Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenase.
Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species. The CmGPD1 gene was expressed in recombinant Escherichia coli with the maltose-binding protein (MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a K(cat)/K(m) value of 195 min(-1) mM(-1) for dihydroxyacetone phosphate whereas K(cat)/K(m) for glycerol-3-phosphate is 0.385 min(-1) mM(-1). In a complementation study, CmGpd1p rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces cerevisiae GPD1DeltaGPD2Delta mutant strain. The overall results indicated that CmGPD1 encodes a functional homologue of S. cerevisiae GPDH.
Lee
Dae-Hee
DH
Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
Kim
Myoung-Dong
MD
Ryu
Yeon-Woo
YW
Seo
Jin-Ho
JH
D000595:Amino Acid Sequence; D002175:Candida / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D005990:Glycerol / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology
D002352:Carrier Proteins; D005656:Fungal Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D005993:Glycerolphosphate Dehydrogenase; D005990:Glycerol