Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase

Biochem J. 1997 Apr 15;323 ( Pt 2)(Pt 2):365-70. doi: 10.1042/bj3230365.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Northern
  • Carbohydrate Dehydrogenases / chemistry
  • Carbohydrate Dehydrogenases / genetics*
  • Carbohydrate Dehydrogenases / metabolism
  • Cloning, Molecular
  • DNA, Complementary / chemistry
  • Humans
  • Kinetics
  • Liver / enzymology
  • Molecular Sequence Data
  • Phosphoglycerate Dehydrogenase
  • Polymerase Chain Reaction
  • Potassium Chloride / pharmacology
  • Pyruvates / pharmacology
  • Rats
  • Sequence Alignment
  • Substrate Specificity

Substances

  • DNA, Complementary
  • Pyruvates
  • phosphohydroxypyruvic acid
  • Potassium Chloride
  • Alcohol Oxidoreductases
  • Carbohydrate Dehydrogenases
  • Phosphoglycerate Dehydrogenase
  • 2-hydroxyglutarate dehydrogenase

Associated data

  • GENBANK/X97772