Photobilirubin II

An improved preparation of photobilirubin II in ammoniacal methanol is described. Evidence is presented which distinguishes between the two structures proposed earlier for photobilirubin II in favour of the cycloheptadienyl structure. Nuclear-Overhauser-enhancement measurements with bilirubin IX alpha and photobilirubin II in dimethyl sulphoxide are complicated by the occurrence of negative and zero effects. The partition coefficient of photobilirubin II between chloroform and phosphate buffer (pH 7.4) is 0.67.     

Crystal structure of a molybdopterin synthase-precursor Z complex: insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency

In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Together, the metal and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enzymes catalyzing the first and second steps of biosynthesis, leading to the formation of precursor Z and MPT, respectively. The second step is catalyzed by the heterotetrameric MPT synthase protein consisting of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer. Previous studies have determined that the conversion of the sulfur- and metal-free precursor Z to MPT by MPT synthase involves the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1' and C-2' positions of precursor Z. Here, we present the crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in its apo form and in complex with precursor Z. A comparison of the two structures reveals conformational changes in a loop that participates in interactions with precursor Z. In the complex, precursor Z is bound by strictly conserved residues in a pocket at the MoaE dimer interface in close proximity of the C-terminal glycine of MoaD. Biochemical evidence indicates that the first dithiolene sulfur is added at the C-2' position.     

The biosynthetic pathway to abscisic acid via ionylideneethane in the fungus Botrytis cinerea

The biosynthetic pathway to abscisic acid (ABA) from isopentenyl diphosphate in the fungus, Botrytis cinerea, was investigated. Labeling experiments with (18)O2 and H2(18)O indicated that all oxygen atoms at C-1, -1, -1' and -4' of ABA were derived from molecular oxygen, and not from water. This finding was inconsistent not only with the known carotenoid pathway via oxidative cleavage of carotenoids, but also with the classical direct pathway via cyclization of farnesyl diphosphate. The fungus produced new C15-compounds, 2E,4E-alpha-ionylideneethane and 2Z,4E-alpha-ionylideneethane, along with 2E,4E,6E-allofarnesene and 2Z,4E,6E-allofarnesene, but did not apparently produce carotenoids except for a trace of phytoene. The C15-compounds labeled with 13C were converted to ABA by the fungus, and the incorporation ratio of 2Z,4E-alpha-ionylideneethane was higher than that of 2E,4E-alpha-ionylideneethane. From these results, it was concluded that farnesyl diphosphate was reduced at C-1, desaturated at C-4, and isomerized at C-2 to form 2Z,4E,6E-allofarnesene before being cyclized to 2Z,4E-alpha-ionylideneethane; the ionylideneethane was then oxidized to ABA with molecular oxygen. This direct pathway via ionylideneethane means that the biosynthetic pathway to fungal ABA, not only before but also after isopentenyl diphosphate, differs from that to ABA in plants, since plant ABA is biosynthesized using the non-mevalonate and carotenoid pathways.     

Two distinct pathways of formation of 4-hydroxynonenal. Mechanisms of nonenzymatic transformation of the 9- and 13-hydroperoxides of linoleic acid to 4-hydroxyalkenals

