Purification and Enzymatic Properties of Glyoxylate Reductase II from Baker's Yeast

Abstract

Glyoxylate reductase II was purified about 2,400-fold from a cell extract of baker's yeast by protamine sulfate treatment, and column chromatographies on DEAE-cellulose, hydroxylapatite, Sephadex G-150, and phosphocellulose. The purified enzyme was electrophoretically homogeneous. The molecular weight was determined to be approximately 65,000 by gel filtration. The enzyme was greatly stabilized by the addition of 20% (v/v) glycerol. It catalyzed the reduction of glyoxylate and hydroxypyruvate and was specific for NADPH as an electron donor, but showed slight affinity towards NADH. The Michaelis constants for glyoxylate, hydroxypyruvate, NADPH, and NADH were found to be 16mM, 1.4 mM, 5.7 microM, and 0.43 mM, respectively. The enzyme was inhibited by p-chloromercuribenzoate (PCMB) and iodoacetate, but inhibition was prevented by dithiothreitol (DTT) or L-cysteine. The reduction of glyoxylate and hydroxypyruvate was not stimulated by anions.

The ORF YNL274c (GOR1) codes for glyoxylate reductase in Saccharomyces cerevisiae

“…The activity of these enzymes towards glyoxylate was two-fold compared to the activity on hydroxypyruvate. This ratio is similar to that measured for the isoenzyme glyoxylate reductase II of S. cerevisiae (Fukuda et al, 1980;Tochikura et al, 1979). The highest activity, an increase of approximately 350-fold on glyoxylate and 160-fold on hydroxypyruvate relative to the control, was measured in the YNL274c overexpressing strain.…”

“…However, in the deletion strain of ORF YNL274c, the activity, 0.2 ± 0.1 nkat/mg protein, was only one-third of the activity in the mother strain. substrates, glyoxylate and hydroxypyruvate, of the baker's yeast glyoxylate reductases (Fukuda et al, 1980;Tochikura et al, 1979) were used. The activity on both glyoxylate and hydroxypyruvate of the CEN.PK2-1D control strain with the empty expression vector was below 1.0 nkat/mg protein.…”

“…The activity on glyoxylate was also measured using NADH as a co-factor instead of NADPH, the latter being the preferred co-factor of glyoxylate reductase isoenzymes in S. cerevisiae (Fukuda et al, 1980;Tochikura et al, 1979). In the strain overexpressing ORF YNL274c the activity was 4% with NADH as a co-factor compared to that with NADPH (11 ± 1 and 230 ± 36 nkat/mg protein, respectively).…”

“…In Saccharomyces cerevisiae, two glyoxylate reductase isoenzymes have been purified (Fukuda et al, 1980;Tochikura et al, 1979) but their physiological role is not known, neither have the corresponding genes been identified. However, a BLAST search of the S. cerevisiae database suggests seven ORFs as possibly coding for glyoxylate reductase.…”

Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis

“…In S. cerevisiae , two glyoxylate reductase isoforms have been purified [23,24] but only one gene, GOR1 , encoding glyoxylate reductase activity has been identified [13]. Deletion of the glyoxylate reductase, GOR1 , led to increased biomass formation after diauxic shift, which was thought to be caused by increased NADPH availability [13].…”