Structure and Biochemical Properties of the Alkene Producing Cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 Bacterium

The production of hydrocarbons in nature has been documented for only a limited set of organisms, with many of the molecular components underpinning these processes only recently identified. There is an obvious scope for application of these catalysts and engineered variants thereof in the future production of biofuels. Here we present biochemical characterization and crystal structures of a cytochrome P450 fatty acid peroxygenase: the terminal alkene forming OleT_JE (CYP152L1) from Jeotgalicoccus sp. 8456. OleT_JE is stabilized at high ionic strength, but aggregation and precipitation of OleT_JE in low salt buffer can be turned to advantage for purification, because resolubilized OleT_JE is fully active and extensively dissociated from lipids. OleT_JE binds avidly to a range of long chain fatty acids, and structures of both ligand-free and arachidic acid-bound OleT_JE reveal that the P450 active site is preformed for fatty acid binding. OleT_JE heme iron has an unusually positive redox potential (−103 mV versus normal hydrogen electrode), which is not significantly affected by substrate binding, despite extensive conversion of the heme iron to a high spin ferric state. Terminal alkenes are produced from a range of saturated fatty acids (C12–C20), and stopped-flow spectroscopy indicates a rapid reaction between peroxide and fatty acid-bound OleT_JE (167 s⁻¹ at 200 μm H₂O₂). Surprisingly, the active site is highly similar in structure to the related P450_BSβ, which catalyzes hydroxylation of fatty acids as opposed to decarboxylation. Our data provide new insights into structural and mechanistic properties of a robust P450 with potential industrial applications.     

Neutral Lipids in the Study of Relationships of Members of the Family Micrococcaceae

The organisms studied were those of the family Micrococcaceae which cannot participate in genetic exchange with Micrococcus luteus and those whose biochemical and physiological characteristics appear to bridge the genera Staphylococcus and Micrococcus. The hydrocarbon compositions of M. luteus ATCC 4698 and Micrococcus sp. ATCC 398 were shown to be similar to those previously reported for many M. luteus strains, consisting of isomers of branched monoolefins in the range C25 to C31. However, Micrococcus sp. ATCC 398 differed somewhat by having almost all C29 isomers (approximately 88% of the hydrocarbon composition). Micrococcus spp. ATCC 401 and ATCC 146 and M. roseus strains ATCC 412, ATCC 416, and ATCC 516 contained the same type of hydrocarbon patterns, but the predominant hydrocarbons were within a lower distribution range (C23 to C27), similar to Micrococcus sp. ATCC 533 previously reported. The chromatographic profile and carbon range of the hydrocarbons of an atypical strain designated M. candicans ATCC 8456 differed significantly from the hydrocarbon pattern presented above. The hydrocarbons were identified as branched and normal olefins in the range C16 to C22. Studies of several different strains of staphylococci revealed that these organisms do not contain readily detectable amounts of aliphatic hydrocarbons. The members of the family Micrococcaceae have been divided into two major groups based on the presence or absence of hydrocarbons. With the exception of M. candicans ATCC 8456, this division corresponded to the separation of these organisms according to their deoxyribonucleic acid compositions.     

Role of CYP27A in cholesterol and bile acid metabolism

The CYP27A gene encodes a mitochondrial cytochrome P450 enzyme, sterol 27-hydroxylase, that is expressed in many different tissues and plays an important role in cholesterol and bile acid metabolism. In humans, CYP27A deficiency leads to cerebrotendinous xanthomatosis. To gain insight into the roles of CYP27A in the regulation of cholesterol and bile acid metabolism, cyp27A gene knockout heterozygous, homozygous, and wild-type littermate mice were studied. In contrast to homozygotes, heterozygotes had increased body weight and were mildly hypercholesterolemic, with increased numbers of lipoprotein particles in the low density lipoprotein size range. Cyp7A expression was not increased in heterozygotes but was in homozygotes, suggesting that parts of the homozygous phenotype are secondary to increased cyp7A expression and activity. Homozygotes exhibited pronounced hepatomegaly and dysregulation in hepatic cholesterol, bile acid, and fatty acid metabolism. Hepatic cholesterol synthesis and synthesis of bile acid intermediates were increased; however, side chain cleavage was impaired, leading to decreased bile salt concentrations in gallbladder bile. Expression of Na-taurocholate cotransporting polypeptide, the major sinusoidal bile salt transporter, was increased, and that of bile salt export pump, the major canalicular bile salt transporter, was decreased. Gender played a modifying role in the homozygous response to cyp27A deficiency, with females being generally more severely affected. Thus, both cyp27A genotype and gender affected the regulation of hepatic bile acid, cholesterol, and fatty acid metabolism.     

