Cloning and expression of a fungal L-arabinitol 4-dehydrogenase gene

L-Arabinitol 4-dehydrogenase (EC 1.1.1.12) was purified from the filamentou s fungus Trichoderma reesei (Hypocrea jecorina). It is an enzyme in the L-a rabinose catabolic pathway of fungi catalyzing the reaction from L-arabinit ol to L-xylulose. The amino acid sequence of peptide fragments was determin ed and used to identify the corresponding gene. We named the gene lad1. It is not constitutively expressed. In a Northern analysis we found it only af ter growth on L-arabinose. The gene was cloned and overexpressed in Sacchar omyces cerevisiae, and the enzyme activity was confirmed in a cell extract. The enzyme consists of 377 amino acids and has a calculated molecular mass of 39,822 Da. It belongs to the family of zinc-binding dehydrogenases and has some amino acid sequence similarity to sorbitol dehydrogenases. It show s activity toward L-arabinitol, adonitol (ribitol), and xylitol with K-m va lues of about 40 mm toward L-arabinitol and adonitol and about 180 mm towar d xylitol. No activity was observed with D-sorbitol, D-arabinitol, and D-ma nnitol. NAD is the required cofactor with a K-m of 180 muM. No activity was observed with NADP.