An NADH-dependent L-xylulose reductase and the corresponding gene were identified from the yeast Ambrosiozyma monospora. The enzyme is part of the yeast pathway for L-arabinose catabolism. A fungal pathway for L-arabinose utilization has been described previously for molds. In this pathway L-arabinose is sequentially converted to L-arabinitol, L-xylulose, xylitol, and D-xylulose and enters the pentose phosphate pathway as D-xylulose 5-phosphate. In molds the reductions are NADPH-linked, and the oxidations are NAD(+)-linked. Here we show that in A. monospora the pathway is similar, i.e. it has the same two reduction and two oxidation reactions, but the reduction by L-xylulose reductase is not performed by a strictly NADPH-dependent enzyme as in molds but by a strictly NADH-dependent enzyme. The ALX1 gene encoding the NADH-dependent L-xylulose reductase is strongly expressed during growth on L-arabinose as shown by Northern analysis. The gene was functionally overexpressed in Saccharomyces cerevisiae and the purified His-tagged protein characterized. The reversible enzyme converts L-xylulose to xylitol. It also converts D-ribulose to D-arabinitol but has no activity with L-arabinitol or adonitol, i.e. it is specific for sugar alcohols where, in a Fischer projection, the hydroxyl group of the C-2 is in the L-configuration and the hydroxyl group of C-3 is in the D-configuration. It also has no activity with C-6 sugars or sugar alcohols. The Km values for L-xylulose and D-ribulose are 9.6 and 4.7 mM, respectively. To our knowledge this is the first report of an NADH-linked L-xylulose reductase.
- D-ARABINITOL DEHYDROGENASE
- XYLOSE REDUCTASE