THE 4-MDA HUMAN BCKD CATALYTIC MACHINE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。哺乳动物支链α-酮酸脱氢酶复合体是线粒体α-酮酸脱氢酶复合体的一员，包括丙酮酸脱氢酶复合体(PDC)、α-酮戊二酸脱氢酶复合体(KGDC)和BCKD复合体，具有相似的结构和功能。哺乳动物BCKD由三个催化组分组成：异四聚体(α2beta2)支链α-酮酸脱羧酶(E1)，Homo-24 Meric二氢硫辛基转移酶(E2)和同源二聚二氢硫辛胺脱氢酶(E3)。E1和E2组分是BCKD复合体所特有的，而E3组分是所有三个α-酮酸脱氢酶复合体共同的。此外，哺乳动物的BCKD含有两种调节酶：一种是特殊的激酶，另一种是特殊的磷酸酶，它们通过磷酸化(失活)/去磷酸化(激活)循环来调节BCKD的活性。BCKD是围绕立方体E2核心组织的，12个副本的E1和数量不详的E3副本、激酶和磷酸酶通过离子相互作用连接到该核心上。BCKD多酶复合体的分子质量估计为4×106道尔顿。遗传性枫糖浆尿病(MSUD)患者的BCKD复合体缺乏。这种代谢障碍会导致有毒的支链α-酮酸的积累，表现为往往致命的酸中毒、神经紊乱和智力低下。对人类突变如何破坏人类BCKD复合体的催化机制的结构性洞察将有助于制定改善MSUD表型的策略。具体目的：1.用冷冻电子显微镜破译人BCKD复合体E2核心的三维结构。2.了解E1和E3在E2支架上的结合拓扑结构和摩尔比。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mammalian branched-chain alpha-ketoacid dehydrogenase (BCKD) complex is a member of the mitochondrial alpha-ketoacid dehydrogenase complexes including pyruvate dehydrogenase complex (PDC), alpha-ketoglutarate dehydrogenase complex (KGDC) and the BCKD complex, having similar structure and function. The mammalian BCKD consists of three catalytic components: a heterotetrameric (alpha2beta2) branched-chain alpha-ketoacid decarboxylase or E1, a homo-24 meric dihydrolipoyl transacylase or E2 and a homodimeric dihydrolipoamide dehydrogenase or E3. E1 and E2 components are specific for the BCKD complex, whereas the E3 component is common to all three alpha-ketoacid dehydrogenase complexes. In addition, the mammalian BCKD contains two regulatory enzymes: a specific kinase and a specific phosphatase that regulate activity of the BCKD through phosphorylation (inactivation) / dephosphorylation (activation) cycles. The BCKD is organized around the cubic E2 core, to which 12 copies of E1, and an unspecified number of copies of E3, the kinase and the phosphatase are attached through ionic interactions. The molecular mass of the BCKD multienzyme complex is estimated to be 4 x 106 daltons. The BCKD complex is deficient in patients inflicted by the inherited Maple Syrup Urine Disease (MSUD). This metabolic block results in the accumulation of toxic branched-chain alpha-ketoacids, manifested by often-fatal acidosis, neurological derangement and mental retardation. Structural insight into how human mutations disrupt the catalytic mechanism of the human BCKD complex will help develop strategies for ameliorating the MSUD phenotype. Specific Aims: 1. To decipher the three-dimensional structure of the E2 core of the human BCKD complex by cryo-EM. 2. To discern the binding topology and molar ratios of E1 and E3 on the E2 scaffold.