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Functional characterization of a novel arachidonic acid 12S-lipoxygenase in the halotolerant bacterium Myxococcus fulvus exhibiting complex social living patterns.

ABSTRACT: Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and mammals. These enzymes are also expressed in lower multicellular organisms but here they are not widely distributed. In bacteria, lipoxygenases rarely occur and evaluation of the currently available bacterial genomes suggested that <0.5% of all sequenced bacterial species carry putative lipoxygenase genes. We recently rescreened the public bacterial genome databases for lipoxygenase-like sequences and identified two novel lipoxygenase isoforms (MF-LOX1 and MF-LOX2) in the halotolerant Myxococcus fulvus. Both enzymes share a low degree of amino acid conservation with well-characterized eukaryotic lipoxygenase isoforms but they involve the catalytically essential iron cluster. Here, we cloned the MF-LOX1 cDNA, expressed the corresponding enzyme as N-terminal hexa-his-tag fusion protein, purified the recombinant enzyme to electrophoretic homogeneity, and characterized it with respect to its protein-chemical and enzymatic properties. We found that M. fulvus expresses a catalytically active intracellular lipoxygenase that converts arachidonic acid and other polyunsaturated fatty acids enantioselectively to the corresponding n-9 hydroperoxy derivatives. The enzyme prefers C20 - and C22 -polyenoic fatty acids but does not exhibit significant membrane oxygenase activity. The possible biological relevance of MF-LOX1 will be discussed in the context of the suggested concepts of other bacterial lipoxygenases.

SUBMITTER: Goloshchapova K

PROVIDER: S-EPMC6612559 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Functional characterization of a novel arachidonic acid 12S-lipoxygenase in the halotolerant bacterium Myxococcus fulvus exhibiting complex social living patterns.

Goloshchapova Kateryna K Stehling Sabine S Heydeck Dagmar D Blum Maximilian M Kuhn Hartmut H

MicrobiologyOpen 20181217 7

Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and mammals. These enzymes are also expressed in lower multicellular organisms but here they are not widely distributed. In bacteria, lipoxygenases rarely occur and evaluation of the currently available bacterial genomes suggested that <0.5% of all sequenced bacterial species carry putative lipoxygenase genes. We recently rescreened the public bacterial genome databases for lipoxygenase-like sequences and ident ...[more]

PMID: 30560563

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Project description:BackgroundChitin is an important biopolymer next to cellulose, extracted in the present study. The exoskeleton of marine bycatch brachyuran crabs, namely Calappa lophos, Dromia dehaani, Dorippe facchino and also from stomatopod Squilla spp. were used to extract chitin through fermentation methods by employing two bacterial strains such as Pseudomonas aeruginosa, Serratia marcescens. The yield of chitin was 44.24%, 37.45%, 11.56% and 27.24% in C. lophos, D. dehaani, D. facchino and Squilla spp. respectively. FT-IR spectra of the produced chitin exhibit peaks which is more or less coherent to that of standard chitin which is further analysed by Scanning Electron Microscope. The quality of produced chitin was assessed through moisture, protein, ash and lipid content analysis ensured that chitin obtained from trash crustaceans are on par with that of standard chitin.ResultsA total of 10 samples were collected from different areas of Jiangsu China for screening of chitinase-producing bacteria. Based on the clearance zone, two of the best samples were chosen for further study. 16S rRNA sequence analysis showed that this strain belongs to genus Myxococcus and species Myxococcus fulvus. Phylogenetic analysis was performed and it shows strain UM01 is a novel bacterial strain. UM01 isolate shows maximum chitinase production at 35 °C and 8 pH. Among all, these colloidal chitins were found to be the best for chitinase production. Three chitinase-producing genes were identified and sequenced by using degenerative plasmid. UMCda gene (chitin disaccharide deacetylase) was cloned into E. coli DH5a by using PET-28a vector, and antagonistic activity was examined against T. reesei.ConclusionTo our knowledge, this is the earliest study report to gene cloning and identification of the chitinase gene in Myxococcus fulvus. Chitinase plays a key role in decomposition and utilization of chitin as a raw material. This research indicates that Myxococcus fulvus UM01 strain is a novel myxobacteria strain and can produce large amounts of chitinase within a short time. The UMCda gene cloned into E. coli DH5a showed a promising effect as antifungal activity. In overall findings, the specific strain UM01 has endowed properties of bioconversation of waste chitin and other biological applications.

Project description:The arachidonic acid lipoxygenase 15B (ALOX15B) orthologs of men and mice form different reaction products when arachidonic acid is used as the substrate. Tyr603Asp+His604Val double mutation in mouse arachidonic acid lipoxygenase 15b humanized the product pattern and an inverse mutagenesis strategy murinized the specificity of the human enzyme. As the mechanistic basis for these functional differences, an inverse substrate binding at the active site of the enzymes has been suggested, but experimental proof for this hypothesis is still pending. Here we expressed wildtype mouse and human arachidonic acid lipoxygenase 15B orthologs as well as their humanized and murinized double mutants as recombinant proteins and analyzed the product patterns of these enzymes with different polyenoic fatty acids. In addition, in silico substrate docking studies and molecular dynamics simulation were performed to explore the mechanistic basis for the distinct reaction specificities of the different enzyme variants. Wildtype human arachidonic acid lipoxygenase 15B converted arachidonic acid and eicosapentaenoic acid to their 15-hydroperoxy derivatives but the Asp602Tyr+Val603His exchange murinized the product pattern. The inverse mutagenesis strategy in mouse arachidonic acid lipoxygenase 15b (Tyr603Asp+His604Val exchange) humanized the product pattern with these substrates, but the situation was different with docosahexaenoic acid. Here, Tyr603Asp+His604Val substitution in mouse arachidonic acid lipoxygenase 15b also humanized the specificity but the inverse mutagenesis (Asp602Tyr+Val603His) did not murinize the human enzyme. With linoleic acid Tyr603Asp+His604Val substitution in mouse arachidonic acid lipoxygenase 15b humanized the product pattern but the inverse mutagenesis in human arachidonic acid lipoxygenase 15B induced racemic product formation. Amino acid exchanges at critical positions of human and mouse arachidonic acid lipoxygenase 15B orthologs humanized/murinized the product pattern with C20 fatty acids, but this was not the case with fatty acid substrates of different chain lengths. Asp602Tyr+Val603His exchange murinized the product pattern of human arachidonic acid lipoxygenase 15B with arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. An inverse mutagenesis strategy on mouse arachidonic acid lipoxygenase 15b (Tyr603Asp+His604Val exchange) did humanize the reaction products with arachidonic acid and eicosapentaenoic acid, but not with docosahexaenoic acid.

Dataset Information

Functional characterization of a novel arachidonic acid 12S-lipoxygenase in the halotolerant bacterium Myxococcus fulvus exhibiting complex social living patterns.

Publications

Functional characterization of a novel arachidonic acid 12S-lipoxygenase in the halotolerant bacterium Myxococcus fulvus exhibiting complex social living patterns.

Similar Datasets

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