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.

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Dihydroxyacetone (DHA), d-glyceraldehyde and l-glyceraldehyde can be reduced using NADPH as a cofactor to form glycerol and NADP. Enzymes catalysing this reaction are generally called NADP:glycerol dehydrogenases. NADP:glycerol dehydrogenase activity is common in moulds and filamentous fungi. Enzymes from different species of filamentous fungi have been purified and characterized. The enzymes purified from Aspergillus niger [1] and Aspergillus nidulans [2] catalyse the reversible reaction from glycerol and NADP to DHA and NADPH. For the A. niger enzyme, an equilibrium constant of 3.1-4.6 · 10 )12 m was estimated for the reaction: The mould Hypocrea jecorina (Trichoderma reesei) has two genes coding for enzymes with high similarity to the NADP-dependent glycerol dehydrogenase. These genes, called gld1 and gld2, were cloned and expressed in a heterologous host. The encoded proteins were purified and their kinetic properties characterized. GLD1 catalyses the conversion of d-glyceraldehyde and l-glyceraldehyde to glycerol, whereas GLD2 catalyses the conversion of dihydroxyacetone to glycerol. Both enzymes are specific for NADPH as a cofactor. The properties of GLD2 are similar to those of the previously described NADP-dependent glycerol-2-dehydrogenases (EC 1.1.1.156) purified from different mould species. It is a reversible enzyme active with dihydroxyacetone or glycerol as substrates. GLD1 resembles EC 1.1.1.72. It is also specific for NADPH as a cofactor but has otherwise completely different properties. GLD1 reduces d-glyceraldehyde and l-glyceraldehyde with similar affinities for the two substrates and similar maximal rates. The activity in the oxidizing reaction with glycerol as substrate was under our detection limit. Although the role of GLD2 is to facilitate glycerol formation under osmotic stress conditions, we hypothesize that GLD1 is active in pathways for sugar acid catabolism such as d-galacturonate catabolism.Abbreviations DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate.