Mattevi A, Obmolova G, Sokatch J R, Betzel C, Hol W G
Department of Chemistry, University of Groningen, The Netherlands.
Proteins. 1992 Aug;13(4):336-51. doi: 10.1002/prot.340130406.
The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A 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 crystallographic R-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 overall B-factor is unusually high: 47 A2. 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 mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, 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 isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms 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 A 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 in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.
恶臭假单胞菌中三种硫辛酰胺脱氢酶之一LipDH Val的三维结构已在2.45 Å分辨率下确定。在20 mM NAD⁺存在下生长的正交晶体，每个不对称单元包含458个残基。一个晶体学二重轴产生了在溶液中观察到的二聚体。对于18216个独立反射以及由3452个蛋白质原子、189个溶剂分子和44个NAD⁺原子组成的模型，最终晶体学R因子为21.8%，而整体B因子异常高：47 Ų。LipDH Val的结构揭示了C末端残基的构象，这些残基“向后”折叠到假定的硫辛酰胺结合区域。通过定点诱变已证明C末端对活性很重要。然而，C末端与催化必需残基之间的距离惊人地大，超过6 Å，C末端的确切作用仍有待阐明。在这种晶体形式中，LipDH Val每个亚基包含一个NAD⁺分子。其腺嘌呤 - 核糖部分占据的位置与谷胱甘肽还原酶结构中的类似。然而，烟酰胺 - 核糖部分远离其在异咯嗪环附近的预期位置并指向溶液。将LipDH Val与棕色固氮菌硫辛酰胺脱氢酶进行比较，每个亚基440个定义明确的Cα原子的均方根差异为1.6 Å。将LipDH Val与谷胱甘肽还原酶进行比较表明，这两种酶在三级和四级结构上存在很大差异。例如，二聚体中的两个亚基相对于彼此偏移了6 Å。因此，LipDH Val证实了谷胱甘肽还原酶和硫辛酰胺脱氢酶在分子结构上令人惊讶的差异，这在棕色固氮菌LipDH中已经观察到。这更值得注意，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。