The mechanism of formation of 4-hydroxy-2E-nonenal (4-HNE) has been a matter of debate since it was discovered as a major cytotoxic product of lipid peroxidation in 1980. Recent evidence points to 4-hydroperoxy-2E-nonenal (4-HPNE) as the immediate precursor of 4-HNE (Lee, S. H., and Blair, I. A. (2000) Chem. Res. Toxicol. 13, 698-702; Noordermeer, M. A., Feussner, I., Kolbe, A., Veldink, G. A., and Vliegenthart, J. F. G. (2000) Biochem. Biophys. Res. Commun. 277, 112-116), and a pathway via 9-hydroperoxylinoleic acid and 3Z-nonenal is recognized in plant extracts. Using the 9- and 13-hydroperoxides of linoleic acid as starting material, we find that two distinct mechanisms lead to the formation of 4-H(P)NE and the corresponding 4-hydro(pero)xyalkenal that retains the original carboxyl group (9-hydroperoxy-12-oxo-10E-dodecenoic acid). Chiral analysis revealed that 4-HPNE formed from 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) retains >90% S configuration, whereas it is nearly racemic from 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9S-HPODE). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid is >90% S when derived from 9S-HPODE and almost racemic from 13S-HPODE. Through analysis of intermediates and products, we provide evidence that (i) allylic hydrogen abstraction at C-8 of 13S-HPODE leads to a 10,13-dihydroperoxide that undergoes cleavage between C-9 and C-10 to give 4S-HPNE, whereas direct Hock cleavage of the 13S-HPODE gives 12-oxo-9Z-dodecenoic acid, which oxygenates to racemic 9-hydroperoxy-12-oxo-10E-dodecenoic acid; by contrast, (ii) 9S-HPODE cleaves directly to 3Z-nonenal as a precursor of racemic 4-HPNE, whereas allylic hydrogen abstraction at C-14 and oxygenation to a 9,12-dihydroperoxide leads to chiral 9S-hydroperoxy-12-oxo-10E-dodecenoic acid. Our results distinguish two major pathways to the formation of 4-HNE that should apply also to other fatty acid hydroperoxides. Slight ( approximately 10%) differences in the observed chiralities from those predicted in the above mechanisms suggest the existence of additional routes to the 4-hydroxyalkenals.     

Biosynthesis of abscisic acid by the direct pathway via ionylideneethane in a fungus, Cercospora cruenta

We examined the biosynthetic pathway of abscisic acid (ABA) after isopentenyl diphosphate in a fungus, Cercospora cruenta. All oxygen atoms at C-1, -1, -1', and -4' of ABA produced by this fungus were labeled with (18)O from (18)O(2). The fungus did not produce the 9Z-carotenoid possessing gamma-ring that is likely a precursor for the carotenoid pathway, but produced new sesquiterpenoids, 2E,4E-gamma-ionylideneethane and 2Z,4E-gamma-ionylideneethane, along with 2E,4E,6E-allofarnesene. The fungus converted these sesquiterpenoids labeled with (13)C to ABA, and the incorporation ratio of 2Z,4E-gamma-ionylideneethane was higher than that of 2E,4E-gamma-ionylideneethane. From these results, we concluded that C. cruenta biosynthesized ABA by the direct pathway via oxidation of ionylideneethane with molecular oxygen following cyclization of allofarnesene. This direct pathway via ionylideneethane in the fungus is consistent with that in Botrytis cinerea, except for the positions of double bonds in the rings of biosynthetic intermediates, suggesting that the pathway is common among ABA-producing fungi.     

Biosynthesis of the pyrimidine moiety of thiamin in Escherichia coli: incorporation of stable isotope-labeled glycines.

Methods are described for the cleavage, extraction, and subsequent gas chromatographic-mass spectrometric analysis of the pyrimidine moiety of thiamin as 2-methyl-4-amino-5-[(ethylthio)methyl]pyrimidine. The methods are of a general nature and can be applied to any system. Using these methods to evaluate the incorporation of 13C-, 15N-, and 2H-labeled glycines into the pyrimidine moiety of thiamin by Escherichia coli, we established that the nitrogen and carbon atoms of glycine are incorporated as a unit into the pyrimidine. 13C- and 15N-labeled glycines are incorporated at greater than 60% but deuterium from [2-(2)H2]glycine was incorporated at only 18%. A detailed analysis of the mass fragmentation pattern of the pyrimidine derivative has established that the glycine nitrogen atom supplies the N-1 of the pyrimidine and that the C-1 and C-2 of the glycine supplies the C-4 and C-6 of the pyrimidine, respectively. This evidence is consistent with the substitution of a C2 unit between the C-5 and C-4 of the 4-aminoimidazole ribonucleotide precursor during the biosynthesis of the pyrimidine moiety of thiamin in E. coli.     

