Pei Xue Yuan, Titman Christopher M, Frank René A W, Leeper Finian J, Luisi Ben F
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Structure. 2008 Dec 10;16(12):1860-72. doi: 10.1016/j.str.2008.10.009.
The pyruvate dehydrogenase multienzyme assembly (PDH) generates acetyl coenzyme A and reducing equivalents from pyruvate in a multiple-step process that is a nexus of central metabolism. We report crystal structures of the Geobacillus stearothermophilus PDH E1p subunit with ligands that mimic the prereaction complex and the postdecarboxylation product. The structures implicate residues that help to orient substrates, nurture intermediates, and organize surface loops so that they can engage a mobile lipoyl domain that receives the acetyl group and shuttles it to the next active site. The structural and enzymatic data suggest that H128beta performs a dual role: first, as electrostatic catalyst of the reaction of pyruvate with the thiamine cofactor; and second, as a proton donor in the second reaction of acetyl group with the lipoate. We also identify I206alpha as a key residue in mediating the conformation of active-site loops. We propose that a simple conformational flip of the H271alpha side chain assists transfer of the acetyl group from thiamine cofactor to lipoyl domain in synchrony with reduction of the dithiolane ring.
丙酮酸脱氢酶多酶复合体（PDH）通过一个多步骤过程从丙酮酸生成乙酰辅酶A和还原当量，该过程是中心代谢的一个枢纽。我们报道了嗜热栖热放线菌PDH E1p亚基与模拟反应前复合物和脱羧后产物的配体的晶体结构。这些结构涉及一些残基，它们有助于使底物定向、培育中间体并组织表面环，以便它们能够与接收乙酰基并将其转运至下一个活性位点的可移动硫辛酰结构域结合。结构和酶学数据表明，H128β发挥双重作用：首先，作为丙酮酸与硫胺素辅因子反应的静电催化剂；其次，作为乙酰基与硫辛酸反应的第二步中的质子供体。我们还确定I206α是介导活性位点环构象的关键残基。我们提出，H271α侧链的一个简单构象翻转有助于乙酰基从硫胺素辅因子同步转移至硫辛酰结构域，同时二硫杂环戊烷环发生还原。
