Insights into the Substrate Specificity of Archaeal Entner-Doudoroff Aldolases: The Structures of Picrophilus torridus 2-Keto-3-deoxygluconate Aldolase and Sulfolobus solfataricus 2-Keto-3-deoxy-6-phosphogluconate Aldolase in Complex with 2-Keto-3-deoxy-6-phosphogluconate.
Insights into the Substrate Specificity of Archaeal Entner-Doudoroff Aldolases: The Structures of Picrophilus torridus 2-Keto-3-deoxygluconate Aldolase and Sulfolobus solfataricus 2-Keto-3-deoxy-6-phosphogluconate Aldolase in Complex with 2-Keto-3-deoxy-6-phosphogluconate.
复制标题
深入了解古菌 Entner-Doudoroff 醛缩酶的底物特异性：Picrophilus torridus 2-Keto-3-deoxygluconate 醛缩酶和 Sulfolobus solfataricus 2-Keto-3-deoxy-6-磷酸葡萄糖酸醛缩酶与 2-Keto-3-deoxy 复合物的结构
作者：
V. Zaitsev;U. Johnsen;Matthias Reher;Marius Ortjohann;G. Taylor;M. Danson;P. Schönheit;S. Crennell
嗜热嗜酸古菌Picrophilus torridus和硫磺硫化叶菌Sulfolobus solfataricus分别通过非磷酸化的Entner-Doudoroff途径和分支的Entner-Doudoroff途径分解代谢葡萄糖。这些Entner-Doudoroff途径的关键酶是醛缩酶、2-酮-3-脱氧葡萄糖酸醛缩酶（KDG-醛缩酶）和2-酮-3-脱氧-6-磷酸葡萄糖酸醛缩酶[KD（P）G-醛缩酶]。来自P. torridus的KDG-醛缩酶（Pt-KDG-醛缩酶）对非磷酸化底物2-酮-3-脱氧葡萄糖酸（KDG）具有高度特异性，而来自S. Solfataricus [Ss-KD（P）G-醛缩酶]是催化KDG和2-酮基-3-脱氧-6-磷酸葡萄糖酸盐（KDPG）裂解的酶，优选KDPG。与更混杂的Ss-KD（P）G-醛缩酶相比，Pt-KDG-醛缩酶对KDG的高特异性的结构基础之前尚未分析。本文报道了在2.35 ℃下，Ss-KD（P）G-醛缩酶与KDPG复合物的结构解析，以及在2.50 ℃下，P. torridus的KDG-醛缩酶的结构解析。通过两种酶的活性位点的叠加，以及随后的定点突变研究，在Ss-KD（P）G-醛缩酶中鉴定出四个氨基酸的网络，即Arg 106、Tyr 132、Arg 237和Ser 241，其与KDPG的带负电荷的磷酸基团相互作用，从而增加酶对KDPG的亲和力。Pt-KDG-醛缩酶中不存在这种KDPG结合网络，这解释了KDPG裂解的低催化效率。
The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolize glucose via a nonphosphorylative Entner-Doudoroff pathway and a branched Entner-Doudoroff pathway, respectively. Key enzymes for these Entner-Doudoroff pathways are the aldolases, 2-keto-3-deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase [KD(P)G-aldolase]. KDG-aldolase from P. torridus (Pt-KDG-aldolase) is highly specific for the nonphosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD(P)G-aldolase from S. solfataricus [Ss-KD(P)G-aldolase] is an enzyme that catalyzes the cleavage of both KDG and 2-keto-3-deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD(P)G-aldolase has not been analyzed before. In this work, we report the elucidation of the structure of Ss-KD(P)G-aldolase in complex with KDPG at 2.35 Å and that of KDG-aldolase from P. torridus at 2.50 Å resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed mutagenesis studies, a network of four amino acids, namely, Arg106, Tyr132, Arg237, and Ser241, was identified in Ss-KD(P)G-aldolase that interact with the negatively charged phosphate group of KDPG, thereby increasing the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-aldolase, which explains the low catalytic efficiency of KDPG cleavage.