Identification in the Mold Hypocrea jecorina of the First Fungal d-Galacturonic Acid Reductase

Abstract

A d-galacturonic acid reductase and the corresponding gene were identified from the mold Hypocrea jecorina (Trichoderma reesei). We hypothesize that the enzyme is part of a fungal d-galacturonic acid catabolic pathway which has not been described previously and which is distinctly different from the bacterial pathway. H. jecorina grown on d-galacturonic acid exhibits an NADPH-dependent d-galacturonic acid reductase activity. This activity is absent when the mold is grown on other carbon sources. The d-galacturonic acid reductase was purified, and tryptic digests of the purified protein were sequenced. The open reading frame of the corresponding gene was then cloned from a cDNA library. The open reading frame was functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was purified, and the enzyme kinetics were characterized. The enzyme converts in a reversible reaction from d-galacturonic acid and NADPH to l-galactonic acid and NADP. The enzyme also exhibits activity with d-glucuronic acid and dl-glyceraldehyde.

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

“…Although a large number of yeasts and molds can use galacturonic acid as a carbon and energy source for growth, knowledge of the metabolic route for D-galacturonic acid catabolism in eukaryotes is surprisingly limited (Kuorelahti et al 2005;Visser et al 1988). It has been concluded that D-galacturonic acid is metabolized through glyceraldehyde and pyruvate (Visser et al 1988).…”

“…It has been concluded that D-galacturonic acid is metabolized through glyceraldehyde and pyruvate (Visser et al 1988). Kuorelahti et al (2005) have recently identified a D-galacturonic acid reductase in the mold Hypocrea jecorina. This enzyme converts D-galacturonic acid and NADPH to L-galactonic acid and NADP + .…”

“…This enzyme converts D-galacturonic acid and NADPH to L-galactonic acid and NADP + . It has been proposed that this enzyme may be involved in a pathway for galacturonic acid metabolism that is distinctly different from the bacterial route, and which would involve L-galactonic acid, 3-dehydro-L-gulonate, L-xylulose, xylitol, D-xylulose, and xylulose 5-phosphate as intermediates (Kuorelahti et al 2005). The NADPH-dependence of the reductase may represent a complication in yeast metabolic-engineering strategies based on this pathway as it implies a need for balancing of the two different cofactor balances NADH/NAD + and NADPH/NADP + .…”

The Missing Link in the Fungal D-Galacturonate Pathway

“…The first two steps of this pathway have been described. In the first step D-galacturonate is reduced to L-galactonate by an NADPH-coupled D-galacturonate reductase (6). In Hypocrea jecorina (Trichoderma reesei) this enzyme activity was induced when grown on D-galacturonate but the activity was absent during growth on other carbon sources.…”

“…In Hypocrea jecorina (Trichoderma reesei) this enzyme activity was induced when grown on D-galacturonate but the activity was absent during growth on other carbon sources. The D-galacturonate reductase gene gar1 is identified and the purified protein characterized (6). In the second step L-galactonate is converted to L-threo-3-deoxy-hexulosonate (2-keto-3-deoxy-L-galactonate) by a dehydratase (7).…”