CYP71B15 (PAD3) catalyzes the final step in camalexin biosynthesis

Camalexin represents the main phytoalexin in Arabidopsis (Arabidopsis thaliana). The camalexin-deficient phytoalexin deficient 3 (pad3) mutant has been widely used to assess the biological role of camalexin, although the exact substrate of the cytochrome P450 enzyme 71B15 encoded by PAD3 remained elusive. 2-(Indol-3-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid (dihydrocamalexic acid) was identified as likely intermediate in camalexin biosynthesis downstream of indole-3-acetaldoxime, as it accumulated in leaves of silver nitrate-induced pad3 mutant plants and it complemented the camalexin-deficient phenotype of a cyp79b2/cyp79b3 double-knockout mutant. Recombinant CYP71B15 heterologously expressed in yeast catalyzed the conversion of dihydrocamalexic acid to camalexin with preference of the (S)-enantiomer. Arabidopsis microsomes isolated from leaves of CYP71B15-overexpressing and induced wild-type plants were capable of the same reaction but not microsomes from induced leaves of pad3 mutants. In conclusion, CYP71B15 catalyzes the final step in camalexin biosynthesis.     

Enzymatic reaction of hydrogen peroxide-dependent peroxygenase cytochrome P450s: kinetic deuterium isotope effects and analyses by resonance Raman spectroscopy

Cytochromes P450SP(alpha) (CYP152B1) and P450BS(beta) (CYP152A1), which are isolated from Sphingomonas paucimobilis and Bacillus subtilis, respectively, belong to the P450 superfamily, but catalyze hydroxylation reactions, in which an oxygen atom from H2O2 is efficiently introduced into fatty acids (e.g., myristic acid). P450SP(alpha) produces the alpha-hydroxylated (alpha-OH) products at 100%, while P450BS(beta) produces alpha- and beta-hydroxylated (beta-OH) products at 33 and 67%, respectively. Using deuterium-substituted fatty acids ([2,2-d2]-myristic acid and d27-myristic acid) as a substrate, the peroxygenase reactions of the two bacterial P450s were investigated. In the P450SP(alpha) reaction, we observed an intermolecular noncompetitive kinetic isotope effect on Vmax (DV = 4.1) when [2,2-d2]-myristic acid was used, suggesting that an isotopically sensitive step involving the alpha-hydrogen of the fatty acid is present in the catalytic cycle. On the other hand, D(V/K) was masked, in sharp contrast to the features of usual monooxygenases P450. The characteristic kinetic features can be interpreted in terms of the faster product formation than the substrate dissociation. A similar kinetic isotope effect was observed [DV = 4.9, D(V/K) approximately 1] for the P450BS(beta) reaction, when d27-myristic acid was used as a substrate, indicating that the reaction mechanism is the same for both peroxygenases. The resonance Raman spectral data of P450BS(beta) in the ferric and ferrous-CO forms in the presence and absence of myristic acid demonstrated that the catalytic pocket of the enzyme is polar, so that the location of the carboxylate of the substrate close to the sixth ligand of the heme could be allowed. On the basis of these results on the kinetic isotope effects and spectroscopy, we discuss the possible mechanisms of the alpha- and beta-hydroxylation of fatty acids catalyzed by peroxygenases P450SP(alpha) and P450BS(beta).     

Isolation and Analysis of Cinnamic Acid 4-Hydroxylase Homologous Genes from a Hybrid Aspen,Populus kitakamiensis

Cinnamic acid 4-hydroxylase (CA4H) is the second enzyme involved in phenylpropanoid biosynthesis and is a member of the cytochrome P-450 superfamily. Three CA4H homologous genes, cyp73a, cyp73b, and cyp73c, and a cDNA clone of cyp73a were isolated from a genomic library and a cDNA library of a hybrid aspen; Populus kitakamiensis, and were characterized. They might be interrupted by two introns each. cyp73a and cyp73b were very similar to each other not only in coding regions but also in non-coding regions. Southern blot analysis showed that four homologous genes for CA4H constructed a small gene family in the diploid genome of P. kitakamiensis. In the promoter regions, there were many common m-element-like sequences in phenylpropanoid biosynthesis genes.     

Factors influencing cell fatty acid composition and A40926 antibiotic complex production in <Emphasis Type="Italic">Nonomuraea</Emphasis> sp. ATCC 39727

A40926 is a glycopeptide antibiotic complex consisting of several structurally related factors. It is produced by fermentation of Nonomuraea sp. ATCC 39727 and the complex components differ in the structure of the fatty acid moiety linked to the aminoglucuronic acid unit. In previous work, we observed that the production of single factors in glycopeptide antibiotic complexes could be selectively enhanced by the addition of suitable precursors to the culture medium. In this contribution, we examine the effects of branched amino acid addition to fermentation of Nonomuraea sp. in a chemically defined minimal medium. The changes in the composition of cell fatty acids correlate to the fatty acid distribution within the A40926 complex in diverse cultivation conditions. Nonomuraea sp. prefers isobutyric, butyric and propionic acids as initiators of fatty acid biosynthesis. The relative amount of the produced fatty acids is significantly influenced by the availability of intermediates or final products from the amino acid catabolic pathways. Antibiotic complex composition closely reflects the cell fatty acid pattern, in agreement with the assumption that the antibiotic fatty acid moieties are synthesized by shortening the chain of cell fatty acids.     

