Enzymes for the NADPH‐dependent reduction of dihydroxyacetone and D‐glyceraldehyde and L‐glyceraldehyde in the mould Hypocrea jecorina

Abstract

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.

Metabolic Engineering of Fungal Strains for Conversion of d -Galacturonate to meso -Galactarate

“…Eukaryotic microorganisms use a reductive pathway in which D-galacturonate is first reduced to L-galactonate by an NAD(P)H-dependent reductase (12,17). In the following steps a dehydratase, aldolase, and reductase convert the L-galactonate to pyruvate and glycerol (9,11,14).In Hypocrea jecorina (anamorph Trichoderma reesei) the gar1 gene codes for a strictly NADPH-dependent D-galacturonate reductase. In Aspergillus niger a homologue gene sequence, gar2, exists; however, a different gene, gaaA, is upregulated during growth on D-galacturonate containing medium (16).…”

“…Eukaryotic microorganisms use a reductive pathway in which D-galacturonate is first reduced to L-galactonate by an NAD(P)H-dependent reductase (12,17). In the following steps a dehydratase, aldolase, and reductase convert the L-galactonate to pyruvate and glycerol (9,11,14).…”

Engineering Filamentous Fungi for Conversion of d -Galacturonic Acid to l -Galactonic Acid

“…1) that includes reactions catalyzed by D-galacturonic acid reductase (10), L-galactonate dehydratase (9), 2-keto-3-deoxy galactonate aldolase (8), and L-glyceraldehyde reductase (11); the intermediates are L-galactonate, 2-keto-3-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate), and L-glyceraldehyde, and the products of the pathway are pyruvate and glycerol. D-Galacturonic acid can induce pectinolytic and D-galacturonic acid catabolic genes in A. niger, regardless of whether D-galacturonic acid is metabolized or not (4,14).…”

The Missing Link in the Fungal D-Galacturonate Pathway

“…In previous communications we described the enzymes and the corresponding genes of D-galacturonate reductase and L-galactonate dehydratase converting D-galacturonate to L-galactonate and L-galactonate to L-threo-3-deoxy-hexulosonate (2-keto-3-deoxy-L-galactonate), respectively (6,7). We also identified an enzyme and corresponding gene for an L-glyceraldehyde reductase (8). The missing link is an L-threo-3-deoxy-hexulosonate aldolase.…”

“…In a subsequent reaction the L-glyceraldehyde could then be converted by a reductase to glycerol. Such a reductase that would use NADPH as a cofactor has been described previously for H. jecorina (8). The missing link in this pathway is an L-threo-3-deoxy-hexulosonate aldolase that would convert L-threo-3-deoxy-hexulosonate to pyruvate and L-glyceraldehyde.…”