Abstract
Phytomonas sp. contains two malate dehydrogenase isoforms, a mitochondrial isoenzyme with a high specificity for oxaloacetate and a glycosomal isozyme that acts on a broad range of substrates (Uttaro, A. D., and Opperdoes, F.R. (1997) Mol. Biochem. Parasitol. 89, 51-59). Here, we show that the low specificity of the latter isoenzyme is the result of a number of recent gene duplications that gave rise to a family of glycosomal 2-hydroxyacid dehydrogenase genes. Two of these genes were cloned, sequenced, and overexpressed in Escherichia coli. Although both gene products have 322 amino acids, share 90.4% identical residues, and have a similar hydrophobicity profile and net charge, their kinetic properties were strikingly different. One isoform behaved as a real malate dehydrogenase with a high specificity for oxaloacetate, whereas the other showed no activity with oxaloacetate but was able to reduce other oxoacids, such as phenyl pyruvate, 2-oxoisocaproate, 2-oxovalerate, 2-oxobutyrate, 2-oxo-4-methiolbutyrate, and pyruvate.
MeSH terms
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Alcohol Oxidoreductases / chemistry
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Alcohol Oxidoreductases / genetics
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Alcohol Oxidoreductases / isolation & purification
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Alcohol Oxidoreductases / metabolism*
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Amino Acid Sequence
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Animals
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Blotting, Southern
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Cloning, Molecular
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Conserved Sequence / genetics*
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Gene Dosage
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Genes, Duplicate / genetics
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Genes, Protozoan / genetics
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Isoenzymes / chemistry
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Isoenzymes / genetics
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Isoenzymes / isolation & purification
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Isoenzymes / metabolism
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Kinetics
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Malate Dehydrogenase / chemistry
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Malate Dehydrogenase / genetics
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Malate Dehydrogenase / isolation & purification
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Malate Dehydrogenase / metabolism
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Microbodies / enzymology*
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Molecular Sequence Data
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Multigene Family / genetics*
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Oxaloacetates / metabolism
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Phylogeny
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Recombinant Proteins / metabolism
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Sequence Alignment
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Sequence Analysis, DNA
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Substrate Specificity
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Trypanosomatina / enzymology*
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Trypanosomatina / genetics
Substances
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Isoenzymes
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Oxaloacetates
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Recombinant Proteins
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Alcohol Oxidoreductases
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2-hydroxyacid dehydrogenase
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Malate Dehydrogenase
Associated data
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GENBANK/AF284096
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GENBANK/AF284097