A novel hyperthermophilic archaeal glyoxylate reductase from Thermococcus litoralis. Characterization, gene cloning, nucleotide sequence and expression in Escherichia coli

Eur J Biochem. 2001 Sep;268(17):4740-7. doi: 10.1046/j.1432-1327.2001.02394.x.


A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified approximately 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of approximately 37 kDa. The optimum pH and temperature for enzyme activity were approximately 6.5 and 90 degrees C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 degrees C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was approximately one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36,807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed.

MeSH terms

  • Alcohol Oxidoreductases / genetics*
  • Alcohol Oxidoreductases / isolation & purification
  • Alcohol Oxidoreductases / metabolism
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Coenzymes / metabolism
  • DNA, Archaeal / analysis
  • Escherichia coli
  • Genomic Library
  • Molecular Sequence Data
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Thermococcus / enzymology*
  • Thermococcus / genetics


  • Coenzymes
  • DNA, Archaeal
  • Recombinant Proteins
  • Alcohol Oxidoreductases
  • glyoxylate reductase

Associated data

  • GENBANK/AB033995