Molecular characterization and chromosomal localization of cytochrome P450 genes involved in the biosynthesis of cyclic hydroxamic acids in hexaploid wheat

The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), are defensive secondary metabolites found in gramineous plants including wheat, maize and rye. cDNAs for five cytochromes P450 (P450s) involved in DIBOA biosynthesis (CYP71C6, CYP71C7v2, CYP71C8v2, CYP71C9v1 and CYP71C9v2) were isolated from seedlings of hexaploid wheat [( Triticum aestivum L. cv. Chinese Spring (2n=6x=42, genomes AABBDD)] by RT-PCR and screening of a cDNA library. CYP71C9v1 and CYP71C9v2 are 97% identical to each other in amino acid and nucleotide sequences. The cloned P450 species showed 76-79% identity at the amino acid level to the corresponding maize P450 species CYP71C1-C4, which are also required for DIBOA biosynthesis. The wheat P450 cDNAs were heterologously expressed in the yeast ( Saccharomyces cerevisiae) strain AH22. Microsome fractions from yeast cells expressing these P450 species catalyzed the same reactions as their maize orthologs. The chromosomes carrying the cyp71C6- C9v1 orthologs were identified by Southern hybridization using aneuploid lines of Chinese Spring wheat. The cyp71C9v1 orthologs were located on the chromosomes of wheat homoeologous group-4. The orthologs of the other P450 genes, cyp71C7v2, cyp71C6 and cyp71C8v2, were located on group-5 chromosomes. The same P450 genes were also present in the three ancestral diploid species of hexaploid wheat, T. monococcum (AA), Aegilops speltoides [BB (approximately SS)] and Ae. squarrosa (DD).     

Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis. Crystallographic,spectroscopic, and mutational studies

Cytochrome P450 isolated from Bacillus subtilis (P450(BSbeta); molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the alpha- and beta-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450(BSbeta) in the substrate-bound form, determined at a resolution of 2.1 A. P450(BSbeta) exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg(242) guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the alpha- and beta-positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg(242) in the same fashion as has been reported for the active site of chloroperoxidase, His(105)-Glu(183), which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450(BSbeta) is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.     

Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.     

Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces

The biosynthetic pathway of the red-pigmented antibiotic, prodigiosin, produced by Serratia sp. is known to involve separate pathways for the production of the monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP) and the bipyrrole, 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) which are then coupled in the final condensation step. We have previously reported the cloning, sequencing and heterologous expression of the pig cluster responsible for prodigiosin biosynthesis in two Serratia sp. In this article we report the creation of in-frame deletions or insertions in every biosynthetic gene in the cluster from Serratia sp. ATCC 39006. The biosynthetic intermediates accumulating in each mutant have been analysed by LC-MS, cross-feeding and genetic complementation studies. Based on these results we assign specific roles in the biosynthesis of MBC to the following Pig proteins: PigI, PigG, PigA, PigJ, PigH, PigM, PigF and PigN. We report a novel pathway for the biosynthesis of MAP, involving PigD, PigE and PigB. We also report a new chemical synthesis of MAP and one of its precursors, 3-acetyloctanal. Finally, we identify the condensing enzyme as PigC. We reassess the existing literature and discuss the significance of the results for the biosynthesis of undecylprodigiosin by the Red cluster in Streptomyces coelicolor A3(2).     

Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the “black box”, remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.     

Biochemical and molecular analyses of gibberellin biosynthesis in fungi

The plant hormone, gibberellin (GA), regulates plant growth and development. It was first isolated as a superelongation-promoting diterpenoid from the fungus, Gibberella fujikuroi. G. fujikuroi uses different GA biosynthetic intermediates from those in plants to produce GA3. Another class of GA-producing fungus, Phaeosphaeria sp. L487, synthesizes GA1 by using the same intermediates as those in plants. A molecular analysis of GA biosynthesis in Phaeosphaeria sp. has revealed that diterpene cyclase and cytochrome P450 monooxygenases were involved in the plant-like biosynthesis of GA1. Fungal ent-kaurene synthase is a bifunctional cyclase. Subsequent oxidation steps are catalyzed by P450s, leading to biologically active GA1. GA biosynthesis in plants is divided into three steps involving soluble enzymes and membrane-bound cytochrome P450. The activation of plant GAs is catalyzed by soluble 2-oxoglutarate-dependent dioxygenases, which is in contrast to the catalysis of fungal GA biosynthesis. This difference suggests that the origin of fungal GA biosynthesis is evolutionally independent of that in plants.     

