A novel NADH-linked l-xylulose reductase in the l-arabinose catabolic pathway of yeast.
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 K(m) 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.
Verho
Ritva
R
VTT Biotechnology, P. O. Box 1500, FIN-02044 VTT, Finland.
Putkonen
Mikko
M
Londesborough
John
J
Penttilä
Merja
M
Richard
Peter
P
D001089:Arabinose / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010427:Pentose Phosphate Pathway; D010428:Pentosephosphates / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry*; D013402:Sugar Alcohols / Q000737:chemistry; D013997:Time Factors; D014996:Xylulose / Q000737:chemistry
D018076:DNA, Complementary; D010428:Pentosephosphates; D013402:Sugar Alcohols; D009243:NAD; D006639:Histidine; D014996:Xylulose; D001089:Arabinose; D013401:Sugar Alcohol Dehydrogenases; C023300:L-xylulose reductase; D010100:Oxygen; C014999:arabitol