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J. Biol. Chem., Vol. 279, Issue 15, 14746-14751, April 9, 2004

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A Novel NADH-linked L-Xylulose Reductase in the L-Arabinose Catabolic Pathway of Yeast^*

Ritva Verho, Mikko Putkonen, John Londesborough, Merja Penttilä, and Peter Richard

From the VTT Biotechnology, P. O. Box 1500, FIN-02044 VTT, Finland

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.

Received for publication, November 17, 2003 , and in revised form, January 12, 2004.

^* This work was supported by the "Sustainable Use of Natural Resources" program of the Academy of Finland and the research program "VTT Industrial Biotechnology" (Academy of Finland; Finnish Centre of Excellence program, 2000–2005, Project 64330). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ583159.

To whom correspondence should be addressed: VTT Biotechnology, Tietotie 2, Espoo, P. O. Box 1500, FIN-02044 VTT, Finland. Tel.: 358-9-456-7190; Fax: 358-9-455-2103; E-mail: Peter.Richard{at}vtt.fi.

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