Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase.
Rat liver d-3-phosphoglycerate dehydrogenase was purified to homogeneity and digested with trypsin, and the sequences of two peptides were determined. This sequence information was used to screen a rat hepatoma cDNA library. Among 11 positive clones, two covered the whole coding sequence. The deduced amino acid sequence (533 residues; Mr 56493) shared closer similarity with Bacillus subtilis 3-phosphoglycerate dehydrogenase than with the enzymes from Escherichia coli, Haemophilus influenzae and Saccharomyces cerevisiae. In all cases the similarity was most apparent in the substrate- and NAD+-binding domains, and low or insignificant in the C-terminal domain. A corresponding 2.1 kb mRNA was present in rat tissues including kidney, brain and testis, whatever the dietary status, and also in livers of animals fed a protein-free, carbohydrate-rich diet, but not in livers of control rats, suggesting transcriptional regulation. The full-length rat 3-phosphoglycerate dehydrogenase was expressed in E. coli and purified. The recombinant enzyme and the protein purified from liver displayed hyperbolic kinetics with respect to 3-phosphoglycerate, NAD+ and NADH, but substrate inhibition by 3-phosphohydroxypyruvate was observed; this inhibition was antagonized by salts. Similar properties were observed with a truncated form of 3-phosphoglycerate dehydrogenase lacking the C-terminal domain, indicating that the latter is not implicated in substrate inhibition or in salt effects. By contrast with the bacterial enzyme, rat 3-phosphoglycerate dehydrogenase did not catalyse the reduction of 2-oxoglutarate, indicating that this enzyme is not involved in human D- or L-hydroxyglutaric aciduria.
Achouri
Y
Y
Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology and Université Catholique de Louvain, B-1200 Brussels, Belgium.
Rider
M H
MH
Schaftingen
E V
EV
Robbi
M
M
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D050543:Phosphoglycerate Dehydrogenase; D016133:Polymerase Chain Reaction; D011189:Potassium Chloride / Q000494:pharmacology; D011773:Pyruvates / Q000494:pharmacology; D051381:Rats; D016415:Sequence Alignment; D013379:Substrate Specificity
D018076:DNA, Complementary; D011773:Pyruvates; C012488:phosphohydroxypyruvic acid; D011189:Potassium Chloride; D000429:Alcohol Oxidoreductases; D002237:Carbohydrate Dehydrogenases; D050543:Phosphoglycerate Dehydrogenase; C023858:2-hydroxyglutarate dehydrogenase