Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Candida albicans contains 10 putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6x(His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, omega-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (omega-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d(3)-dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the omega- to the (omega-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein.     

The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands

Cytochromes P450 of the CYP2C and CYP4A gene subfamilies metabolize arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) and to 19- and 20-hydroxyeicosatetraenoic acids (HETEs), respectively. Abundant functional studies indicate that EETs and HETEs display powerful and often opposing biological activities as mediators of ion channel activity and regulators of vascular tone and systemic blood pressures. Incubation of 8,9-, 11,12-, and 14,15-EETs with microsomal and purified forms of rat CYP4A isoforms led to rapid NADPH-dependent metabolism to the corresponding 19- and 20-hydroxylated EETs. Comparisons of reaction rates and catalytic efficiency with those of arachidonic and lauric acids showed that EETs are one of the best endogenous substrates so far described for rat CYP4A isoforms. CYP4A1 exhibited a preference for 8,9-EET, whereas CYP4A2, CYP4A3, and CYP4A8 preferred 11,12-EET. In general, the closer the oxido ring is to the carboxylic acid functionality, the higher the rate of EET metabolism and the lower the regiospecificity for the EET omega-carbon. Analysis of cis-parinaric acid displacement from the ligand-binding domain of the human peroxisome proliferator-activated receptor-alpha showed that omega-hydroxylated 14,15-EET bound to this receptor with high affinity (K(i) = 3 +/- 1 nm). Moreover, at 1 microm, the omega-alcohol of 14,15-EET or a 1:4 mixture of the omega-alcohols of 8,9- and 11,12-EETs activated human and mouse peroxisome proliferator-activated receptor-alpha in transient transfection assays, suggesting a role for them as endogenous ligands for these orphan nuclear receptors.     

Purification of the multienzyme complex for fatty acid oxidation from Pseudomonas fragi and reconstitution of the fatty acid oxidation system

The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3,trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxyacyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240,000. Two types of subunits, with molecular weights of 73,000 and 42,000, were identified. The complex was composed of two copies each of the 73,000- and 42,000-Da subunits. The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.     

Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy. CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid, at a rate of 130 mol (mol P450)⁻¹ min⁻¹. Furthermore, CYP199A2 also oxidized quinoline-6-carboxylic acid, although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2, C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids.     

Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2

The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.     

Oxidation of fatty alcohols to acids in the caecum of a gourami (Trichogaster cosby).

Oxidation of fatty alcohols to acids in gourami caeca was investigated by measuring the reduction of NAD+ and the formation of labeled hexadecanoic acid from [1(-14)C]hexadecanol. Virtually all dehydrogenase activity is in the microsomal fraction. Maximal activity is obtained with NAD+ as cofactor whereas with NADP+ 60% of that activity is obtained. The enzyme is rather specific for long chain alcohols and 2 NADH are formed for each molecule of hexadecanol oxidized to acid. It is stabilized by mercaptoethanol, and completely inhibited by p-chloromercuribenzoate. The activity is optimal at pH 9.5. At higher pH, small amounts of aldehyde are found. The first reaction in the sequence, fatty alcohol leads to aldehyde leads to acid seems to occur under the more physiological condition at a much slower rate than the second reaction so that free aldehyde is not detected. Addition of palmitic acid indicated an uncompetitive product inhibition. The oxidation of alcohol to acid is reversible only to a very minor extent even in the presence of NADPH, CoA, ATP and Mg2+. Location, activity and properties of the enzyme are in agreement with the earlier observation from dietary experiments that in the gourami fatty alcohols of wax esters are oxidized to acids in the course of absorption.     