Free radical oxidation of 15-(S)-hydroxyeicosatetraenoic acid with the Fenton reagent: characterization of an epoxy-alcohol and cytotoxic 4-hydroxy-2E-nonenal from the heptatrienyl radical pathway

The oxidation of (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-(S)-HETE, 1a) with the Fenton reagent (Fe2+/EDTA/H2O2) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with 75% substrate consumption after 1 h to give a mixture of products, one of which was identified as (2E,4S)-4-hydroxy-2-nonenal (3a, 18% yield). Methylation of the mixture with diazomethane allowed isolation of another main product which could be identified as methyl (5Z,8Z,13E)-11,12-trans-epoxy-15-hydroxy-5,8,13-eicosatrienoate (2a methyl ester, 8% yield). A similar oxidation carried out on (15-(2)H)-15-HETE (1b) indicated complete retention of the label in 2b methyl ester and 3b, consistent with an oxidation pathway involving as the primary event H-atom abstraction at C-10. Overall, these results support the recently proposed role of 1a as a potential precursor of the cytotoxic gamma-hydroxyalkenal 3a and disclose a hitherto unrecognized interconnection between 1a and the epoxy-alcohol 2a, previously implicated only in the metabolic transformations of the 15-hydroperoxy derivative of arachidonic acid.     

The structure of a molybdopterin precursor. Characterization of a stable, oxidized derivative

An oxidized pterin species, termed compound Z, has been isolated from molybdenum cofactor-deficient mutants of Escherichia coli and shown to be the direct product of oxidation of a molybdopterin precursor which accumulates in these mutants. The complete structural characterization of compound Z has been accomplished. A carbonyl function at C-1' of the 6-alkyl side chain can be reacted with 2,4-dinitrophenylhydrazine to yield a phenylhydrazone and can be reduced with borohydride, producing a mixture of two enantiomers, each with a hydroxyl group on C-1'. Compound Z contains one phosphate/pterin and no sulfur. The phosphate group is insensitive to alkaline phosphatase and to a number of phosphodiesterases but is quantitatively released as inorganic phosphate by mild acid hydrolysis. From 31P and 1H NMR of compound Z it was inferred that the phosphate is bound to C-2' and C-4' of a 4-carbon side chain, forming a 6-membered cyclic structure. Mass spectral analysis showed an MH+ ion with an exact mass of 344.0401 corresponding to the molecular formula C10H11N5O7P, confirming the proposed structure.     

Stereoselective oxidation of regioisomeric octadecenoic acids by fatty acid dioxygenases

Seven Z-octadecenoic acids having the double bond located in positions 6Z to 13Z were photooxidized. The resulting hydroperoxy-E-octadecenoic acids [HpOME(E)] were resolved by chiral phase-HPLC-MS, and the absolute configurations of the enantiomers were determined by gas chromatographic analysis of diastereoisomeric derivatives. The MS/MS/MS spectra showed characteristic fragments, which were influenced by the distance between the hydroperoxide and carboxyl groups. These fatty acids were then investigated as substrates of cyclooxygenase-1 (COX-1), manganese lipoxygenase (MnLOX), and the (8R)-dioxygenase (8R-DOX) activities of two linoleate diol synthases (LDS) and 10R-DOX. COX-1 and MnLOX abstracted hydrogen at C-11 of (12Z)-18:1 and C-12 of (13Z)-18:1. (11Z)-18:1 was subject to hydrogen abstraction at C-10 by MnLOX and at both allylic positions by COX-1. Both allylic hydrogens of (8Z)-18:1 were also abstracted by 8R-DOX activities of LDS and 10R-DOX, but only the allylic hydrogens close to the carboxyl groups of (11Z)-18:1 and (12Z)-18:1. 8R-DOX also oxidized monoenoic C(14)-C(20) fatty acids with double bonds at the (9Z) position, suggesting that the length of the omega end has little influence on positioning for oxygenation. We conclude that COX-1 and MnLOX can readily abstract allylic hydrogens of octadecenoic fatty acids from C-10 to C-12 and 8R-DOX from C-7 and C-12.     

