Mechanism and stereochemistry in the sequential enzymatic saturation of the two double bonds in cholesta-4,6-dien-3-one.

The mechanism and stereochemistry in connection with enzymatic conversion of cholesta-4,6-dien-3-one into cholestanol was studied. Rat and mouse liver microsomes are able to catalyze NADPH-dependent sequential saturation of the two double bonds. Evidence was obtained that the saturation of the delta 6-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 7-position of the steroid (mainly 7 beta-position), followed by addition of a proton to the 6 alpha-position (mainly trans addition). The saturation of the delta 4-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 5 alpha-position of the steroid followed by addition of a proton to the 4 beta-position (trans addition). The reduction of the 3-oxo group was found to involve transfer of a hydride ion from the B-side of the cofactor NADPH to the 3 alpha-position of the steroid. The results are in accord with the contention that the enzymatic saturation of the two double bonds involves a polarization of the 3-oxo group making C-7 electrophilic and C-6 nucleophilic in connection with the saturation of the delta 6-double bond and C-5 electrophilic and C-4 nucleophilic in connection with the saturation of the delta 4-double bond.     

OH-induced free radicals in uridine studied by a method combining ESR, spin-trapping, and liquid chromatography

Free radicals produced by the reactions of OH radicals with uridine were investigated by a method combining ESR, spin-trapping, and liquid chromatography. A N2O-saturated aqueous solution of uridine, containing 2-methyl-2-nitrosopropane as a spin-trap, was X-irradiated and the resulting spin-adducts were separated by gel permeation chromatography and reverse-phase HPLC. ESR and uv-absorbance spectra obtained from the separated spin-adducts show that 5-yl and 6-yl radicals are produced by OH addition to the 5,6 double bond of the base moiety. It is also shown that radicals due to H abstraction from the sugar moiety at the C-4' and C-5' positions are produced.     

The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes

We report for the first time the complete structure and sequence of the trichothecene biosynthesis gene cluster (i.e. Tri5-cluster) from Fusarium graminearum F15, a strain that produces 3-acetyldeoxynivalenol (3-ADON). A putative tyrosinase and polysaccharide deacetylase gene flank the Tri5-cluster: the number of pathway genes between them is less than half the total number of steps necessary for 3-ADON biosynthesis. In comparison with partial Tri5-cluster sequences of strains with 15-acetyldeoxynivalenol and 4-acetylnivalenol chemotypes, the Tri5-cluster from strain F15 contains three genes that are apparently unnecessary for the biosynthesis of 3-ADON (i.e. Tri8 and Tri3, which are expressed, and pseudo-Tri13, which is not expressed). In addition, the Tri7 gene was missing from the cluster. Recombinant TRI3 protein showed limited trichothecene C-15 acetylase activity. In contrast, recombinant TRI8 protein displayed no C-3 deacetylase activity, suggesting that the loss or alteration of function contribute directly to the chemotype difference.     

Design and syntheses of putative bioactive taxanes

Reduction of 5 alpha-hydroxy-7 beta,9 alpha,10 beta-triacetoxy-4(20), 11(12)-taxadien-13-one 1 with activated zinc in glacial acetic acid led to rearranged products, including compounds with double bonds at C3-C4, C10-C11 or with an epoxide at C11-C12. Molecular modeling studies suggested that addition of a side chain at C-20 or C-5 of the taxanes with a C3-C4 double bond might lead to bioactivity. Semi-syntheses and results of bioactivities are discussed.     

Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols

Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.     

Inhibition of sterol synthesis by delta 5-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450

Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.     

Cytochrome P450 homolog is responsible for C-N bond formation between aglycone and deoxysugar in the staurosporine biosynthesis of Streptomyces sp. TP-A0274

The staurosporine biosynthetic gene cluster in Streptomyces sp. TP-A0274 consists of 15 sta genes. In the cluster, it was predicted that staN, which shows high similarity to cytochrome P450 is involved in C-N bond formation between the nitrogen at N-12 of aglycone and the carbon at C-5' of deoxysugar. The staN disruptant produced holyrine A instead of staurosporine. The structure of holyrine A is aglycone linking to 2,3,6-trideoxy-3-aminoaldohexose between N-13 and C-1' of deoxysugar. Holyrine A was converted to staurosporine by the staD disruptant. These results indicate that StaN, cytochrome P450 is responsible for C-N bond formation. This is the first example of C-N bond formation catalyzed by cytochrome P450. In addition, holyrine A was confirmed to be an intermediate of staurosporine biosynthesis, which suggests that the N- and O-methylation at C-3' and C-4' takes place after the formation of the C-N bond between C-5' and N-12 in the biosynthetic pathway.     

