytically decomposed into 6-chlorohydroxyquinol (6-CHQ). Based on this, a tentative reaction mechanism for the photo-biodegradation 2,4,6-TCP was proposed.

Decarboxylation of fatty acids to terminal alkenes by cytochrome P450 compound I.

OleT(JE), a cytochrome P450, catalyzes the conversion of fatty acids to terminal alkenes using hydrogen peroxide as a cosubstrate. Analytical studies with an eicosanoic acid substrate show that the enzyme predominantly generates nonadecene and that carbon dioxide is the one carbon coproduct of the reaction. The addition of hydrogen peroxide to a deuterated substrate-enzyme (E-S) complex results in the transient formation of an iron(IV) oxo π cation radical (Compound I) intermediate which is spectroscopically indistinguishable from those that perform oxygen insertion chemistries. A kinetic isotope effect for Compound I decay suggests that it abstracts a substrate hydrogen atom to initiate fatty acid decarboxylation. Together, these results indicate that the initial mechanism for alkene formation, which does not result from oxygen rebound, is similar to that widely suggested for P450 monooxygenation reactions.

Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes.

This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex.

Acyl-Carbon Bond Cleaving Cytochrome P450 Enzymes: CYP17A1, CYP19A1 and CYP51A1.

Cytochrome P450 (P450 or CYP) enzymes in their resting state contain the heme-iron in a high-spin FeIII state. Binding of a substrate to a P450 enzyme allows transfer of the first electron, producing a Fe(II) species that reacts with oxygen to generate a low-spin iron superoxide intermediate (FeIII-O-O•) ready to accept the second electron to produce an iron peroxy anion intermediate (a, FeIII-O-O-). In classical monooxygenation reactions, the peroxy anion upon protonation fragments to form the reactive Compound I intermediate (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). However, when the substrate projects a carbonyl functionality, of the type b, at the active site as is the case for reactions catalyzed by CYP17A1, CYP19A1 and CYP51A1, the peroxy anion (FeIII-O-O-) is trapped, yielding a tetrahedral intermediate (c) that fragments to an acyl-carbon cleavage product (d plus an acid). Analogous acyl-carbon cleavage reactions are also catalyzed by certain hepatic P450s and CYP125A1 from Mycobacterium tuberculosis. A further improvisation on the theme is provided by aldehyde deformylases that convert long-chain aliphatic aldehydes to hydrocarbons. CYP17A1 is involved in the biosynthesis of corticoids as well as androgens. The flux toward these two classes of hormones seems to be regulated by cytochrome b 5, at the level of the acyl-carbon cleavage reaction. It is this regulation of CYP17A1 that provides a safety mechanism, ensuring that during corticoid biosynthesis, which requires 17α-hydroxylation by CYP17A1, androgen formation is avoided (Fig. 4.1).

How UV photolysis accelerates the biodegradation and mineralization of sulfadiazine (SD).

Sulfadiazine (SD), one of broad-spectrum antibiotics, exhibits limited biodegradation in wastewater treatment due to its chemical structure, which requires initial mono-oxygenation reactions to initiate its biodegradation. Intimately coupling UV photolysis with biodegradation, realized with the internal loop photobiodegradation reactor, accelerated SD biodegradation and mineralization by 35 and 71 %, respectively. The main organic products from photolysis were 2-aminopyrimidine (2-AP), p-aminobenzenesulfonic acid (ABS), and aniline (An), and an SD-photolysis pathway could be identified using C, N, and S balances. Adding An or ABS (but not 2-AP) into the SD solution during biodegradation experiments (no UV photolysis) gave SD removal and mineralization rates similar to intimately coupled photolysis and biodegradation. An SD biodegradation pathway, based on a diverse set of the experimental results, explains how the mineralization of ABS and An (but not 2-AP) provided internal electron carriers that accelerated the initial mono-oxygenation reactions of SD biodegradation. Thus, multiple lines of evidence support that the mechanism by which intimately coupled photolysis and biodegradation accelerated SD removal and mineralization was through producing co-substrates whose oxidation produced electron equivalents that stimulated the initial mono-oxygenation reactions for SD biodegradation.

Peroxygenase reactions catalyzed by cytochromes P450

Cytochromes P450 (P450s) catalyze monooxygenation of a wide range of less reactive organic molecules under mild conditions. By contrast with the general reductive oxygen activation pathway of P450s, an H2O2-shunt pathway does not require any supply of electrons and protons for the generation of a highly reactive intermediate (compound I). Because the low cost of H2O2 allows us to use it in industrial-scale synthesis, the H2O2-shunt pathway is an attractive process for monooxygenation reactions. This review focuses on the P450-catalyzed monooxygenation of organic molecules using H2O2 as the oxidant.

