Cloning and expression of a fungal L-arabinitol 4-dehydrogenase gene.
L-Arabinitol 4-dehydrogenase (EC ) 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 microM. No activity was observed with NADP.
Richard
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P
VTT Biotechnology, P.O. Box 1500, Espoo, FIN-02044 VTT, Finland. Peter.Richard@vtt.fi
Londesborough
J
J
Putkonen
M
M
Kalkkinen
N
N
Penttilä
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D013401:Sugar Alcohol Dehydrogenases; C440029:L-arabinitol 4-dehydrogenase