Ishikura Shuhei, Isaji Tomoya, Usami Noriyuki, Nakagawa Junichi, El-Kabbani Ossama, Hara Akira
Laboratory of Biochemistry, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, 502-8585, Gifu, Japan.
Chem Biol Interact. 2003 Feb 1;143-144:543-50. doi: 10.1016/s0009-2797(02)00217-x.
L-Xylulose reductase (XR) catalyzes the oxidoreduction between xylitol and L-xylulose in the uronate cycle. The enzyme has been shown to be identical to diacetyl reductase, an enzyme that reduces alpha-dicarbonyl compounds. XR belongs to the short-chain dehydrogenase/reductase family, and shows high sequence identity with mouse lung carbonyl reductase (MLCR), an enzyme that reduces 3-ketosteroids but not sugars. In this study, we have confirmed the roles of Ser136, Tyr149 and Lys153 of XR as the catalytic triad by drastic loss of activity resulting from the mutagenesis of S136A, Y149F and K153M in rat XR. We have also constructed several mutant XRs, in which putative substrate binding residues from rat XR were substituted with those found in the corresponding positions of MLCR, in order to identify amino acids responsible for the different substrate recognition of the enzymes. While single mutants at positions 137, 143, 146, 190 and 191 caused little or moderate change in substrate specificity, a double mutant (N190V and W191S) and triple mutant (Q137M, L143F and H146L) resulted in almost loss of activity for only the sugars. In addition, the triple mutant exhibited 3-ketosteroid reductase activity, which was further enhanced by quintuple mutagenesis of the above five residues. These results suggest the importance of the size and hydrophobicity of the five residues for substrate recognition by XR and MLCR. Furthermore, the mutant enzymes containing a Q137M mutation were stable against cooling, which provides a structural mechanism of the cold inactivation that is a characteristic of the rodent XR.
L-木酮糖还原酶（XR）催化糖醛酸循环中木糖醇和L-木酮糖之间的氧化还原反应。该酶已被证明与双乙酰还原酶相同，双乙酰还原酶是一种还原α-二羰基化合物的酶。XR属于短链脱氢酶/还原酶家族，与小鼠肺羰基还原酶（MLCR）具有高度的序列同一性，MLCR是一种还原3-酮类固醇而非糖类的酶。在本研究中，我们通过大鼠XR中S136A、Y149F和K153M突变导致的活性急剧丧失，证实了XR的Ser136、Tyr149和Lys153作为催化三联体的作用。我们还构建了几种突变型XR，其中大鼠XR中假定的底物结合残基被MLCR相应位置的残基取代，以确定负责酶不同底物识别的氨基酸。虽然137、143、146、190和191位的单突变对底物特异性影响很小或有中等程度的改变，但双突变体（N190V和W191S）和三突变体（Q137M、L143F和H146L）仅对糖类几乎丧失活性。此外，三突变体表现出3-酮类固醇还原酶活性，通过上述五个残基的五重诱变进一步增强。这些结果表明这五个残基的大小和疏水性对于XR和MLCR识别底物的重要性。此外，含有Q137M突变的突变酶对冷却稳定，这为啮齿动物XR特有的冷失活提供了一种结构机制。