Synthesis of 14β-cholesta-5,7-dien-3β-ol

5, 7-Cholestadien-3β-ol was transformed into 14β-cholesta-5, 7-dien- 3β-ol in six steps. The inversion of the stereochemistry at C-14 was obtained by a selective protection of the Δ⁵ and the elaboration of the Δ⁷ double bond.     

Deuterium transfer from [1,1-2-H] ethanol during metabolism of bile acids and cyclohexanone in the isolated perfused rat liver.

Deuterium transfer from [1,1-2-H]ethanol (95 atoms % excess) to reducible substrates was studied in the isolated perfused rat liver. The dueterium excess in cyclohexanol formed from cyclohexanone was somewhat lower (49 atoms%) than found under conditions in vivo, and this was also true of the deuterium excess in lithocholic acid formed from 3-oxo-5beta-cholanoic acid. These results may reflect a slower rate of ethanol oxidation in the isolated organ than in vivo. Cycloserine decreased the dueterium transfer to both substrates, whereas addition of lactate and malate resulted in an increased deuterium excess in cyclohexanol and a decreased deuterium excess in lithocholic acid. Addition of heavy water to the perfusion fluid resulted in labelling at C-3 of lithocholic acid formed from 3-oxo-5beta-cholanoic acid, and at C-3, C-4 and C-5 of 3alpha-hydroxy-5alpha-cholanoic acid formed from 3-oxo-4-cholenoic acid. The deuterium excess of hydrogens derived from NADPH (at C-3 and C-5) was approximately the same as that of hydrogen derived directly from water (at C-4). Thus, the hydrogen of NADPH is extensively exchanged with protons of water, which explains the dilution of deuterium with protium during the transfer from [1,1-2-H]ethanol via NADPH to the bile acids. The labelling at C-5 in the reduction of the 4,5-double bond indicates that different pools of NADPH are used for reduction of this double bond and the 3-oxo group, since in a previous study it was shown that deuterium is transferred from [1,1-2-H]ethanol only in the latter reaction.     

High resolution X-ray structure of dTDP-glucose 4,6-dehydratase from Streptomyces venezuelae

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in some macrolide antibiotics. In Streptomyces venezuelae, there are seven genes required for the biosynthesis of this unusual sugar. One of the genes, desIV, codes for a dTDP-glucose 4,6-dehydratase, which is referred to as DesIV. The reaction mechanisms for these types of dehydratases are quite complicated with proton abstraction from the sugar 4'-hydroxyl group and hydride transfer to NAD+, proton abstraction at C-5, and elimination of the hydroxyl group at C-6 of the sugar, and finally return of a proton to C-5 and a hydride from NADH to C-6. Here we describe the cloning, overexpression, and purification, and high resolution x-ray crystallographic analysis to 1.44 A of wild-type DesIV complexed with dTDP. Additionally, for this study, a double site-directed mutant protein (D128N/E129Q) was prepared, crystallized as a complex with NAD+ and the substrate dTDP-glucose and its structure determined to 1.35 A resolution. In DesIV, the phenolate group of Tyr(151) and O(gamma) of Thr(127) lie at 2.7 and 2.6 A, respectively from the 4'-hydroxyl group of the dTDP-glucose substrate. The side chain of Asp(128) is in the correct position to function as a general acid for proton donation to the 6'-hydroxyl group while the side chain of Glu(129) is ideally situated to serve as the general base for proton abstraction at C-5. This investigation provides further detailed information for understanding the exquisite chemistry that occurs in these remarkable enzymes.     

Steroid structural requirements for high affinity binding to human sex steroid binding protein (SBP)

The sex steroid binding protein (SBP) which binds androgens circulating in the blood of man has been examined to determine the structural requirements for high affinity binding. SBP was purified partially and the ability of each of more than 150 steroids to compete with [³H]dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) for binding to SBP was assessed.Binding was enhanced by reduction of the Δ⁴ double bond to 5α-dihydro, addition of a methyl group at C-4 and in one case unsaturation at C-14, 15. Affinity was always reduced by modifications of the C-17β hydroxy. Binding was also severely decreased by deletion of the keto moiety at C-3; however, relatively high affinity was retained by an alcohol or an unsubstituted pyrazole group at C-3. Certain alpha surface substitutions such as 17α-ethinyl had limited effects on binding; whereas, other modifications such as 7α-methyl or 17α-methyl caused significant reduction in binding. Most modifications at C-2, 6, 9 or 11 also impaired affinity, and the 5β steroids had reduced affinity.     

