The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution.
The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution.
复制标题
恶臭假单胞菌硫辛酰胺脱氢酶与 NAD 复合的精制晶体结构，分辨率为 2.45 A。
作者：
Mattevi,A;Obmolova,G;Sokatch,JR;Betzel,C;Hol,WG
恶臭假单胞菌中存在的三种硫辛酰胺脱氢酶之一 LipDH Val 的三维结构已以 2.45 Å 分辨率测定。在 20 mM NAD+ 存在下生长的斜方晶体，每个不对称单元含有 458 个残基。晶体学双倍轴产生在溶液中观察到的二聚体。对于 18,216 个独特的反射和由 3,452 个蛋白质原子、189 个溶剂分子和 44 个 NAD+ 原子组成的模型，最终的晶体学 R 因子为 21.8%，而总体 B 因子异常高； 47 Å2。LipDH Val 的结构揭示了 C 末端残基的构象，这些残基“返回”到假定的硫辛酰胺结合区域。通过定点诱变，C 末端已被证明对活性很重要。然而，C 末端与催化必需残基的距离惊人地大，超过 6 Å，并且 C 末端的确切作用仍需要阐明。 在这种晶体形式中，LipDH Val 每个亚基含有一个 NAD+ 分子。其腺嘌呤-核糖部分占据与谷胱甘肽还原酶结构中类似的位置。然而，烟酰胺-核糖部分远离异咯嗪环附近的预期位置，并指向溶液中。将 LipDH Val 与固氮菌硫辛酰胺脱氢酶进行比较，每个亚基 440 个明确定义的 Cα 原子的 rms 差异为 1.6 Å。将 LipDH Val 与谷胱甘肽还原酶进行比较，发现两种酶的三级和四级结构存在很大差异。例如，二聚体中的两个亚基彼此偏移 6 Å。因此，LipDH Val 证实了谷胱甘肽还原酶和硫辛酸酰胺脱氢酶之间分子结构上的惊人差异，这些差异已经在维氏固氮菌LipDH 中观察到。这是更值得注意的，因为活性位点位于亚基界面并且在所有三种酶中实际上是相同的。 © 1992 Wiley-Liss, Inc.
The three‐dimensional structure of one of the three lipoamide dehydroge‐nases occurring inPseudomonas putida, LipDH Val, has been determined at 2.45 Å resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2‐fold axis generates the dimer which is observed in solution. The final crystallographicR‐factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+atoms, while the overallB‐factor is unusually high; 47 Å2.The structure of LipDH Val reveals the conformation of the C‐terminal residues which fold “back” into the putative lipoamide binding region. The C‐terminus has been proven to be important for activity by site‐directed mutagene‐sis. However, the distance of the C‐terminus to the catalytically essential residues is surprisingly large, over 6 Å, and the precise role of the C‐terminus still needs to be elucidated.In this crystal form LipDH Val contains one NAD+molecule per subunit. Its adenine‐ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide‐ribose moiety is far removed from its expected position near the isoallox‐azine ring and points into solution.Comparison of LipDH Val withAzotobacter vinelandiilipoamide dehydrogenase yields an rms difference of 1.6 Å for 440 well defined Cαatoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 Å with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed inAzotobacter vinelandiiLipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes. © 1992 Wiley‐Liss, Inc.