The biosynthesis of D-ring fatty acid esters of estriol

Biological esterification with fatty acids is a feature that is now known to be common to most steroids. The esterification of estradiol in the D-ring at the 17 beta-hydroxyl leads to a family of extremely active estrogens. Similarly, esterification of the weaker estrogen, estriol (E3), has an even greater impact on its hormonal potency. We have recently shown that synthetic long chain esters of E3 at either 16 alpha- or 17 beta- are highly potent estrogens. The estrogenic activity of the synthetic E3 esters led us to determine whether E3 is biologically esterified, and if so, to characterize the resulting esters. Incubation of E3 with rat lung, a tissue which is highly active in esterifying estradiol, produces a nonpolar metabolite which upon saponification is converted back into E3. There was no evidence for the formation of a diester. Purification by high performance liquid chromatography separates the non-polar metabolite into two peaks, one the C-16 alpha- (approximately 60%) and the other the C-17 beta-ester (approximately 40%). The two fractions were further purified and characterized; each is a mixture of fatty acid esters of E3. The composition of the C-16 alpha- and the C-17 beta-fatty acid esters of E3 is identical. The predominant fatty acids are arachidonate, 34%, palmitate, 26%, followed by oleate 14%, linoleate 13%, stearate 8%, and palmitoleate 5%. The similarity of the esters at C-16 and C-17 may indicate that the fatty acid precursor for the acyltransferase is the same for both hydroxyl groups. It may also suggest that the same enzyme esterifies both positions in the D-ring. Since synthetic estriol fatty acid esters are extremely potent and long-lived estrogens, the enzymatic esterification of estriol produces powerful estrogens with considerable physiological potential.     

Synthesis and structural identification of four dihydroxy acids and 11,12-leukotriene C4 derived from 11,12-leukotriene A4

Using a partially purified 12-lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)-12-hydroperoxy-5,8,10,14-icosate traenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C-12 was determined as S by chiral-phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (11,12-leukotriene A4) and (5Z,7Z,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (7-cis-11,12-leukotriene A4). 11,12-Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)-(12S)-5,12-dihydroxy-6,8,10,14-i cosatetraenoic acid and two isomers of (14Z)-(12S)-11,12-dihydroxy-5,7,9,14-icosatetraenoic acid. Upon incubation with rat liver glutathione S-transferase, 11,12-leukotriene A4 was converted to 11,12-leukotriene C4, a spasmogenic compound.     

Analysis of carotenoid composition in petals of calendula (Calendula officinalis L.)

Nineteen carotenoids were identified in extracts of petals of orange- and yellow-flowered cultivars of calendula (Calendula officinalis L.). Ten carotenoids were unique to orange-flowered cultivars. The UV-vis absorption maxima of these ten carotenoids were at longer wavelengths than that of flavoxanthin, the main carotenoid of calendula petals, and it is clear that these carotenoids are responsible for the orange color of the petals. Six carotenoids had a cis structure at C-5 (C-5'), and it is conceivable that these (5Z)-carotenoids are enzymatically isomerized at C-5 in a pathway that diverges from the main carotenoid biosynthesis pathway. Among them, (5Z,9Z)-lycopene (1), (5Z,9Z,5'Z,9'Z)-lycopene (3), (5'Z)-gamma-carotene (4), and (5'Z,9'Z)-rubixanthin (5) has never before been identified. Additionally, (5Z,9Z,5'Z)-lycopene (2) has been reported only as a synthesized compound.     

Mechanistic and stereochemical studies of a unique dehydration catalyzed by CDP-4-keto-6-deoxy-D-glucose-3-dehydrase: a pyridoxamine 5'-phosphate dependent enzyme isolated from Yersinia pseudotuberculosis.