Structure-Activity Relationships of Abscisic Acid Analogs Based on the Induction of Freezing Tolerance in Bromegrass (Bromus inermis Leyss) Cell Cultures

The induction of freezing tolerance in bromegrass (Bromus inermis Leyss) cell culture was used to investigate the activity of absisic acid (ABA) analogs. Analogs were either part of an array of 32 derived from systematic alterations to four regions of the ABA molecule or related, pure optical isomers. Alterations were made to the functional group at C-1 (acid replaced with methyl ester, aldehyde, or alcohol), the configuration at C-2, C-3 (cis double bond replaced with trans double bond), the bond order at C-4, C-5 (trans double bond replaced with a triple bond), and ring saturation (C-2′, C-3′ double bond replaced with a single bond so that the C-2′ methyl and side chain were cis). All deviations in structure from ABA reduced activity. A cis C-2, C-3 double bond was the only substituent absolutely required for activity. Overall, acids and esters were more active than aldehydes and alcohols, cyclohexenones were more active than cyclohexanones, and dienoic and acetylenic analogs were equally active. The activity associated with any one substituent was, however, markedly influenced by the presence of other substituents. cis, trans analogs were more active than their corresponding acetylenic analogs unless the C-1 was an ester. Cyclohexenones were more active than cyclohexanones regardless of oxidation level at C-1. An acetylenic side chain decreased the activity of cyclohexenones but increased the activity of cyclohexanones relative to their cis, trans counterparts. Trends suggested that for activity the configuration at C-1′ has to be the same as in (S)-ABA, in dihydro analogs the C-2′-methyl and the side chain must be cis, small positional changes of the 7′-methyl are tolerable, and the C-1 has to be at the acid oxidation level.     

The functional importance of structural features of ergosterol in yeast.

As an approach to the study of the relationship between the structure of sterols and their capacity to function in the lipid leaflet of membranes, various sterols were examined for their ability to support the growth of anaerobic Saccharomyces cerevisiae. A marked dependence on precise structural features was observed in growth-response and morphology. Of the chemical groups which distinguish ergosterol, the main sterol of S. cerevisiae, the hydroxyl group at C-3 was obligatory, and the other groups were found to be of the following relative importance: 24beta-methyl-delta22-grouping greater than 24beta-methyl group greater than delta5,7-diene system = delta5-bond approximately or equal to no double bond. Methyl groups at C-4 and C-14 were inconsistent with activity. Consequently, the data strongly suggest that the normal biosynthetic processes removal of methyl groups from the nucleus and introduction of one in the side chain are of functional significance. A double bond between C-17 and C-20 joining the steroidal side chain to the nucleus had no deleterious effect on the growth process but only if C-22 was trans-oriented to C-13. In the cis-case no growth at all proceeded. This means the natural sterol probably acts functionally in the form of its preferred conformer in which C-22 is to the right ("right-handed") in the usual view. Since the placing of a substituent (OH or CH3) in the molecule at C-20 in such a way that it appears on the front side in the right-handed conformer completely destroyed activity, the sterol apparently presents its front face to protein or phospholipid when complexing occurs.     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris

alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.     

Preparation and antigenic properties of androsterone-7-BSA conjugate.

The 7-carboxymethoximino derivative of androsterone (1) has been prepared from dehydroisoandrosterone-17-ethyleneketal by a sequence involving inversion at C-3, introduction of a carbonyl at C-7, and reduction of the double bond at C-5. The substance was condensed with BSA by the carbodiimide procedure to afford a conjugate which produced anti-androsterone antiserum in innoculated rabbits. The antiserum is sufficiently active to be useful in radioimmunoassay procedures.     

Phosphorylation of Charge Isomers (Components) of Human Myelin Basic Protein: Identification of Phosphorylated Sites

Myelin basic protein isolated from normal human brain was resolved into its various components (charge isomers) by CM-52 column chromatography. Two of the components C-1 and C-4, were phosphorylated in vitro with a soluble preparation of brain protein kinase C. For each component, the peptides phosphorylated were identified. In both components a major site of phosphorylation was found at Ser7 in the N-terminal portion of the protein. Both the specific activity and the rate of phosphorylation were greatest at this site in both components when compared with the other sites. The rate of phosphorylation of peptide 5-13 was approximately 10 times greater than that of any of the other peptides derived from C-1, while the rate of phosphorylation of peptide 5-13 derived from C-4 was 10-20 times greater than that of any of the other peptides derived from C-4. In addition, peptide 5-13, which contained a major phosphorylation site in both C-1 and C-4, was phosphorylated at a faster rate in C-4 (460 cpm/nM/min) compared with C-1 (285 cpm/nM/min). Both the specific activity and the rate data presented in the present communication were correlated with the proportion of beta-structure in a previous study. In that study, C-1, which contained about 13% beta-structure before phosphorylation, increased to approximately 40% after phosphorylation. Construction of a model peptide of this N-terminal region, which included the phosphorylation site at Ser7, demonstrated that the beta-structure was stabilized by electrostatic interactions between the phosphate on Ser7 and the guanidyl groups of Arg5 and Arg9.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sterol C-14 reductase encoded by the Neurospora crassa erg-3 gene: essential charged and polar residues identified by site-specific mutagenesis

