Cloning and Expression of a Fungal L-Arabinitol 4-Dehydrogenase Gene

Peter Richard, John Londesborough, Mikko Putkonen, Nisse Kalkkinen, Merja Penttilä

Research output: Contribution to journalArticleScientificpeer-review

82 Citations (Scopus)

Abstract

L-Arabinitol 4-dehydrogenase (EC 1.1.1.12) was purified from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). It is an enzyme in the L-arabinose catabolic pathway of fungi catalyzing the reaction from L-arabinitol to L-xylulose. The amino acid sequence of peptide fragments was determined 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 after growth on L-arabinose. The gene was cloned and overexpressed in Saccharomyces 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 shows activity toward L-arabinitol, adonitol (ribitol), and xylitol with K m values of about 40 mM toward L-arabinitol and adonitol and about 180 mM toward xylitol. No activity was observed with D-sorbitol, D-arabinitol, and D-mannitol. NAD is the required cofactor with a Km of 180 μM. No activity was observed with NADP.

Original languageEnglish
Pages (from-to)40631-40637
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number44
DOIs
Publication statusPublished - 2 Nov 2001
MoE publication typeA1 Journal article-refereed

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