Expression of a metagenome-derived fumarate reductase from marine microorganisms and its characterization
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Enzyme Activators metabolism MeSH
- Escherichia coli genetics MeSH
- Gene Expression MeSH
- Fumarates metabolism MeSH
- Phylogeny MeSH
- Magnesium metabolism MeSH
- Kinetics MeSH
- Cloning, Molecular MeSH
- Hydrogen-Ion Concentration MeSH
- Succinic Acid metabolism MeSH
- Metagenome * MeSH
- Molecular Sequence Data MeSH
- Oxidation-Reduction MeSH
- Recombinant Proteins genetics metabolism MeSH
- Sequence Analysis, DNA MeSH
- Sequence Homology, Amino Acid MeSH
- Enzyme Stability MeSH
- Succinate Dehydrogenase chemistry genetics metabolism MeSH
- Temperature MeSH
- Aquatic Organisms MeSH
- Chromatography, High Pressure Liquid MeSH
- Zinc metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Enzyme Activators MeSH
- Fumarates MeSH
- Magnesium MeSH
- Succinic Acid MeSH
- Recombinant Proteins MeSH
- Succinate Dehydrogenase MeSH
- Zinc MeSH
A potential novel fumarate reductase gene designated frd1A was isolated by screening a marine metagenomic library through a sequence-based strategy. Sequence analyses indicated that Frd1A and other putative fumarate reductases were closely related. The putative fumarate reductase gene was subcloned into a pETBlue-2 vector and expressed in Escherichia coli Tuner(DE3)pLacІ cells. The recombinant protein was purified to homogeneity. Functional characterization by high-performance liquid chromatography demonstrated that the recombinant Frd1A protein could catalyze the hydrogenation of fumarate to succinate acid. The Frd1A protein displayed an optimal activity at pH 7.0 and 28 °C, which could be stimulated by adding metal ions such as Zn(2+) and Mg(2+). The Frd1A enzyme showed a comparable affinity and catalytic efficiency under optimal reaction conditions: k m =0.227 mmol/L, v max= 29.9 U/mg, and k cat/k m=5.44 × 10(4) per mol/s. The identification of Frd1A protein underscores the potential of marine metagenome screening for novel biomolecules.
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Metab Eng. 2011 May;13(3):328-35 PubMed
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Mol Biol Evol. 2011 Oct;28(10):2731-9 PubMed
PLoS Pathog. 2011 Oct;7(10):e1002287 PubMed
Mol Biochem Parasitol. 2002 Jul;122(2):189-200 PubMed
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Anal Biochem. 1987 Nov 1;166(2):368-79 PubMed
Biochim Biophys Acta. 2002 Jan 17;1553(1-2):140-57 PubMed
Mar Drugs. 2010 Mar 15;8(3):608-28 PubMed
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