UV Photolysis for Accelerating Pyridine Biodegradation

Pyridine, a nitrogen-containing heterocyclic compound, is slowly biodegradable, and coupling biodegradation with UV photolysis is a potential means to accelerate its biotransformation and mineralization. The initial steps of pyridine biodegradation involve mono-oxygenation reactions that have molecular oxygen and an intracellular electron carrier as cosubstrates. We employed an internal circulation baffled biofilm reactor for pyridine biodegradation following three protocols: direct biodegradation (B), biodegradation after photolysis (P+B), and biodegradation with succinic acid added (B+S). Succinic acid was the main UV-photolysis product from pyridine, and its catabolic oxidation generates internal electron carriers that may accelerate the initial steps of pyridine biodegradation. Compared with direct biodegradation of pyridine (B), the removal rate for the same concentration of photolyzed pyridine (P+B) was higher by 15 to 43%, depending on the initial pyridine concentrations (increasing through the range of 130 to 310 mg/L). Adding succinic acid alone (B+S) gave results similar to P+B, which supports that succinic acid was the main agent for accelerating the pyridine biodegradation rate. In addition, protocols P+B and B+S were similar in terms of increasing pyridine mineralization over 10 h: 84% and 87%, respectively, which were higher than with protocol B (72%). The positive impact of succinic acid-whether added directly or produced via UV photolysis-confirms that its catabolism, which produced intracellular electron carriers, accelerated the initial steps of pyridine biotransformation.

Understanding the mechanism of cytochrome P450 3A4: recent advances and remaining problems

Cytochromes P450 (CYPs) represent a diverse group of heme-thiolate proteins found in almost all organisms. CYPs share a common protein fold but differ in substrate selectivity and catalyze a wide variety of monooxygenation reactions via activation of molecular oxygen. Among 57 human P450s, the 3A4 isoform (CYP3A4) is the most abundant and the most important because it metabolizes the majority of administered drugs. A remarkable feature of CYP3A4 is its extreme promiscuity in substrate specificity and cooperative substrate binding, which often leads to undesirable drug-drug interactions and toxic side effects. Owing to its importance in drug development and therapy, CYP3A4 has been the most extensively studied mammalian P450. In this review we provide an overview on recent progress and remaining problems in the CYP3A4 research.

Compound I Is the Reactive Intermediate in the First Monooxygenation Step during Conversion of Cholesterol to Pregnenolone by Cytochrome P450scc: EPR/ENDOR/Cryoreduction/Annealing Studies

Cytochrome P450scc (CYP11A1) catalyzes conversion of cholesterol (CH) to pregnenolone, the precursor to all steroid hormones. This process proceeds via three sequential monooxygenation reactions: two stereospecific hydroxylations with formation first of 22R-hydroxycholesterol (22-HC) and then 20α,22R-dihydroxycholesterol (20,22-DHC), followed by C20-C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc-cholesterol complex. This approach is fully validated by the demonstration that the cryoreduced ternary complex of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol to form 22-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77 K produces predominantly the hydroperoxy-ferriheme P450scc intermediate, along with a minor fraction of peroxo-ferriheme intermediate that converts into a new hydroperoxo-ferriheme species at 145 K. This behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol complex exhibits an efficient proton delivery network, with an ordered water molecule H-bonded to the distal oxygen of the dioxygen ligand. During annealing of the hydroperoxy-ferric P450scc intermediates at 185 K, they convert to the primary product complex in which CH has been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate decays with a large solvent kinetic isotope effect, as expected when proton delivery to the terminal O leads to formation of Compound I (CpdI). (1)H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe(III) is coordinated to the 22R-O(1)H of 22-HC, where the (1)H is derived from substrate and exchanges to D after annealing at higher temperatures. These observations establish that CpdI is the agent that hydroxylates CH, rather than the hydroperoxy-ferric heme.

Impacts of Diversification of Cytochrome P450 on Plant Metabolism

Cytochrome P450 monooxygenases (P450s) catalyze a wide variety of monooxygenation reactions in primary and secondary metabolism in plants. The share of P450 genes in each plant genome is estimated to be up to 1%. This implies that the diversification of P450 has made a significant contribution to the ability to acquire the emergence of new metabolic pathways during land plant evolution. The P450 families conserved universally in land plants contribute to their chemical defense mechanisms. Several P450s are involved in the biosynthesis and catabolism of plant hormones. Species-specific P450 families are essential for the biosynthetic pathways of phytochemicals such as terpenoids and alkaloids. Genome wide analysis of the gene clusters including P450 genes will provide a clue to defining the metabolic roles of orphan P450s. Metabolic engineering with plant P450s is an important technology for large-scale production of valuable phytochemicals such as medicines.

