Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.
In: European Journal of Biochemistry, Jg. 271 (2004-08-01), Heft 15, S. 3115-3126
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Zugriff:
Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 °C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme. [ABSTRACT FROM AUTHOR]
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Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.
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Autor/in / Beteiligte Person: | Maloney, Alan P. ; Callan, Susan M. ; Murray, Patrick G. ; Tuohy, Maria G. |
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Zeitschrift: | European Journal of Biochemistry, Jg. 271 (2004-08-01), Heft 15, S. 3115-3126 |
Veröffentlichung: | 2004 |
Medientyp: | academicJournal |
ISSN: | 0014-2956 (print) |
DOI: | 10.1111/j.1432-1033.2004.04230.x |
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