THE 4-MDA HUMAN BCKD CATALYTIC MACHINE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。哺乳动物支链α-酮酸脱氢酶复合体（BCKD）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由三种催化组分组成：异四聚体（α 2 β 2）支链α-酮酸脱羧酶或E1、同24梅里克二氢硫辛酰转酰酶或E2和同二聚体二氢硫辛酰胺脱氢酶或E3。E1和E2组分对BCKD复合物是特异性的，而E3组分对所有三种α-酮酸脱氢酶复合物是共同的。此外，哺乳动物BCKD含有两种调节酶：特异性激酶和特异性磷酸酶，其通过磷酸化（失活）/去磷酸化（活化）循环调节BCKD的活性。BCKD围绕立方E2核心组织，E1的12个拷贝和E3的未指定数量的拷贝，激酶和磷酸酶通过离子相互作用连接。BCKD多酶复合物的分子量估计为4 × 106道尔顿。BCKD复合体在患有遗传性枫糖浆尿病（MSUD）的患者中缺乏。这种代谢障碍导致有毒支链α-酮酸的积累，表现为经常致命的酸中毒、神经紊乱和智力迟钝。对人类突变如何破坏人类BCKD复合物的催化机制的结构洞察将有助于开发改善MSUD表型的策略。具体目标：1。目的：通过冷冻电镜技术研究人BCKD复合体E2核心的三维结构。2.辨别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.