The CYP701B1 of Physcomitrella patens is an ent-kaurene oxidase that resists inhibition by uniconazole-P

The moss Physcomitrella patens produces both ent-kaurene and ent-kaurenoic acid, which are intermediates of gibberellin biosynthesis in flowering plants. The CYP701 superfamily of cytochrome P450s functions as ent-kaurene oxidases in the biosynthesis of ent-kaurenoic acid. A candidate gene encoding ent-kaurene oxidase in P. patens, CYP701B1, was cloned and heterologously expressed in yeast to examine enzyme activities in vitro. The recombinant CYP701B1 protein catalyzed the oxidation reaction from ent-kaurene to ent-kaurenoic acid. CYP701B1 activity was highly resistant to the ent-kaurene oxidase inhibitor uniconazole-P (IC(50) 64 μM), even though the activity of Arabidopsis ent-kaurene oxidase (CYP701A3) was sensitive (IC(50) 0.26 μM).     

过氧化氢驱动的细胞色素P450酶研究进展

细胞色素P450酶在自然界中广泛存在,能催化多种类型的氧化反应,在有机合成和生物化工方面具有重要的应用潜力。尽管大多数P450酶通常需要辅酶和复杂的电子传递体系协助活化氧分子,一些P450酶也可以利用过氧化氢作为末端氧化剂,这极大地简化了催化循环,为P450酶的合成应用提供了一条新的简便途径。本文系统地介绍了几类过氧化氢驱动的P450酶催化体系,包括脂肪酸羟化酶P450SPα和P450BSβ、脂肪酸脱羧酶P450OleTJE、人工改造的羟化酶P450BM3和P450cam突变体、以及基于底物误识别策略的P450-H2O2体系。通过分析催化反应机制,本文探讨了P450-H2O2催化体系在目前存在的挑战和可能的解决途径,并对其进一步应用前景进行了展望。     

Cytochrome P450 monooxygenase from Clostridium acetobutylicum: a new alpha-fatty acid hydroxylase

Cytochrome P450 monooxygenase from the anaerobic microorganism Clostridium acetobutylicum (CYP152A2) has been produced in Escherichia coli. CYP152A2 was shown to bind a broad range of saturated and unsaturated fatty acids and corresponding methyl esters and demonstrated a high peroxygenase activity of up to 200min(-1) with myristic acid. Although a high concentration of hydrogen peroxide of 200microM was necessary for high activities of the enzyme, it led to a fast enzyme inactivation within 2-4min. This might reflect the natural function of CYP152A2 as a rapid hydrogen peroxide scavenging enzyme. In two different reconstituted systems with NADPH, CYP152A2 was able to convert 10 times more substrate, if provided with flavodoxin and flavodoxin reductase from E. coli and even 30-40 times more substrate with the CYP102A1-reductase from Bacillus megaterium. According to the clear preference for hydroxylation at alpha-position, CYP152A2 can be referred to as fatty acid alpha-hydroxylase.     

Genetic analysis of biosurfactant production in Ustilago maydis

The dimorphic basidiomycete Ustilago maydis produces large amounts of surface-active compounds under conditions of nitrogen starvation. These biosurfactants consist of derivatives of two classes of amphipathic glycolipids. Ustilagic acids are cellobiose lipids in which the disaccharide is O-glycosidically linked to 15,16-dihydroxyhexadecanoic acid. Ustilipids are mannosylerythritol lipids derived from acylated beta-d-mannopyranosyl-d-erythritol. Whereas the chemical structure of these biosurfactants has been determined, the genetic basis for their biosynthesis and regulation is largely unknown. Here we report the first identification of two genes, emt1 and cyp1, that are essential for the production of fungal extracellular glycolipids. emt1 is required for mannosylerythritol lipid production and codes for a protein with similarity to prokaryotic glycosyltransferases involved in the biosynthesis of macrolide antibiotics. We suggest that Emt1 catalyzes the synthesis of mannosyl-d-erythritol by transfer of GDP-mannose. Deletion of the gene cyp1 resulted in complete loss of ustilagic acid production. Cyp1 encodes a cytochrome P450 monooxygenase which is highly related to a family of plant fatty acid hydroxylases. Therefore we assume that Cyp1 is directly involved in the biosynthesis of the unusual 15,16-dihydroxyhexadecanoic acid. We could show that mannosylerythritol lipid production is responsible for hemolytic activity on blood agar, whereas ustilagic acid secretion is required for long-range pheromone recognition. The mutants described here allow for the first time a genetic analysis of glycolipid production in fungi.