Investigation of Substrate Binding and Product Stereochemistry Issues in Two Linoleate 9‐Lipoxygenases

Boeglin, William E.; Itoh, Aya; Zheng, Yuxiang; Coffa, Gianguido; Howe, Gregg A.; Brash, Alan R.

doi:10.1007/s11745-008-3230-1

Cited by 33 publications

(24 citation statements)

References 39 publications

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“…The tomato CYP74C3 plasmid was s-cis conformation 9 a gift of Dr. Gregg Howe (Michigan State University) and was expressed and purified as described (19). The guayule CYP74A2 in plasmid pET28b was a gift of Drs.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3

Brash¹,

Boeglin²,

Stec

et al. 2013

Journal of Biological Chemistry

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Background: Allene oxides involved in cyclopentenone biosynthesis are extremely labile and have eluded full stereochemical assignment. Results: We identify a novel Z-allene oxide that unexpectedly rearranges to a cyclopentenone. Conclusion: Other natural allene oxides are assigned the E configuration and can be easily distinguished from the Z-allene oxide by NMR. Significance: Unequivocal determination of the unknown allene oxide configuration is documented and helps elucidate the cyclization chemistry.

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Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3

Brash¹,

Boeglin²,

Stec

et al. 2013

Journal of Biological Chemistry

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“…The same mode of binding has been described for 13S-LOXs (53, 54), 9R-LOXs (23,44), and Mn-LOX (51). On the other hand, a number of linoleate 9S-LOX and arachidonate 8S-and 5S-LOX cannot metabolize such substrates (55)(56)(57) and have been thought to bind fatty acids with the carboxylic end buried in the active site pocket. The production of the bisallylic product from esterified substrate has also been reported for Mn-LOX (51).…”

Section: Experiments With Labeled [(11s)-mentioning

confidence: 64%

A Bisallylic Mini-lipoxygenase from Cyanobacterium Cyanothece sp. That Has an Iron as Cofactor

Andreou¹,

Göbel²,

Hámberg

et al. 2010

Journal of Biological Chemistry

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Lipoxygenases are enzymes that are found ubiquitously in higher animals and plants, but have only recently been identified in a number of bacteria. The genome of the diazotrophic unicellular cyanobacterium Cyanothece sp. harbors two genes with homology to lipoxygenases. Here we describe the isolation of one gene, formerly named csplox2. It was cloned, and the protein was expressed in Escherichia coli and purified. The purified enzyme belongs to the group of prokaryotic mini lipoxygenases, because it had a molecular mass of 65 kDa. Interestingly, it catalyzed the conversion of linoleic acid, the only endogenously found polyunsaturated fatty acid, primarily to the bisallylic hydroperoxide 11R-hydroperoxyoctadecadienoic acid. This product had previously only been described for the manganese lipoxygenase from the take all fungus, Gaeumannomyces graminis. By contrast, CspLOX2 was shown to be an iron lipoxygenase. In addition, CspLOX2 formed a mixture of typical conjugated lipoxygenase products, e.g. 9R-and 13S-hydroperoxide. The conversion of linoleic acid took place with a maximum reaction rate of 31 s ؊1 . Incubation of the enzyme with [(11S)-2 H]linoleic acid led to the formation of hydroperoxides that had lost the deuterium label, thus suggesting that CspLOX2 catalyzes antarafacial oxygenation as opposed to the mechanism of manganese lipoxygenase. CspLOX2 could also oxidize diarachidonylglycerophosphatidylcholine with similar specificity as the free fatty acid, indicating that binding of the substrate takes place with a "tail-first" orientation. We conclude that CspLOX2 is a novel iron mini-lipoxygenase that catalyzes the formation of bisallylic hydroperoxide as the major product.

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“…The protein was expressed in Escherichia coli BL21 (DE3) cells and purifi ed by nickel affi nity chromatography according to a previously published protocol ( 22 ).…”

Section: Expression and Purifi Cation Of Human 15-lox-1 And Arabidopsmentioning

confidence: 99%

“…With the requirement that the LOX enzyme used in these preparations should have very high catalytic activity (which our available preparations of mammalian 5-LOX did not), we switched to the use of recombinant Arabidopsis AtLOX1 as the arachidonate 5-LOX ( 22 ). This enzyme is a homolog of the potato 5-LOX ( 22 ) that is capable of LTA epoxide synthesis and has been used as a model for the study of 5-LOX-catalyzed leukotriene synthesis in earlier studies ( 7,29,30 ).…”

Section: Leukotriene Epoxide Formation From Enantiomeric Hpete Substrmentioning

confidence: 99%

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Jin

Zheng²,

Boeglin

et al. 2013

Journal of Lipid Research

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The biosynthetic pathway, unraveled three decades ago, is a model for related transformations to anti-infl ammatory mediators including the lipoxins, eoxins, resolvins, protectins, and maresins ( 2-5 ). 5-Lipoxygenase (5-LOX) catalyzes the fi rst two steps in leukotriene biosynthesis, namely the stereospecifi c oxygenation of arachidonic acid to 5 S -hydroperoxy-eicosatetraenoic acid (5 S -HPETE), and dehydration of the hydroperoxide to LTA 4 , the allylic 5,6-trans -epoxide from which the bioactive leukotrienes are derived ( Fig. 1 ) ( 6-9 ). The importance of this pivotal intermediate is refl ected in the extensive efforts directed toward perfecting the total synthesis of LTA 4 and of closely related 5,6-epoxide isomers (as reviewed in Refs. 10-12 ).LTA 4 is highly unstable in physiological buffer at pH 7.4, with a half-life estimated as approximately 3 s at 25°C and 18 s at 4°C ( 13 ). Despite this instability, LTA 4 was detected as an evanescent intermediate in short-term incubations of arachidonic acid with human leukocytes ( 6 ), and the radiolabeled product was isolated and, as the methyl ester derivative, shown to have similar chromatographic mobility and hydrolysis under acidic conditions as a synthetic standard ( 14 ). The stereochemistry has heretofore not been established directly on the biosynthetic product but was deduced from an understanding of the biosynthetic pathway and by study of the reactions of synthetic LTA 4 and related isomers in transformations to the stable, bioactive leukotrienes LTB 4 and LTC 4 ( 15-17 ). Herein we report the isolation and purifi cation of LTA epoxides as products of LOX enzymes in quantities suffi cient for NMR analysis of the stereoconfi gurations. The isolation method Abstract Leukotriene (LT)A 4 and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA 4 is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide confi guration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities suffi cient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15 S -hydroperoxy-eicosatetraenoic acid (15 S -HPETE) to the LTA analog 14 S ,15 S -trans -epoxy-eicosa-5 Z ,8 Z ,10 E ,12 E -tetraenoate, confi rming the proposed structure of eoxin A 4 . Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5 S -HPETE to the trans epoxide LTA 4 and converts 5 R -HPETE to the cis epoxide 5-epi -LTA 4 , establishing substrate chirality as a determinant of the cis or trans epoxide confi guration.The results are reconciled...

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Investigation of Substrate Binding and Product Stereochemistry Issues in Two Linoleate 9‐Lipoxygenases

Cited by 33 publications

References 39 publications

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3

A Bisallylic Mini-lipoxygenase from Cyanobacterium Cyanothece sp. That Has an Iron as Cofactor

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

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