Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus:: Insight into thermostability and cofactor binding
Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus:: Insight into thermostability and cofactor binding
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DOI：
10.1021/bi049736p
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发表时间：
2004-05-11
期刊：
影响因子：
2.9
通讯作者：
Bahnson, BJ
中科院分区：
文献类型：
--
作者：
Ceccarelli, C;Liang, ZX;Bahnson, BJ
用X射线衍射法测定了嗜热脂肪芽孢杆菌菌株LLD-R(HtADH)依赖NAD(+)的乙醇脱氢酶的晶体结构，其分辨率为2.35埃。其同源四聚体htADH的结构与细菌和古生菌的同源四聚体乙醇脱氢酶(ADHS)以及哺乳动物的二聚体ADHS高度同源。每个酶亚基有一个催化锌原子和一个结构锌原子。该酶是一种缺少烟酰胺腺嘌呤二核苷酸(NAD(+))辅因子但含有锌配位底物类似物三氟乙醇的二元复合体。二元络合物结构为开放构象，类似于无结合辅因子的ADH结构。通过与来自大肠杆菌的同源中温酶(同源的55%同源性)，依赖于NAD(+)的乙醇脱氢酶的比较，提出了对htADH热稳定性重要的特征。为了深入了解NAD(+)结合引发的构象变化，用高效液相色谱-电喷雾质谱仪研究了在NAD(+)存在和不存在的情况下htADH的胺氢-氢-氢交换。当分析这些蛋白衍生的多肽的氚参入时，发现21个多肽中有9个在与NAD(+)结合时，其中9个的氚参入水平有所下降，另外4个多肽的交换速度较慢。除了一个例外(肽编号8)，所有被结合的NAD(+)改变的肽都不与酒精底物结合口袋接触。此外，位于NAD(+)结合口袋外的多肽5和8在NAD(+)结合时显示变化，这是值得注意的。这表明辅因子结合引起的从开放构象到封闭构象的转变对蛋白质的结构和动力学有一些长期的影响。
The crystal structure of NAD(+)-dependent alcohol dehydrogenase from Bacillus stearothermophilus strain LLD-R (htADH) was determined using X-ray diffraction data at a resolution of 2.35 Angstrom The structure of homotetrameric htADH is highly homologous to those of bacterial and archaeal homotetrameric alcohol dehydrogenases (ADHs) and also to the mammalian dimeric ADHs. There is one catalytic zinc atom and one structural zinc atom per enzyme subunit. The enzyme was crystallized as a binary complex lacking the nicotinamide adenine dinucleotide (NAD(+)) cofactor but including a zinc-coordinated substrate analogue trifluoroethanol. The binary complex structure is in an open conformation similar to ADH structures without the bound cofactor. Features important for the thermostability of htADH are suggested by a comparison with a homologous mesophilic enzyme (55% identity), NAD(+)-dependent alcohol dehydrogenase from Escherichia coli. To gain insight into the conformational change triggered by NAD(+) binding, amide hydrogen-deuterium exchange of htADH, in the presence and absence of NAD(+), was studied by HPLC-coupled electrospray mass spectrometry. When the deuteron incorporation of the protein-derived peptides was analyzed, it was found that 9 of 21 peptides show some decrease in the level of deuteron incorporation upon NAD(+) binding, and another 4 peptides display slower exchange rates. With one exception (peptide number 8), none of the peptides that are altered by bound NAD(+) are in contact with the alcohol-substrate-binding pocket. Furthermore, peptides 5 and 8, which are located outside the NAD(+)-binding pocket, are notable by displaying changes upon NAD(+) binding. This suggests that the transition from the open to the closed conformation caused by cofactor binding has some long-range effects on the protein structure and dynamics.