CYP52A23 from Candida albicans and its Substrate Preference for Fatty Acid Hydroxylation.

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.

Inhibitory effect of α-terpinyl acetate on cytochrome P450 2B6 enzymatic activity.

Human cytochrome P450 2B6 is an important hepatic enzyme for the metabolism of xenobiotics and clinical drugs. Recently, more attention has been paid to P450 2B6 because of the increasing number of drugs it metabolizes. It has been known to interact with terpenes, the major constituents of the essential oils used for various medicinal purposes.

In vitro functional analysis of human cytochrome P450 2A13 genetic variants: P450 2A13*2, *3, *4, and *10.

Humans possess three cytochrome P450 enzymes in the 2A subfamily (2A6, 2A7, and 2A13). P450 2A13 is mainly expressed in the human trachea and lung, whereas P450 2A6 is found in human liver. The P450 2A13 enzyme may be considered as the primary enzyme responsible for metabolic activation of many tobacco-specific carcinogens.

Terfenadine metabolism of human cytochrome P450 2J2 containing genetic variations (G312R, P351L and P115L).

The human cytochrome P450 2J2 is involved in several metabolic reactions, including the oxidation of important therapeutics and epoxidation of endogenous arachidonic acid. At least ten genetic variations of P450 2J2 have been identified, but their effects on enzymatic activity have not been clearly characterized. Here, we evaluated the functional effects of three genetic variations of P450 2J2 (G312R, P351L, and P115L).

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP in the affinity chromatography process.

Characterization of a Biflaviolin Synthase CYP158A3 from and Its Role in the Biosynthesis of Secondary Metabolites.

produces clinically useful drugs such as avermectins and oligomycins. Its genome contains approximately 33 cytochrome P450 genes and they seem to play important roles in the biosynthesis of many secondary metabolites. The gene from encodes CYP158A3.

Structural insights into the binding of lauric acid to CYP107L2 from Streptomyces avermitilis.

Streptomyces avermitilis is an actinobacterium known to produce clinically useful macrolides including avermectins. CYP107L2 from S. avermitilis shares a high sequence similarity with the PikC (CYP107L1) from S.

Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue.

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1.

Heterologous expression and characterization of CYP61A1 from dandruff-causing Malassezia globosa.

Malassezia globosa is pathogenic fungus that causes skin disorders including dandruff in humans. Many yeast cytochrome CYP enzymes are involved in the biosynthesis of sterols and are considered major targets of azole antifungal agents. Here, we report on the expression and characterization of the MGL_0310 gene product (CYP61A1), a sterol C-22 desaturase in M.

Functional characterization of CYP107W1 from Streptomyces avermitilis and biosynthesis of macrolide oligomycin A.

Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C.

Functional Significance of Cytochrome P450 1A2 Allelic Variants, P450 1A2*8, *15, and *16 (R456H, P42R, and R377Q).

P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 1A2*8, R456H; *15, P42R; *16, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli.

Directed-evolution analysis of human cytochrome P450 2A6 for enhanced enzymatic catalysis.

Cytochrome P450 2A6 (P450 2A6) is the major enzyme responsible for the oxidation of coumarin, nicotine, and tobacco-specific nitrosamines in human liver. In this study, the catalytic turnover of coumarin oxidation was improved by directed-evolution analysis of P450 2A6 enzyme. A random mutant library was constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the P450 2A6 gene and individual mutant clones were screened for improved catalytic activity in analysis of fluorescent coumarin 7-hydroxylation.

Expression and Characterization of Truncated Recombinant Human Cytochrome P450 2J2.

The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a Ni(2+)-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction.

Carbon-carbon bond cleavage in activation of the prodrug nabumetone.

Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA).

Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.

Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M.

Heterologous expression and characterization of the sterol 14α-demethylase CYP51F1 from Candida albicans.

Lanosterol 14α-demethylase (CYP51F1) from Candida albicans is known to be an essential enzyme in fungal sterol biosynthesis. Wild-type CYP51F1 and several of its mutants were heterologously expressed in Escherichia coli, purified, and characterized. It exhibited a typical reduced CO-difference spectrum with a maximum at 446 nm.

Bioactivation of Aromatic Amines by Human CYP2W1, An Orphan Cytochrome P450 Enzyme.

The human genome contains approximately 13 orphan cytochrome P450 (P450, CYP) genes, of which the apparent function or substrate has not been identified. However, they seem to possess their own biological relevance in some tissues or developmental stages. Here, we characterized the heterologously expressed CYP2W1, an orphan P450 enzyme.

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

Candida albicans is responsible for serious fungal infections in humans. Analysis of its genome identified NCP1 gene coding for a putative NADPH-P450 reductase (NPR) enzyme. This enzyme appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence.

Regioselective oxidation of lauric acid by CYP119, an orphan cytochrome P450 from Sulfolobus acidocaldarius.

Archaebacteria Sulfolobus acidocaldarius contains the highly thermophilic cytochrome P450 enzyme (CYP119). CYP119 possesses stable enzymatic activity at up to 85 degrees C. However, this enzyme is still considered as an orphan P450 without known physiological function with endogenous or xenobiotic substrates.

Differential Display Analysis of 2,3,7,8-Tetrachlorodibenzo--dioxin Identified Induction of Ras-related Nuclear Protein Binding Protein2 (RanBP2) Gene.

TCDD (2,3,7,8-tetrachlorodibenzo--dioxin) and related halogenated aromatic hydrocarbons elicit a diverse spectrum of biochemical and toxic responses in laboratory animals and mammalian cells in culture. Toxicity and carcinogenicity of TCDD is well established but the molecular mechanism is still poorly understood. Here, we found the noble responsive genes to TCDD using the differential display analysis.