Abstract
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 Km 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.
| Original language | English |
|---|---|
| Pages (from-to) | 14746-14751 |
| Number of pages | 6 |
| Journal | Journal of Biological Chemistry |
| Volume | 279 |
| Issue number | 15 |
| DOIs | |
| Publication status | Published - 9 Apr 2004 |
| MoE publication type | A1 Journal article-refereed |
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Keywords
- reductase
- L-Xylulose reductase
- yeasts
- genes
- gene identification
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A Novel NADH-linked L-Xylulose Reductase in the L-Arabinose Catabolic Pathway of Yeast. / Verho, Ritva; Putkonen, Mikko; Londesborough, John; Penttilä, Merja; Richard, Peter.
In: Journal of Biological Chemistry, Vol. 279, No. 15, 09.04.2004, p. 14746-14751.Research output: Contribution to journal › Article › Scientific › peer-review
TY - JOUR
T1 - A Novel NADH-linked L-Xylulose Reductase in the L-Arabinose Catabolic Pathway of Yeast
AU - Verho, Ritva
AU - Putkonen, Mikko
AU - Londesborough, John
AU - Penttilä, Merja
AU - Richard, Peter
PY - 2004/4/9
Y1 - 2004/4/9
N2 - 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 Km 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.
AB - 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 Km 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.
KW - reductase
KW - L-Xylulose reductase
KW - yeasts
KW - genes
KW - gene identification
UR - http://www.scopus.com/inward/record.url?scp=2442438033&partnerID=8YFLogxK
U2 - 10.1074/jbc.M312533200
DO - 10.1074/jbc.M312533200
M3 - Article
C2 - 14736891
AN - SCOPUS:2442438033
VL - 279
SP - 14746
EP - 14751
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 15
ER -