Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae

Abstract

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (K m ‫؍‬ 46 M; k cat /K m ‫؍‬ 52,100 s ؊1 M ؊1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (K m ‫؍‬ 1.44 mM; k cat /K m ‫؍‬ 1,790 s ؊1 M ؊1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.

Endogenous NADPH‐dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

“…XR, XDH and XK activities were measured in the reference strain CEN.PK 113-7A and in the two constructed strains TMB3265(XYL2, XKS1 ) and TMB3267(XYL2, XKS1, GRE3 ) ( Table 2). XR activity with NADPH as co-factor was 5-6 mU/mg protein in CEN.PK 113-7A and TMB3265(XYL2, XKS1 ), similar to previously reported (Kuhn et al, 1995;Träff et al, 2001), whereas no XR activity was detected with NADH as co-factor. In TMB3267, overexpressing GRE3, the XR activity with NADPH was increased four times.…”

“…In continuous culture with glucose and xylose, ethanol, glycerol, xylulose but also xylitol were formed. Xylitol formation can be circumvented by deleting the GRE3 gene coding for an unspecific aldo-keto reductase (Kuhn et al, 1995;Garay-Arroyo and Covarrubias, 1999) and xylitol production has been successfully reduced in recombinant S. cerevisiae producing XI from Thermus thermophilus (Träff et al, 2001). A deletion of GRE3 might influence the xylitol yield also in strains overproducing XR and XDH.…”

“…Gre3p has been purified and it is strictly NADPH dependent (Kuhn et al, 1995;Ford and Ellis, 2001;Jeong et al, 2001;Petrash et al, 2001), in contrast to XR from P. stipitis, which uses both NADH and NADPH (Rizzi et al, 1989), and the kinetic properties of Gre3p have been characterized. The K m for xylose suggests that this protein is a xylose reductase with the ability to reduce several other compounds, including arabinose, glyceraldehydes, methylglyoxal and β-keto esters (Kuhn et al, 1995;Rodríguez et al, 2000;Ford and Ellis, 2001;Petrash et al, 2001). The GRE3 gene has been shown to be induced by stress (Aguilera and Prieto, 2001) and to be regulated by the HOG pathway (Garay-Arroyo and Covarrubias, 1999).…”

“…The GRE3 gene has been shown to be induced by stress (Aguilera and Prieto, 2001) and to be regulated by the HOG pathway (Garay-Arroyo and Covarrubias, 1999). The K m values in xylose reduction of the protein encoded by endogenous GRE3 are in the same range as P. stipitis XR for xylose (28 and 44 mM, respectively) (Kostrzynska et al, 1998;Kuhn et al, 1995) and NADPH (13 µM and 11 µM) (Kuhn et al, 1995;Kostrzynska et al, 1998). Besides the GRE3 gene product which is capable of xylose reduction, S. cerevisiae also harbours a gene encoding a XDH (Richard et al, 1999), so that S. cerevisiae has a potential initial xylose pathway.…”

“…In the present investigation, the GRE3 gene was expressed at three different levels (deleted, wildtype and overexpressed) in combination with the XYL1, XYL2 and XKS1 genes to investigate the impact of the strictly NADPH-dependent Gre3p (Kuhn et al, 1995;Ford and Ellis, 2001;Petrash et al, 2001) on xylose fermentation by recombinant S. cerevisiae strains.…”