On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant

Cristea, Mirela; Oliw, Ernst H.

doi:10.1194/jlr.m600505-jlr200

Cited by 14 publications

(15 citation statements)

References 42 publications

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“…1B. Analysis of the products during the rapid and almost linear increase in UV absorbance at 235 nm, which is attributable mainly to the formation of 15R-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HPETE) (26), showed that 13-hydroperoxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (13-HPETE) accounted for ?10-14% of the products during the upper part of the linear phase. This number was below 1% already at the peak of UV absorbance and was ,0.1% at later time points (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…20:4n-6 is a poor substrate of Mn-LOX and required large amounts of enzyme for rapid transformation (25,26). Oxidation of 20:4n-6 by Mn-LOX and analysis of the products are illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The heated transfer capillary was set at 315jC, the ion isolation width at 1.5 amu, and the collision energy at 25- Normal phase (NP)-HPLC with MS/MS analysis was performed on silica with an analytical column (Kromasil-100SI; 250 3 2 mm, 5 mm, 100 Å ), which was eluted at 0.3 ml/min with 5% isopropanol in hexane with 0.05 ml/l acetic acid (Constrametric 3200 pump; LDC). The effluent was combined with isopropanolwater (3:2; 0.2 ml/min) from a second pump (Surveyor MS pump) as described (26). The combined effluents were introduced by electrospray into the ion trap mass spectrometer (LTQ).…”

Section: Hplc-ms/msmentioning

confidence: 99%

“…The first goal was to confirm that the fatty acids bind "tail-first" in the active site of Mn-LOX during rearrangement in the same way as during oxygenation (26). The second goal was to determine the influence of the Gly316Ala mutant on the rearrangement reaction.…”

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confidence: 99%

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Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase

Oliw

2008

Journal of Lipid Research

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Manganese lipoxygenase (Mn-LOX) catalyzes the rearrangement of bis-allylic S-hydroperoxides to allylic Rhydroperoxides, but little is known about the reaction mechanism. 1-Linoleoyl-lysoglycerophosphatidylcholine was oxidized in analogy with 18:2n-6 at the bis-allylic carbon with rearrangement to C-13 at the end of lipoxygenation, suggesting a "tail-first" model. The rearrangement of bisallylic hydroperoxides was influenced by double bond configuration and the chain length of fatty acids. The Gly316Ala mutant changed the position of lipoxygenation toward the carboxyl group of 20:2n-6 and 20:3n-3 and prevented the bis-allylic hydroperoxide of 20:3n-3 but not 20:2n-6 to interact with the catalytic metal. The oxidized form, Mn III -LOX, likely accepts an electron from the bis-allylic hydroperoxide anion with the formation of the peroxyl radical, but rearrangement of 11-hydroperoxyoctadecatrienoic acid by Mn-LOX was not reduced in D 2 O (pD 7.5), and aqueous Fe 31 did not transfer 11S-hydroperoxy-9Z,12Z,15Z-octadecatrienoic acid to allylic hydroperoxides. Mutants in the vicinity of the catalytic metal, Asn466Leu and Ser469Ala, had little influence on bis-allylic hydroperoxide rearrangement. In conclusion, Mn-LOX transforms bis-allylic hydroperoxides to allylic by a reaction likely based on the positioning of the hydroperoxide close to Mn 31 and electron transfer to the metal, with the formation of a bis-allylic peroxyl radical, b-fragmentation, and oxygenation under steric control by the protein.-Oliw, E. H. Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase. J. Lipid Res. 2008. 49: 420-428.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Hplc-ms/msmentioning

confidence: 99%

mentioning

confidence: 99%

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Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase

Oliw

2008

Journal of Lipid Research

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“…The effluents from the column were combined with isopropyl alcohol/water (3/2) in a ratio of ~2:1 from a second HPLC pump (21) and then introduced by electrospray into a linear ion trap mass spectrometer (LTQ, Thermo) with analysis of carboxylate anions. The ion isolation width was set at 5 for anions of HPOME (m/z 313→ full scan) in the first selection and 1.5 at the final selection of MS 3 analysis of HPOME (m/z 313 → 295 → full scan).…”

Section: Analytical Cp-hplc Separations With In-line Lc-ms Analysismentioning

confidence: 99%

Chiral Phase-HPLC Separation of Hydroperoxyoctadecenoic Acids and Their Biosynthesis by Fatty Acid Dioxygenases

Oliw

Wennman

2014

Advanced Protocols in Oxidative Stress III

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PostprintThis is the accepted version of a paper published in Methods in Molecular Biology. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the original published paper (version of record):Oliw, E., Wennman, A. (2015) Chiral phase-HPLC separation of hydroperoxyoctadecenoic acids and their biosynthesis by fatty acid dioxygenases.. A stationary phase, Reprosil Chiral NR, was found to resolve these hydroperoxy fatty acids with 1-hydroperoxy-2-propene and with 3-hydroperoxy-1-propene elements so that the S hydroperoxy fatty acids consistently eluted before the R stereoisomers. The chiral selector has not been disclosed, but it is described as an aromatic chiral phase with π-donor and π-acceptor groups of Pirkle type. The MS 3 spectra of the hydroperoxides showed characteristic fragments, which were influenced by the distance between the hydroperoxy and the carboxyl groups and the relative position of the double bond. Methods in Molecular BiologyOctadecenoic fatty acids can be oxidized by fungal and bacterial dioxygenases to hydroperoxides with cis or trans double bond configuration. Steric analysis of the hydroperoxy metabolites can be performed by this method, and it can also be used for preparative purposes.

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A novel class of fungal lipoxygenases

Heshof

Jylhä

Haarmann

et al. 2013

Appl Microbiol Biotechnol

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Lipoxygenases (LOXs) are well-studied enzymes in plants and mammals. However, fungal LOXs are less studied. In this study, we have compared fungal LOX protein sequences to all known characterized LOXs. For this, a script was written using Shell commands to extract sequences from the NCBI database and to align the sequences obtained using Multiple Sequence Comparison by Log-Expectation. We constructed a phylogenetic tree with the use of Quicktree to visualize the relation of fungal LOXs towards other LOXs. These sequences were analyzed with respect to the signal sequence, C-terminal amino acid, the stereochemistry of the formed oxylipin, and the metal ion cofactor usage. This study shows fungal LOXs are divided into two groups, the Ile- and the Val-groups. The Ile-group has a conserved WRYAK sequence that appears to be characteristic for fungal LOXs and has as a C-terminal amino acid Ile. The Val-group has a highly conserved WL-L/F-AK sequence that is also found in LOXs of plant and animal origin. We found that fungal LOXs with this conserved sequence have a Val at the C-terminus in contrast to other LOXs of fungal origin. Also, these LOXs have signal sequences implying these LOXs will be expressed extracellularly. Our results show that in this group, in addition to the Gaeumannomyces graminis and the Magnaporthe salvinii LOXs, the Aspergillus fumigatus LOX uses manganese as a cofactor.

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On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant

Cited by 14 publications

References 42 publications

Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase

Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase

Chiral Phase-HPLC Separation of Hydroperoxyoctadecenoic Acids and Their Biosynthesis by Fatty Acid Dioxygenases

A novel class of fungal lipoxygenases

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