Izard T, Sarfaty S, Westphal A, de Kok A, Hol W G
Howard Hughes Medical Institute, University of Washington, Seattle 98195-7742, USA.
Protein Sci. 1997 Apr;6(4):913-5. doi: 10.1002/pro.5560060419.
Members of the family of 2-oxoacid dehydrogenase multienzyme complexes catalyze the oxidative decarboxylation of alpha-keto acids and are among the most remarkable enzymatic machineries in the living cell. These multienzyme complexes combine a highly symmetric (cubic or icosahedral) core with a dynamic and flexible arrangement of numerous subunits and domains surrounding the core. The center of the complex is formed by either 24 or 60 copies of dihydrolipoamide acetyltransferase (E2)-a multidomain enzyme. The hollow icosahedral cores are composed of 60 identical subunits of the catalytic domain of E2 with a molecular weight of about 1.8 million Da. Bipyramidal crystals suitable for X-ray diffraction of the icosahedral core of the pyruvate dehydrogenase multienzyme complex from Enterococcus faecalis were grown up to 0.7 mm in each dimension. The crystals belong to space group R32 with a = b = 244.3 A (hexagonal setting), and have a solvent content of 73%. The asymmetric unit contains one-third of the molecule, i.e., 20 of the 60 subunits. Initial X-ray crystallographic data to 7 A resolution were collected at cryotemperatures at synchrotron facilities. Interestingly, the diffraction was improved significantly upon rehydrating dehydrated crystals and extended to 4.2 A.
2-氧代酸脱氢酶多酶复合物家族的成员催化α-酮酸的氧化脱羧反应，是活细胞中最显著的酶机制之一。这些多酶复合物将高度对称（立方或二十面体）的核心与围绕核心的众多亚基和结构域的动态灵活排列相结合。复合物的中心由24个或60个二氢硫辛酰胺乙酰转移酶（E2，一种多结构域酶）的拷贝形成。空心二十面体核心由60个相同的E2催化结构域亚基组成，分子量约为180万道尔顿。适合对粪肠球菌丙酮酸脱氢酶多酶复合物二十面体核心进行X射线衍射的双锥体晶体，在每个维度上生长到了0.7毫米。这些晶体属于空间群R32，a = b = 244.3 Å（六方晶系），溶剂含量为73%。不对称单元包含分子的三分之一，即60个亚基中的20个。在同步辐射设施的低温下收集了分辨率达7 Å的初始X射线晶体学数据。有趣的是，对脱水晶体进行再水化后，衍射显著改善，并扩展到了4.2 Å。
