General biochemical synthesis of oxygenated prostaglandins E

Sih, Charles J.; Ambrus, Gábor; Foss, Paul; Lai, Chin-Chun

doi:10.1021/ja01041a065

Cited by 31 publications

(4 citation statements)

References 2 publications

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“…P450 monooxygenases that oxygenate carboxylic acids with high chemo-, regio-, and stereoselectivity are widely distributed (2)(3)(4)(5), and metabolites formed from such reactions are of significant structural and physiological importance (6,7). P450 BM-3 3 (CYP102) and its mutant F87A used in this study are water-soluble heme enzymes of M r 120 kDa which contain a FAD-and FMN-dependent reductase domain on the same polypeptide chain.…”

mentioning

confidence: 99%

A Continuous Spectrophotometric Assay for P450 BM-3, a Fatty Acid Hydroxylating Enzyme, and Its Mutant F87A

Schwaneberg

Schmidt‐Dannert

Schmitt

et al. 1999

Analytical Biochemistry

145

109

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mentioning

confidence: 99%

A Continuous Spectrophotometric Assay for P450 BM-3, a Fatty Acid Hydroxylating Enzyme, and Its Mutant F87A

Schwaneberg

Schmidt‐Dannert

Schmitt

et al. 1999

Analytical Biochemistry

145

109

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“…This enzyme (diol synthase) metabolizes linoleate sequentially to 8-HPODE 1 and (7S,8S)-dihydroxyoctadeca-(9Z,12Z)-dienoic acid (21,22). Mycelia of G. graminis also contain NADPH-dependent monooxygenases, which oxygenate polyunsaturated fatty acids at the 2 and 3 carbons to R-hydroxy fatty acids (23)(24)(25). We now report that filtrate of the culture medium of G. graminis contains a linoleate dioxygenase, which appears to be secreted by the fungus in large quantities.…”

mentioning

confidence: 99%

Manganese Lipoxygenase

Oliw

1998

Journal of Biological Chemistry

165

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A linoleic acid (13R)-lipoxygenase was purified to homogeneity from the culture medium of Gä umannomyces graminis, the take-all fungus, by hydrophobic interaction, cation exchange, lectin affinity, and size-exclusion chromatography. The purified dioxygenase lacked light absorption between 300 and 700 nm. Gel filtration indicated an apparent molecular mass of ϳ135 kDa in 6 M urea and ϳ160 kDa in buffer. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was heterogeneous in size and consisted of diffuse protein bands of 100 -140 kDa. Treatment with glycosidases for N-and O-linked oligosaccharides yielded a distinct protein of ϳ73 kDa on SDS-PAGE. Atomic emission spectroscopy indicated 0.5-1.0 manganese atom/enzyme molecule. The isoelectric point was ϳ9.7, and the enzyme was active between pH 5 and 11 with optimum activity at pH 7.0. For molecular oxygen, K m was 30 M and V max 10 mol mg . The enzyme oxidized linolenic acid twice as fast as linoleic acid. The main products were identified by mass spectrometry as 13-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic and 13-hydroperoxy-(9Z,11E)-octadecadienoic acids, respectively. After reduction of the hydroperoxide, steric analysis of methyl 13-hydroxyoctadecadienoate by chiral high performance liquid chromatography yielded one enantiomer (>95%), which co-eluted with the R-stereoisomer of methyl (13R,13S)-hydroxyoctadecadienoate. Arachidonic and dihomogammalinolenic acids were not substrates, while oxygen consumption, UV analysis, and mass spectrometric analysis indicated that ␥-linolenic acid was oxygenated both at C-11 and C-13. The enzyme was active at 60°C and after treatment with 6 M urea. It was strongly inhibited by 10 -50 M concentrations of eicosatetraynoic acid and a lipoxygenase inhibitor (N-(3-phenoxycinnamyl)acetohydroxamic acid), but many other lipoxygenase inhibitors (100 M) were without effect. We conclude that, after deglycosylation, the enzyme has the same size on SDS-PAGE as mammalian and marine lipoxygenases, but it differs from all previously described lipoxygenases in three ways. It is secreted, it forms (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid, and it contains manganese.

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“…In the field of pesticides, studies of metabolism of halogenated aromatic hydrocarbons by microorganisms (472) and structures of metabolites of aldrin and dieldrin ( 473) have been aided by mass spectrometry. Metabolism of prostaglandins has received considerable attention with the use of mass spectrometry (474)(475)(476)(477)(478)(479). For example, the structures of the major urinary metabolites of prostaglandin E2 in man (474) and in guinea pig (476) and of prostaglandin F2q in man (475) and in the rat (478) have been determined.…”

Section: Sulfur-containingmentioning

confidence: 99%

Mass spectrometry

DeJongh¹

1970

Anal. Chem.

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General biochemical synthesis of oxygenated prostaglandins E

Cited by 31 publications

References 2 publications

A Continuous Spectrophotometric Assay for P450 BM-3, a Fatty Acid Hydroxylating Enzyme, and Its Mutant F87A

A Continuous Spectrophotometric Assay for P450 BM-3, a Fatty Acid Hydroxylating Enzyme, and Its Mutant F87A

Manganese Lipoxygenase

Mass spectrometry

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