Biotransformation of N-Heterocyclic Compounds 1-Phenylpyrazole and 1-Phenylpyrrole by Escherichia coli (pDTG141) Expressing Naphthalene Dioxygenase of Pseudomonas sp. Strain NCIB 9816-4

Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 is a multicomponent enzyme system that carries out the initial step in the degradation of naphthalene. This enzyme has broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. 1-Phenylpyrazole and 1-phenylpyrrole were tested for biotransformation by NDO. High-performance liquid chromatography (HPLC) analysis showed two metabolites from each substrate. Liquid chromatography/ Mass Spectroscopy (LC/MS) analysis indicated the molecular weight of metabolites M1 and M2 of 1-phenylpyrazole were increased by 16 and 34, respectively, which could be produced from enzymatic monooxygenation and dioxgenation reactions. HPLC and LC/MS analyses of metabolites M3 and M4 from 1-phenylpyrrole showed increased molecular weights of 16 and 32, as compared to parent compounds, proposing addition of one and two atoms of oxygen to substrate, respectively Metabolite structures were confirmed by 1H- nuclear magnetic resonance (NMR) and 13C-NMR analyses. Metabolites M1 and M2 from 1-phenylpyrazole were determined as 1-phenyl-1Hpyrazol-4-ol and (1H-pyrazol-1-yl)cyclohexa-4′,6′-diene-cis-2′,3′-diol, respectively. Metabolite M3 from 1-phenylpyrrole was confirmed as (1H-pyrrol-1-yl)benzene-2′,3′-diol. NDO enzyme could be used to produce a novel synthon from N-heterocylic compounds, useful for synthesis of many biologically active compounds.

Structural Basis for Three-step Sequential Catalysis by the Cholesterol Side Chain Cleavage Enzyme CYP11A1

Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20α,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the "soft" recognition of the 22HC 3β-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1-22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.

Control of the stereo-selectivity of styrene epoxidation by cytochrome P450 BM3 using structure-based mutagenesis

The potential of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium for biocatalysis and biotechnological application is widely acknowledged. The catalytic and structural analysis of the Ala82Phe mutant of P450 BM3 has shown that filling a hydrophobic pocket near the active site improved the binding of small molecules, such as indole (see Huang et al., J. Mol. Biol., 2007, 373, 633) and styrene. In this paper, additional mutations at Thr438 are shown to decrease the binding of and catalytic activity towards laurate, whereas they significantly increased the stereo-specificity of styrene epoxidation. Production of R-styrene oxide with 48% and 64% e.e., respectively, was achieved by the Ala82Phe-Thr438Leu and Ala82Phe-Thr438Phe mutants. These structure-based mutants of P450 BM3 illustrate the promise of rational design of synthetically useful biocatalysts for regio- and stereo- specific mono-oxygenation reactions.

Oxidation of Hydrocarbons with Tetrabutylammonium Oxone Catalyzed by β-Tribrominated meso-Tetraphenylporphyrinatomanganese(III) Acetate in the Presence of N-Bases

Different manganese porphyrins and related systems such as Schiff bases have been used extensively in chemical modeling of biological monooxygenation reactions. The biomimetic oxidation of hydrocarbons and nitrogenand sulfur-containing compounds with different oxygen donors such as PhIO, NaOCl, H2O2, periodate, amine N-oxides and Bu4NHSO5 (TBAO) have been extensively studied. [1-20] Iron and manganese porphyrins bearing halogen substituents on the periphery of the porphyrin ring have been shown to be particularly efficient for epoxidation and hydroxylation reactions.[21-23] Such porphyrins are also more resistant to degradation via free-radical attack or direct oxidation of porphyrin ring than those containing electron-donating substituents.[1,24-25] The employment of nitrogenous donors in metalloporphyrin systems for mimicking the oxygenation function of P-450 has led to marked improvement in selectivities and turnover rates in the epoxidation of alkenes.[18,26-27] Despite the favorable employment of Oxone® (2KHSO5·KHSO4·K2SO4) in the Mn porphyrin catalyzed oxygenation of hydrocarbons,[28-29] attempts at using TBAO as an oxygen source in association with porphyrin catalysts has been unsuccessful until 2002.[18] Oxidation of hydrocarbons with TBAO in the presence of different electron-rich and electron-deficient MnIII meso-tetra(halogensubstituted aryl)porphyrins have been reported. The present work is the first report on using electron deficient Mn porphyrins bearing halogen atoms at β-positions (β-tri-brominated meso-tetraphenylporphyrinato-manganese(III) acetate, Mn(TPPBr3) OAc, as catalyst for oxidation of hydrocarbons with TBAO in the presence of different nitrogen donors as co-catalyst.