Purification and characterization of mannitol dehydrogenase and identification of the corresponding cDNA from the head blight fungus, Gibberella zeae (Fusarium graminearum).
The mannitol-2-dehydrogenase (MtDH) from Gibberella zeae was purified and the corresponding cDNA identified. Purification of MtDH was accomplished using a combination of ammonium sulfate fractionation, anion exchange and dye-ligand chromatography. Final purification was achieved following electroelution from a native gel. Molecular mass determination based on SDS-PAGE indicated that the denatured protein was 29 kDa. Native protein mass was determined to be 110 kDa using gel permeation chromatography, indicating a tetrameric form. The pH optima for mannitol oxidation and fructose reductase activities were 9.0, and 7.0, respectively. Activity with sorbitol as the substrate was 21% of activity with mannitol. Kinetic parameters were determined by direct-linear plots of enzyme activity vs. substrate concentrations. Fructose concentrations above 600 mM and NADPH concentrations above 0.3 mM caused substrate inhibition. Comparisons of predicted amino acid sequences of several fungal MtDHs indicated high conservation within the phyla. A possible role for MtDH in generation of turgor pressure for forcible ascospore discharge is discussed.
Trail
Frances
F
Department of Plant Biology, Michigan State University, East Lansing, MI 48824-1312, USA. trail@msu.edu
Xu
Haixin
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D005591:Chemical Fractionation / Q000379:methods; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005632:Fructose / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005874:Gibberella / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D008354:Mannitol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013012:Sorbitol / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D005632:Fructose; D008353:Mannitol; D013012:Sorbitol; D008354:Mannitol Dehydrogenases