Sterol C-14 reductase catalyses the reduction of the Delta(14,15) bond in intermediates in the sterol biosynthesis pathway using NADPH as a cofactor. We have undertaken a systematic site-directed mutational analysis of all the conserved charged and potentially proton-donating residues of the sterol C-14 reductase from Neurospora crassa. The effect of each mutation was determined using an in vivo assay based on the complementation of the corresponding N. crassa mutant ( erg-3). The non-complementing mutations were also tested in the erg24 mutant of Saccharomyces cervisiae. The results are discussed with reference to the predicted topology of the enzyme and to its proposed catalytic mechanism, which involves addition of a proton from an appropriately positioned charged or polar residue to the substrate double bond, followed by addition of hydride ion from NADPH.     

Characterization of two polyketide methyltransferases involved in the biosynthesis of the antitumor drug mithramycin by Streptomyces argillaceus

A DNA chromosomal region of Streptomyces argillaceus ATCC 12596, the producer organism of the antitumor polyketide drug mithramycin, was cloned. Sequence analysis of this DNA region, located between four mithramycin glycosyltransferase genes, showed the presence of two genes (mtmMI and mtmMII) whose deduced products resembled S-adenosylmethionine-dependent methyltransferases. By independent insertional inactivation of both genes nonproducing mutants were generated that accumulated different mithramycin biosynthetic intermediates. The M3DeltaMI mutant (mtmMI-minus mutant) accumulated 4-demethylpremithramycinone (4-DPMC) which lacks the methyl groups at carbons 4 and 9. The M3DeltaM2 (mtmMII-minus mutant) accumulated 9-demethylpremithramycin A3 (9-DPMA3), premithramycin A1 (PMA1), and 7-demethylmithramycin, all of them containing the O-methyl group at C-4 and C-1', respectively, but lacking the methyl group at the aromatic position. Both genes were expressed in Streptomyces lividans TK21 under the control of the erythromycin resistance promoter (ermEp) of Saccharopolyspora erythraea. Cell-free extracts of these clones were precipitated with ammonium sulfate (90% saturation) and assayed for methylation activity using different mithramycin intermediates as substrates. Extracts of strains MJM1 (expressing the mtmMI gene) and MJM2 (expressing the mtmMII gene) catalyzed efficient transfer of tritium from [(3)H]S-adenosylmethionine into 4-DPMC and 9-DPMA3, respectively, being unable to methylate other intermediates at a detectable level. These results demonstrate that the mtmMI and mtmMII genes code for two S-adenosylmethionine-dependent methyltransferases responsible for the 4-O-methylation and 9-C-methylation steps of the biosynthetic precursors 4-DPMC and 9-DPMA3, respectively, of the antitumor drug mithramycin. A pathway is proposed for the last steps in the biosynthesis of mithramycin involving these methylation events.     

Oxidation of methyl derivatives of pteridin-4-one, lumazine and related pteridines by bovine milk xanthine oxidase.

1. Pteridin-4-ones, methylated at nitrogen or carbon, N-methylated lumazines and related oxopteridines were studied as substrates of a highly purified bovine milk xanthine oxidase (xanthine : oxygen oxidoreductase, EC 1.2.3.2). 2. The enzyme can oxidise at high rates both uncharged and anionic substrates. Variation of enzymic activity with pH is mainly due to pH-dependent changes in the active enzymic center. 3. Milk xanthine oxidases at different stages of purification convert pteridin-4-one into the 4,7-dione (compound 13 in this article). 4. Methylation at C-6 in the pyrazine moiety enhances enzymic attack at C-2 in the pyrimidine ring. N-Methylation may increase or reduce rates of oxidation. 5. For oxidation at C-2, the most favorable form of the substrate bears a double bond at C(2) = N(3). Attack at C-7 is enhanced strongly in structures bearing a double bond at C(6) = C(7). 6. In general, pteridines react with xanthine oxidase as non-hydrated molecules. However, oxidation of 8-methyllumazine at C-7 may take place by dehydrogenation of the 7-CHOH group of the covalently hydrated molecule.