CDP-4-keto-6-deoxy-D-glucose-3-dehydrase (E1) purified from Yersinia pseudotuberculosis is a pyridoxamine 5'-phosphate (PMP) dependent enzyme which catalyzes the C-O bond cleavage at C-3 of a CDP-4-keto-6-deoxy-D-glucose substrate, a key step in the formation of 3,6-dideoxyhexoses. Since enzyme E1 utilizes the PMP cofactor in a unique manner, it is essential to establish its role in E1 catalysis. When an incubation was conducted in [18O]H2O, incorporation of 18O into positions C-3 and C-4 of the recovered substrate was observed. This result not only provided the evidence necessary to reveal the reversibility of E1 catalysis but also lent credence to the formation of a delta 3,4-glucoseen intermediate. In view of E1 catalysis being initiated by a C-4' deprotonation of the PMP-substrate complex, the stereochemical course of this step was examined using chemically synthesized (4'S)- and (4'R)-[4'-3H]PMP as probes. Our results clearly demonstrated that the stereochemistry of this deprotonation is pro-S specific, which is in agreement with the stereochemical consistency found with other vitamin B6 phosphate dependent enzymes. The fact that reprotonation at C-4' of the PMP-delta 3,4-glucoseen complex in the reverse direction of E1 catalysis was also found to be pro-S stereospecific strongly suggested that enzyme E1, like most of its counterparts, has the si face of its cofactor-substrate complex exposed to solvent and accessible to active-site catalytic groups as well.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arachidonate 12-lipoxygenase purified from porcine leukocytes by immunoaffinity chromatography and its reactivity with hydroperoxyeicosatetraenoic acids

Arachidonate 12-lipoxygenase was purified to near homogeneity from the cytosol fraction of porcine leukocytes by ammonium sulfate fractionation, DEAE-cellulose chromatography, and immunoaffinity chromatography using a monoclonal antibody against the enzyme. The purified enzyme was unstable (half-life of about 24 h at 4 degrees C) but was markedly protected from the inactivation by storage in the presence of ferrous ion or in the absence of air. The lag phase which was observed before the start of the enzyme reaction was abolished by the presence of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. An apparent substrate inhibition was observed with arachidonic acid and other active substrates; however, the substrate concentration curve was normalized by the presence of 0.03% Tween 20. Arachidonic acid was transformed to the omega-9 oxygenation product 12-hydroperoxy-5Z,8Z,10Z,14Z-eicosatetraenoic acid. C-12 oxygenation also occurred with 5-hydroxy- and 5-hydroperoxyeicosatetraenoic acids; the respective maximal velocities were 60 and 150% of the rate with arachidonic acid. Octadecaenoic acids were also good substrates. gamma-Linolenic acid was oxygenated in the omega-9 position (C-10), while linoleic and alpha-linolenic acids were subject to omega-6 oxygenation (C-13). A far more complex reaction was observed using 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid as substrate. Reaction occurred at 70% of the rate with arachidonic acid. The dihydroperoxy and dihydroxy products were identified by their UV absorption spectra, high performance liquid chromatography, and gas chromatography-mass spectrometry. Among these products, (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicos atetraenoic acid and (14R,15S)-erythro-dihydroperoxy-5Z,8Z,10E, 12E-eicosatetraenoic acid were produced in larger amounts than the (8R)- and (14S,15S)-threo isomers, respectively; these products were attributed to 8- and 14-oxygenation of the 15-hydroperoxy acid. Furthermore, formation of 14,15-leukotriene A4 was inferred from the characteristic pattern of its hydrolysis products comprised of equal amounts of (8R,15S)- and (8S,15S)-dihydroxy-5Z,9E,11E,13E-eicosatetraenoi c acids together with smaller amounts of (14R,15S)-erythro- and (14S,15S)-threo-dihydroxy-5Z,8Z,10E,12E-eicosate traenoic acids. Thus, both lipoxygenase and leukotriene synthase activities were demonstrated with the homogeneous preparation of porcine leukocyte 12-lipoxygenase.     

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1A1: a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA.

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.     

Biosynthesis of phycobilins. 3(Z)-phycoerythrobilin and 3(Z)-phycocyanobilin are intermediates in the formation of 3(E)-phycocyanobilin from biliverdin IX alpha

