A Novel L‑Xylulose Reductase Essential for L‑Arabinose Catabolism in Trichoderma reesei

Plant cell walls consist of the polysaccharides cellulose, different hemicelluloses, and pectins and the complex polymer lignin. While cellulose is a linear β-1,4-linked d-glucose polymer, the structure and composition of hemicelluloses and pectins are more diverse. Following d-xylose, l-arabinose is the second most abundant pentose in hemicelluloses and pectins. It is present as a single residue or a short side chain in arabinoxylans or as larger branched side chains in the form of arabinan or arabinogalactan in pectins.1,2 Though the enzymatic steps for the catabolism of l-arabinose were described in the 1960s,3 most of the genes encoding the enzymes of this five-step pathway were only characterized recently.4 One reason for this might be that an l-arabinose pathway is not found in the fungal model organism Saccharomyces cerevisiae. Recently, considerable efforts were undertaken to fully elucidate this pathway for introduction of such a trait into S. cerevisiae to allow a complete conversion of plant biomass to, e.g., advanced biofuels or other biorefinery products.5,6 Degradation of l-arabinose in fungi usually consists of four oxidoreductive reactions and a final phosphorylation step, distinguishing this path from the different pathways for bacterial l-arabinose catabolism. The last two reactions of the fungal l-arabinose pathway are shared with the d-xylose catabolic pathway (Figure ​(Figure1).1). The bacterial isomerase pathway consists of an l-arabinose isomerase, ribulokinase, and l-ribulose phosphate-4-epimerase, while the enzyme sequence of the oxidative pathway consists of l-arabinose dehydrogenase, l-arabinolactonase, l-arabonate dehydratase, l-2-keto-3-deoxy-arabonate dehydratase, and 2,5-dioxovalerate dehydrogenase, the end product being α-ketoglutarate. In a modification of this oxidative pathway, l-2-keto-3-deoxy-arabonate is split by an aldolase into pyruvate and glycoaldehyde.7,8 Most of the genes and proteins involved in the fungal l-arabinose pathway were characterized in the two ascomycetes Aspergillus niger and Trichoderma reesei.4 In T. reeseil-arabinose reduction is mediated by the NADPH specific d-xylose reductase XYL1, which is the major reductase activity for the reduction of both pentoses d-xylose and l-arabinose.9,10 In A. niger, this NADPH-dependent reduction is accomplished by an l-arabinose specific LarA and a d-xylose specific XyrA.11 The subsequent steps are mediated by l-arabitol 4-dehydrogenase,12,13l-xylulose reductase,14 xylitol dehydrogenase,15 and xylulose kinase.16 Figure 1 Fungal l-arabinose degrading pathway represented by enzymes of A. niger and T. reesei. The first three specific steps of the fungal l-arabinose catabolism lead to xylitol, the first common intermediate of the l-arabinose and d-xylose pathway. Xylitol ... Enzymes with l-xylulose reductase activity are found within the short chain dehydrogenase and reductase family17 and participate in the glucuronic acid/uronate cycle of mammals. In humans, LXR deficiency causes pentosuria, a clinically benign condition that results in large amounts of l-xylulose in the urine of such patients.18 The first fungal l-xylulose reductase, ALX1, was identified in the yeast Ambrosiozyma monospora and, interestingly, is NADH-dependent.19 Although an enzyme with l-xylulose reductase (LXR1) was described for T. reesei,20 its functional characterization showed that it is actually a d-mannitol 2-dehydrogenase.21,22 Only recently was a true l-xylulose reductase LxrA identified in A. niger. Its deletion resulted in an almost complete loss of the NADPH specific l-xylulose reductase activity but had an only small effect on the growth on l-arabinose as the carbon source, explained by the presence of a NADH-dependent l-xylulose reductase activity.14 However, deletion of the LxrA homologue LXR4 in T. reesei showed that this gene is not involved in the oxidoreductive catabolism of l-arabinose but of d-galactose.23 To clone putative LXRs involved in l-arabinose catabolism in T. reesei, we made use of the fact that all LXRs identified to date are found within the group of short chain dehydrogenases and reductases. Consequently, we screened the T. reesei genome database for SDRs encoding genes and reduced the number of LXR candidates by selecting for highly conserved fungal LXRs that are expressed in the presence of l-arabinose. Functional analysis identified a novel NADPH-dependent l-xylulose reductase that is involved in l-arabinose catabolism in T. reesei, which is different from the case for the previously described enzymes.