Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization
Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization
复制标题
DOI：
10.1016/j.bpc.2008.10.003
复制
发表时间：
2009-01-01
影响因子：
3.8
通讯作者：
Neira, Jose L.
中科院分区：
文献类型：
--
作者：
Martinez-Rodriguez, Sergio;Encinar, Jose A.;Neira, Jose L.
二氢嘧啶酶参与嘧啶降解的还原途径，催化5，6-二氢尿嘧啶和5，6-二氢胸腺嘧啶的环酰胺键（-CO-NH-）可逆水解为相应的N-氨甲酰-β-氨基酸。这种酶是商业生产D-氨基酸的有吸引力的候选者，D-氨基酸用于生产半合成β-内酰胺、抗病毒剂、人工甜味剂、肽激素和杀虫剂。我们已经获得了二氢嘧啶酶从苜蓿中华根瘤菌（SmelDhp）在锌离子的存在下的晶体结构，但我们还没有能够获得良好的衍射晶体在其缺席。然后，离子在蛋白质结构及其稳定性中的作用仍有待阐明。在这项工作中，SmelDhp的稳定性和结构进行了研究，在没有和存在下的锌。在其不存在的情况下，蛋白质在pH接近6.0时获得四聚体功能结构，其在pH接近9.0时稳定，如从荧光和CD得出的结论。通过具有非天然结构的单体中间体发生化学变性。锌的添加导致：（i）螺旋结构的增加，和芳香残基环境的变化;和（ii）更高的热稳定性。然而，化学变性仍然通过单体中间体发生。这是第一个乙内酰脲酶的稳定性和二级结构的变化后，加入锌的描述和解释，和少数几个例子之一，其中锌专门改变二级螺旋结构和环境中的一些芳香族残基的蛋白质，保持不变的四级结构。(c)2008 Elsevier B. V.保留所有权利。
Dihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (-CO-NH-) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbarnoyl-beta-amino acids. This enzyme is an attractive candidate for commercial production of D-amino acids, which are used in the production of semi-synthetic beta-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH similar to 6.0, which is stable up to pH similar to 9.0, as concluded from fluorescence and CD. Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure. (c) 2008 Elsevier B.V. All rights reserved.