An enzyme extract from the phycocyanin-containing unicellular rhodophyte, Cyanidium caldarium, reductively transforms biliverdin IX alpha to phycocyanobilin, the chromophore of phycocyanin, in the presence of NADPH. Unpurified cell extract forms both 3(E)-phycocyanobilin, which is identical to the major pigment that is released from phycocyanin by methanolysis, and 3(Z)-phycocyanobilin, which is obtained as a minor methanolysis product. After removal of low molecular weight material from the cell extract, only 3(Z)-phycocyanobilin is formed. 3(E)-Phycocyanobilin formation from biliverdin IX alpha, and the ability to isomerize 3(Z)-phycocyanobilin to 3(E)-phycocyanobilin, are reconstituted by the addition of glutathione to the incubation mixture. Partially purified protein fractions derived from the initial enzyme extract form 3(Z)-phycocyanobilin plus two additional, violet colored bilins, upon incubation with NADPH and biliverdin IX alpha. Further purified protein fractions produce only the violet colored bilins from biliverdin IX alpha. One of these bilins was identified as 3(Z)-phycoerythrobilin by comparative spectrophotometry, reverse-phase high pressure liquid chromatography, and 1H NMR spectroscopy. A C. caldarium protein fraction catalyzes the conversion of 3(Z)-phycoerythrobilin to 3(Z)-phycocyanobilin. This fraction also catalyzes the conversion of 3(E)-phycoerythrobilin to 3(E)-phycocyanobilin. The conversion of phycoerythrobilins to phycocyanobilins requires neither biliverdin nor NADPH. The synthesis of phycoerythrobilin and its conversion to phycocyanobilin by extracts of C. caldarium, a species that does not contain phycoerythrin, indicates that phycoerythrobilin is a biosynthetic precursor to phycocyanobilin. The enzymatic conversion of the ethylidine group from the Z to the E configuration suggests that the E-isomer is the precursor to the protein-bound chromophore.     

一株能水解巴卡亭ⅢC-10位乙酰基的成团泛菌的筛选和鉴定

10-脱乙酰巴卡亭Ⅲ是合成紫杉醇和多烯紫杉醇的前体。以巴卡亭Ⅲ为底物,结合TLC、HPLC、HPLC-MS分析方法,通过设计专门的筛选方法筛选产酶菌株,得到一株巴卡亭ⅢC-10位脱乙酰基酶产生菌株Z1-56。以形态特征、生理生化特征、16S rDNA序列分析作为菌株的鉴定手段,Z1-56被鉴定为成团泛菌(Pantoea agglomerans),首次发现成团泛菌产生巴卡亭ⅢC-10-脱乙酰酶。     

Metabolism of 6,9,12-octadecatrienoic acid in the red alga Lithothamnion corallioides: mechanism of formation of a conjugated tetraene fatty acid.

Incubation of [1-14C]6(Z),9(Z),12(Z)-octadecatrienoic acid with an enzyme preparation from the red alga Lithothamnion corallioides Crouan led to the formation of two new compounds, i.e. the conjugated tetraene 6(Z),8(E),10(E),12(Z)-octadecatetraenoic acid and the bis-allylic hydroxy acid 11(R)-hydroxy-6(Z),9(Z),12(Z)-octadecatrienoic acid. These two compounds were formed by independent pathways and were not interconvertible by the enzyme preparation. Experiments with stereospecifically deuteriated 6,9,12-octadecatrienoic acids demonstrated that formation of 6,8,10,12-octadecatetraenoic acid was accompanied by loss of the pro-S and pro-R hydrogens from C-8 and C-11, respectively, whereas formation of 11-hydroxy-6,9,12-octadecatrienoic acid proceeded with loss of the pro-S hydrogen from C-11. Biosynthesis of 6,8,10,12-octadecatetraenoic acid was dioxygen-dependent and was accompanied by production of hydrogen peroxide. A number of artificial electron acceptors supported formation of 6,8,10,12-octadecatetraenoic acid under anaerobic conditions. The existence in Lithothamnion corallioides of a fatty acid oxidase that catalyzes the oxidation of certain poly-unsaturated fatty acids into conjugated tetraene fatty acids is postulated.     

(S)-12-hydroxygeranylgeraniol-derived diterpenes from the brown alga Bifurcaria bifurcata

Four novel diterpenes were isolated from the brown alga Bifurcaria bifurcata collected off the Atlantic coast from Morocco, and their structures established by spectral and chemical methods. These compounds are acyclic diterpenes derived from (S)-12-hydroxygeranylgeraniol. One of them is its dehydration product at C-12, while the others are its oxidation derivatives: the methyl ester of the acid at C-1 and two stereoisomers (Z and E) of the aldehyde at C-1. These results are discussed from a chemotaxonomic point of view.