Verho Ritva, Putkonen Mikko, Londesborough John, Penttilä Merja, Richard Peter
VTT Biotechnology, P. O. Box 1500, FIN-02044 VTT, Finland.
J Biol Chem. 2004 Apr 9;279(15):14746-51. doi: 10.1074/jbc.M312533200. Epub 2004 Jan 21.
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.
从单孢酒香酵母中鉴定出一种依赖NADH的L-木酮糖还原酶及其相应基因。该酶是酵母L-阿拉伯糖分解代谢途径的一部分。此前已描述了霉菌利用L-阿拉伯糖的真菌途径。在该途径中，L-阿拉伯糖依次转化为L-阿拉伯糖醇、L-木酮糖、木糖醇和D-木酮糖，并以5-磷酸-D-木酮糖的形式进入磷酸戊糖途径。在霉菌中，还原反应与NADPH相关，氧化反应与NAD(+)相关。在这里我们表明，在单孢酒香酵母中该途径是相似的，即它具有相同的两个还原反应和两个氧化反应，但L-木酮糖还原酶的还原反应不是由像霉菌中那样严格依赖NADPH的酶进行的，而是由严格依赖NADH的酶进行的。如Northern分析所示，编码依赖NADH的L-木酮糖还原酶的ALX1基因在L-阿拉伯糖上生长期间强烈表达。该基因在酿酒酵母中进行了功能过表达，并对纯化的带有His标签的蛋白质进行了表征。这种可逆酶将L-木酮糖转化为木糖醇。它还将D-核糖醇转化为D-阿拉伯糖醇，但对L-阿拉伯糖醇或阿东糖醇没有活性，即它对费歇尔投影中C-2羟基为L-构型且C-3羟基为D-构型的糖醇具有特异性。它对C-6糖类或糖醇也没有活性。L-木酮糖和D-核糖醇的K(m)值分别为9.6和4.7 mM。据我们所知，这是关于依赖NADH的L-木酮糖还原酶的首次报道。
