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

P Richard*, J Londesborough, M Putkonen, N Kalkkinen, M Penttila

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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 K-m of 180 muM. No activity was observed with NADP.

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

Keywords

  • YEAST PICHIA-STIPITIS
  • SACCHAROMYCES-CEREVISIAE
  • XYLOSE REDUCTASE
  • XYLITOL-DEHYDROGENASE
  • ASPERGILLUS-NIGER
  • SEQUENCE
  • PROTEINS
  • PURIFICATION

Cite this