Human CYP4F3s are the main catalysts in the oxidation of fatty acid epoxides

CYP4F isoforms are involved in the oxidation of important cellular mediators such as leukotriene B4 (LTB4) and prostaglandins. The proinflammatory agent LTB4 and cytotoxic leukotoxins have been associated with several inflammatory diseases. We present evidence that the hydroxylation of Z 9(10)-epoxyoctadecanoic, Z 9(10)-epoxyoctadec-Z 12-enoic, and Z 12(13)-epoxyoctadec-Z 9-enoic acids and that of monoepoxides from arachidonic acid [epoxyeicosatrienoic acid (EET)] is important in the regulation of leukotoxin and EET activity. These three epoxidized derivatives from the C18 family (C18-epoxides) were converted to 18-hydroxy-C18-epoxides by human hepatic microsomes with apparent Km values of between 27.6 and 175 microM. Among recombinant P450 enzymes, CYP4F2 and CYP4F3B catalyzed mainly the omega-hydroxylation of C18-epoxides with an apparent Vmax of between 0.84 and 15.0 min(-1), whereas the apparent Vmax displayed by CYP4F3A, the isoform found in leukocytes, ranged from 3.0 to 21.2 min(-1). The rate of omega-hydroxylation by CYP4A11 was experimentally found to be between 0.3 and 2.7 min(-1). CYP4F2 and CYP4F3 exhibited preferences for omega-hydroxylation of Z 8(9)-EET, whereas human liver microsomes preferred Z 11(12)-EET and, to a lesser extent, Z 8(9)-EET. Moreover, vicinal diol from both C18-epoxides and EETs were omega-hydroxylated by liver microsomes and by CYP4F2 and CYP4F3. These data support the hypothesis that the human CYP4F subfamily is involved in the omega-hydroxylation of fatty acid epoxides. These findings demonstrate that another pathway besides conversion to vicinal diol or chain shortening by beta-oxidation exists for fatty acid epoxide inactivation.     

Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

Molecular basis for the omega-regiospecificity of the CYP4A2 and CYP4A3 fatty acid hydroxylases

The CYP4A fatty acid omega-hydroxylases are involved in important physiological processes such as the regulation of vascular pressure. A previous study of chimeras of the rat CYP4A2 and CYP4A3 enzymes established that the regiochemistry of fatty acid hydroxylation is determined by the first 119 amino acid residues (Hoch, U., Zhang. Z. P., Kroetz, D. L., and Ortiz de Montellano, P. R. (2000) Arch. Biochem. Biophys. 373, 63-71). The role of the individual amino acid differences in this region has therefore been examined by site-specific mutagenesis to determine which residues actually control the omega- versus (omega-1)-regiospecificity. The results indicate that regiospecificity is controlled by the presence or absence of a three-residue insert (Ser-114, Gly-115, Ile-116) in CYP4A3 and by the residue at position 119 (CYP4A3 numbering). Furthermore, analysis of the absolute stereochemistry of the 11-hydroxylauric acid product indicates that this stereochemistry is not very sensitive to changes in the residues that line the substrate access channel. These results define a model of the specificity determinants of an important class of cytochrome P450 enzymes.     

Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis

The oxidizing activity of CYP109B1 from Bacillus subtilis was reconstituted in vitro with various artificial redox proteins including putidaredoxin reductase and putidaredoxin from Pseudomonas putida, truncated bovine adrenodoxin reductase and adrenodoxin, flavodoxin reductase and flavodoxin from Escherichia coli, and two flavodoxins from B. subtilis (YkuN and YkuP). Binding and oxidation of a broad range of chemically different substrates (fatty acids, n-alkanes, primary n-alcohols, terpenoids like (+)-valencene, α- and β-ionone, and the steroid testosterone) were investigated. CYP109B1was found to oxidize saturated fatty acids (conversion up to 99%) and their methyl and ethyl esters (conversion up to 80%) at subterminal positions with a preference for the carbon atoms C11 and C12 counted from the carboxyl group. For the hydroxylation of primary n-alcohols, the ω_?2 position was preferred. n-Alkanes were not accepted as substrates by CYP109B1. Regioselective hydroxylation of terpenoids α-ionone (～70% conversion) and β-ionone (～ 91% conversion) yielded the allylic alcohols 3-hydroxy-α-ionone and 4-hydroxy-β-ionone, respectively. Furthermore, indole was demonstrated to inhibit fatty acid oxidation.     

Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via beta-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor beta-oxidized in mitochondria. We thus examined whether omega-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their omega-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 +/- 0.5 and 1.46 +/- 0.30 nmol product formed/min/mg protein, respectively (mean +/- SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal omega-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%), whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed omega-hydroxylation of 3-hydroxystearate, whereas CYP4F3b, CYP4F12, and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were K(m) = 55 microM and V(max) = 8.33 min(-1), whereas those for 3-hydroxypalmitate were K(m) = 56.4 microM and V(max) = 14.2 min(-1). CYP4F11 kinetic values resembled those of native microsomes, with K(m) = 53.5 microM and V(max) = 13.9 min(-1) for 3-hydroxystearate and K(m) = 105.8 microM and V(max) = 70.6 min(-1) for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to omega-hydroxylated 3-OHDCA precursors in human liver and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted omega-hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid beta-oxidation increases circulating 3-OHFA levels.     

Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C_18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C_14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.     

Oxidation of monohalogenated ethanes and n-chlorinated alkanes by whole cells of Nitrosomonas europaea.

We have investigated the substrate specificity of ammonia monooxygenase in whole cells of the nitrifying bacterium Nitrosomonas europaea for a number of aliphatic halogenated hydrocarbons. To determine the effect of the halogen substituent and carbon chain length on substrate reactivity, we measured the rates of oxidation of the monohalogenated ethanes (fluoroethane, chloroethane, bromoethane, and iodoethane) and n-chlorinated C1 to C4 alkanes by whole cells of N. europaea. For monohalogenated ethanes, acetaldehyde was the major organic product and little or none of any of the alternate predicted products (2-halogenated alcohols) were detected. The maximum rate of haloethane oxidation increased with decreasing halogen molecular weight from iodoethane to chloroethane (19 to 221 nmol/min per mg of protein). In addition, the amount of substrate required for the highest rate of haloethane oxidation increased with decreasing halogen molecular weight. For the n-chlorinated alkanes, the rate of dechlorination, as measured by the appearance of the corresponding aldehyde product, was greatest for chloroethane and decreased dramatically for chloropropane and chlorobutane (118, 4, and 8 nmol of aldehyde formed per min per mg of protein, respectively). The concentration profiles for halocarbon oxidation by ammonia monooxygenase showed apparent substrate inhibition when ammonia was used as the reductant source. When hydrazine was used as the electron donor, no substrate inhibition was observed, suggesting that the inhibition resulted from reductant limitation.     

What's new in enzymatic halogenations

The halogenation of thousands of natural products occurs during biosynthesis and often confers important functional properties. While haloperoxidases had been the default paradigm for enzymatic incorporation of halogens, via X+ equivalents into organic scaffolds, a combination of microbial genome sequencing, enzymatic studies and structural biology have provided deep new insights into enzymatic transfer of halide equivalents in three oxidation states. These are (1) the halide ions (X-) abundant in nature, (2) halogen atoms (X*), and (3) the X+ equivalents. The mechanism of halogen incorporation is tailored to the electronic demands of specific substrates and involves enzymes with distinct redox coenzyme requirements.     

Omega oxidation of fatty acids and the pathway of 3-hydroxybutyric acid formation

Pathways followed by the carbons of long chain fatty acids in their conversion to 3-hydroxybutyric acid were traced and the contribution of ω-oxidation to fatty acid oxidation was determined in the cellular environment where ketone body formation occurs. 1-¹⁴C-, 2-¹⁴C-, and ω-¹⁴C-labeled fatty acids were injected into alloxan-induced diabetic rats in ketosis. 3-Hydroxybutyric acid was isolated from their urines and degraded. About 1.2 to 1.4 times as much ¹⁴C was found in carbon 1 as carbon 3 of 3-hydroxybutyric acid when the 1-¹⁴C-labeled fatty acids were injected and in carbon 2 as carbon 4 when the 2-¹⁴C-labeled fatty acids were injected. There was about 4 times as much incorporation into carbon 4 as carbon 2 of 3-hydroxybutyric acid formed from the ω-¹⁴C-labeled fatty acids. This means that 50% or more of the fatty acids were oxidized, so that the terminal two carbons of the fatty acids were converted to acetoacetyl-CoA without acetyl-CoA as an intermediate. Incorporation of ¹⁴C into carbons 1 and 2 of the hydroxybutyric acid reflects the distribution of ¹⁴C in acetyl-CoA. Incorporation into carbon 1 was very small when the ω-¹⁴C-labeled fatty acids were substrate. This means that ω-oxidation of fatty acids makes, at most, a small contribution to the formation of the acetyl-CoA pool from which acetoacetate is derived.     

Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study

Lipid peroxidation plays an important role in cell membrane damage. We investigated the effect of lipid peroxidation on the properties of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) lipid bilayers using molecular dynamics simulations. We focused on four main oxidation products of linoleic acid with either a hydroperoxide or an aldehyde group: 9-trans, cis-hydroperoxide linoleic acid, 13-trans, cis-hydroperoxide linoleic acid, 9-oxo-nonanoic acid, and 12-oxo-9-dodecenoic acid. These oxidized chains replaced the sn-2 linoleate chain. The properties of PLPC lipid bilayers were characterized as a function of the concentration of oxidized lipids, with concentrations from 2.8% to 50% for each oxidation product. The introduction of oxidized functional groups in the lipid tail leads to an important conformational change in the lipids: the oxidized tails bend toward the water phase and the oxygen atoms form hydrogen bonds with water and the polar lipid headgroup. This conformational change leads to an increase in the average area per lipid and, correspondingly, to a decrease of the bilayer thickness and the deuterium order parameters for the lipid tails, especially evident at high concentrations of oxidized lipid. Water defects are observed in the bilayers more frequently as the concentration of the oxidized lipids is increased. The changes in the structural properties of the bilayer and the water permeability are associated with the tendency of the oxidized lipid tails to bend toward the water interface. Our results suggest that one mechanism of cell membrane damage is the increase in membrane permeability due to the presence of oxidized lipids.     

Structural basis and mechanism of the inhibition of the type-3 copper protein tyrosinase from Streptomyces antibioticus by halide ions

The inhibition of the type-3 copper enzyme tyrosinase by halide ions was studied by kinetic and paramagnetic (1)H NMR methods. All halides are inhibitors in the conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) with apparent inhibition constants that follow the order I(-) < F(-) < Cl(-) < Br(-) at pH 6.80. The results show that the inhibition arises from the interaction of halide with both the oxidized (affinity F(-) > Cl(-) > Br(-) > I(-)) and reduced (affinity I(-) > Br(-) > Cl(-) > F(-)) enzyme. The paramagnetic (1)H NMR of the oxidized enzyme complexed with the halides is consistent with a direct interaction of halide with the type-3 site and shows that the (Cu-His(3))(2) coordination occurs in all halide-bound species. It is surmised that halides bridge both of the copper ions in the active site. Fluoride and chloride are shown to bind only to the low pH form of oxidized tyrosinase, explaining the strong pH dependence of the inhibition by these ions. We further show that p-toluic acid and the bidentate transition state analogue, Kojic acid, displace chloride from the oxidized active site, whereas the monodentate substrate analogue, p-nitrophenol, forms a ternary complex with the enzyme and the chloride ion. On the basis of the experimental results, a model is formulated for the inhibitor action and for the reaction of diphenols with the oxidized enzyme.     

Structural determination of the substrate specificities and regioselectivities of the rat and human fatty acid omega-hydroxylases

The substrate and regiospecificities of the known CYP4A enzymes from rat (CYP4A1, -4A2, -4A3, and -4A8) and human (CYP4A11) have been determined using lauric (C12), myristic (C14), palmitic (C16), oleic (C18:1), and arachidonic (C20:4) acids. The CYP4A2 and CYP4A8 cDNAs required to complete the enzyme set were cloned from a rat kidney library. All five proteins were expressed in Escherichia coli and were purified with the help of a six-histidine tag at the carboxyl terminus. Two complementary CYP4A2-CYP4A3 chimeras fused at residue 119 (CYP4A2) and 122 (CYP4A3) were constructed to explore the roles of the 18 amino acid differences between the parent proteins in determining their catalytic profiles. The chimera in which the first 119 amino acids are from CYP4A2 indicates that the first 120 amino acids control the substrate specificity. The chimera in which the first 122 amino acids are from CYP4A3 is inactive due to a defect in electron transfer to the heme group. The highest activity for lauric acid was obtained with CYP4A1 and CYP4A8, but for all the proteins the activity decreased with increasing fatty acid chain length. The fact that none of the rat and human CYP4A enzymes exhibits a high activity with arachidonic acid appears to limit their role as catalysts for the physiologically important conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE).