| 1. |
- Alberti, Jean-Christophe, et al.
(författare)
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A functional role identified for conserved charged residues at the active site entrance of lipoxygenase with double specificity
- 2016
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 123, s. 167-173
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Tidskriftsartikel (refereegranskat)abstract
- Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of free polyunsaturated fatty acids, producing 9-hydroperoxides or 13-hydroperoxides from linoleic and alpha-linolenic acids, and are called 9-LOX or 13-LOX, respectively. Some LOXs produce both 9- and 13- hydroperoxides. The models proposed to explain the reaction mechanism specificity fail to explain the "double specificity" character of these LOXs. In this study, we used the olive LOX1 with double specificity to investigate the implication of the charged residues R265, R268, and K283 in the orientation of the substrate into the active site. These residues are present in a conserved pattern around the entrance of the active site. Our results show that these residues are involved in the penetration of the substrate into the active site: this positive patch could capture the carboxylate end of the substrate, and then guide it into the active site. Due to its position on alpha 2 helix, the residue K283 could have a more important role, its interaction with the substrate facilitating the motions of residues constituting the "cork of lipoxygenases" or the alpha 2 helix, by disrupting putative hydrogen and ionic bonds.
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| 2. |
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| 4. |
- Chen, Yang, et al.
(författare)
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Crystal structure of linoleate 13R-manganese lipoxygenase in complex with an adhesion protein.
- 2016
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Ingår i: Journal of Lipid Research. - 0022-2275 .- 1539-7262. ; 57:8, s. 1574-1588
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of 13R-manganese lipoxygenase (MnLOX) of Gaeumannomyces graminis (Gg) in complex with zonadhesin of Pichia pastoris was solved by molecular replacement. Zonadhesin contains β-strands in two subdomains. A comparison of Gg-MnLOX with the 9S-MnLOX of Magnaporthe oryzae (Mo) shows that the protein fold and the geometry of the metal ligands are conserved. The U-shaped active sites differ mainly due to hydrophobic residues of the substrate channel. The volumes and two hydrophobic side pockets near the catalytic base may sanction oxygenation at C-13 and C-9, respectively. Gly-332 of Gg-MnLOX is positioned in the substrate channel between the entrance and the metal center. Replacements with larger residues could restrict oxygen and substrate to reach the active site. C18 fatty acids are likely positioned with C-11 between Mn(2+)OH2 and Leu-336 for hydrogen abstraction and with one side of the 12Z double bond shielded by Phe-337 to prevent antarafacial oxygenation at C-13 and C-11. Phe-347 is positioned at the end of the substrate channel and replacement with smaller residues can position C18 fatty acids for oxygenation at C-9. Gg-MnLOX does not catalyze the sequential lipoxygenation of n-3 fatty acids in contrast to Mo-MnLOX, which illustrates the different configurations of their substrate channels.
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| 5. |
- Chen, Yang, et al.
(författare)
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Purification and site-directed mutagenesis of linoleate 9S-dioxygenase-allene oxide synthase of Fusarium oxysporum confirms the oxygenation mechanism
- 2017
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Ingår i: Archives of Biochemistry and Biophysics. - : ELSEVIER SCIENCE INC. - 0003-9861 .- 1096-0384. ; 625-626, s. 24-29
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Tidskriftsartikel (refereegranskat)abstract
- Plants and fungi form jasmonic acid from a-linolenic acid. The first two steps of biosynthesis in plants occur by sequential transformation by 13S-Iipoxygenase and allene oxide synthase (AOS). The biosynthesis in fungi may follow this classical scheme, but the only fungal AOS discovered so far are cytochromes P450 (CYP) fused to 8- and 9-dioxygenases (DOX). In the present report, we purified recombinant 9S-DOX-AOS of Fusarium oxysporum from cell lysate by cobalt affinity chromatography to near homogeneity and studied key residues by site-directed mutagenesis. Sequence homology with 8R-DOX-linoleate diol synthases (8R-DOX-LDS) suggested that Tyr414 catalyzes hydrogen abstraction and that Cys1051 forms the heme thiolate ligand. Site-directed mutagenesis (Tyr414Phe; Cys1051Ser) led to loss of 9S-DOX and 9S-AOS activities, respectively, but other important residues in the CYP parts of 5,8 and 7,8-LDS or 9R-AOS were not conserved. The UV-visible spectrum of 9S-DOX-AOS showed a Soret band at 409 nm, which shifted to 413 nm in the Cys1051Ser mutant. The 9S-AOS of the Tyr414Phe mutant transformed 9S-hydroperoxides of alpha-linolenic and linoleic acids to allene oxides/alpha-ketols, but it did not transform 13-hydroperoxides. We conclude that 9S- and 8R-DOX catalyze hydrogen abstraction at C-11 and C-8, respectively, by homologous Tyr residues.
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| 8. |
- Cristea, Mirela, 1976-
(författare)
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Expression of Manganese Lipoxygenase and Site-Directed Mutagenesis of Catalytically Important Amino Acids : Studies on Fatty Acid Dioxygenases
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast Pichia pastoris with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).Mn-lipoxygenase is an R-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis. Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.
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| 9. |
- Cristea, Mirela, et al.
(författare)
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Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative manganese ligands
- 2005
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 434:1, s. 201-211
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Tidskriftsartikel (refereegranskat)abstract
- Manganese lipoxygenase is secreted by the fungus Gaeumannomyces graminis. We expressed the enzyme in Pichia pastoris, which secreted approximately 30 mg Mn-lipoxygenase/L culture medium in fermentor. The recombinant lipoxygenase was N- and O-glycosylated (80-100 kDa), contained approximately 1 mol Mn/mol protein, and had similar kinetic properties (K(m) approximately 7.1 microM alpha-linolenic acid and V(max) 18 nmol/min/microg) as the native Mn-lipoxygenase. Mn-lipoxygenase could be quantitatively converted, presumably by secreted Pichia proteases, to a smaller protein (approximately 67 kDa) with retention of lipoxygenase activity (K(m) approximately 6.4 microM alpha-linolenic acid and V(max) approximately 12 nmol/min/microg). Putative manganese ligands were investigated by site-directed mutagenesis. The iron ligands of soybean lipoxygenase-1 are two His residues in the sequence HWLNTH, one His residue and a distant Asn residue in the sequence HAAVNFGQ, and the C-terminal Ile residue. The homologous sequences of Mn-lipoxygenase are H274VLFH278 and H462HVMN466QGS, respectively, and the C-terminal amino acid is Val-602. The His274Gln, His278Glu, His462Glu, and the Val-602 deletion mutants of Mn-lipoxygenase were inactive, and had lost >95% of the manganese content. His-463, Asn-466, and Gln-467 did not appear to be critical for Mn-lipoxygenase activity, as His463Gln, Asn466Gln, Asn466Leu, and Gln467Asn mutants metabolized alpha-linolenic acid to 11- and 13-hydroperoxylinolenic acids. We conclude that His-274, His-278, His-462, and Val-602 likely coordinate manganese.
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