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

A novel NADH-dependent glyoxylate reductase has been found in a hyperthermo philic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophili c archaea. NADH-dependent glyoxylate reductase was purified approximate to 560-fold from a crude extract of the hyperthermophile by five successive co lumn chromatographies and preparative PAGE. The molecular mass of the purif ied enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodi mer with a subunit molecular mass of approximate to 37 kDa. The optimum pH and temperature for enzyme activity were approximate to6.5 and 90 degreesC, respectively. The enzyme was extremely thermostable; the activity was stab le up to 90 degreesC. The glyoxylate reductase catalyzed the reduction of g lyoxylate and hydroxypyravate, and the relative activity for hydroxypyruvat e was approximate to one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron d onor compared with NADH. The K-m values for glyoxylate, hydroxypyruvate, an d 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 nucl eotide sequence of the a oxylate reductase a ne was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular m ass of 36 807. The amino-acid sequence of the T. litoralis enzyme showed hi gh similarity with those of probable dehydrogenases in Pyrococcus horikoshi i and P. abyssi. The purification of the enzyme from recombinant E. coli wa s much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed.