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首页> 外文期刊>ACS catalysis >Conformational Sampling of the Intrinsically Disordered C-Terminal Tail of DERA Is Important for Enzyme Catalysis
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Conformational Sampling of the Intrinsically Disordered C-Terminal Tail of DERA Is Important for Enzyme Catalysis

机译：Dera的本质上无序的C末端尾部的构象取样对于酶催化很重要

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2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes the reversible conversion of acetaldehyde and glyceraldehyde-3-phosphate into deoxyribose-5-phosphate. DERA is used as a biocatalyst for the synthesis of drugs such as statins and is a promising pharmaceutical target due to its involvement in nucleotide catabolism. Despite previous biochemical studies suggesting the catalytic importance of the C-terminal tyrosine residue found in several bacterial DERAs, the structural and functional basis of its participation in catalysis remains elusive because the electron density for the last eight to nine residues (i.e., the C-terminal tail) is absent in all available crystal structures. Using a combination of NMR spectroscopy and molecular dynamics simulations, we conclusively show that the rarely studied C-terminal tail of E. coli DERA (ecDERA) is intrinsically disordered and exists in equilibrium between open and catalytically relevant closed states, where the C-terminal tyrosine (Y259) enters the active site. Nuclear Overhauser effect distance restraints, obtained due to the presence of a substantial closed state population, were used to derive the solution-state structure of the ecDERA closed state. Real-time NMR hydrogen/deuterium exchange experiments reveal that Y259 is required for efficiency of the proton abstraction step of the catalytic reaction. Phosphate titration experiments show that, in addition to the phosphate-binding residues located near the active site, as observed in the available crystal structures, ecDERA contains previously unknown auxiliary phosphate-binding residues on the C-terminal tail which could facilitate in orienting Y259 in an optimal position for catalysis. Thus, we present significant insights into the structural and mechanistic importance of the ecDERA C-terminal tail and illustrate the role of conformational sampling in enzyme catalysis.
机译：2-脱氧-5-磷酸醛糖酶（DERA）催化乙醛和甘油醛-3-磷酸盐中的可逆转化成脱氧-5-磷酸盐。 Dera用作生物催化剂，用于合成他汀类药物，并且是由于其参与核苷酸分解代谢而具有有前途的药物靶标。尽管先前的生化研究表明，在几种细菌DERAS中发现的C末端酪氨酸残基的催化重要性，其参与催化的结构和功能仍然是难以捉摸的，因为最后八至九个残留物的电子密度（即，C-终端尾部）在所有可用的晶体结构中不存在。使用NMR光谱和分子动力学模拟的组合，我们得出结论地表明，大肠杆菌DERA（ECDERA）的很少研究的C末端尾部是本质上的，并且在开放和催化相关的封闭状态之间的平衡中存在于C末端酪氨酸（Y259）进入活跃点。由于存在大量闭合状态群而导致的核传承效应距离限制来得出ecdera闭合状态的溶液状态结构。实时NMR氢气/氘交换实验表明，Y259是催化反应质子抽象步骤的效率所必需的。磷酸盐滴定实验表明，除了位于活性位点附近的磷酸盐结合残留物之外，如在可用晶体结构中观察到的，ECDERA含有先前未知的辅助磷酸盐结合残留物在C末端尾部，这可以有助于定向Y259催化的最佳位置。因此，我们对ECDERA C末端尾部的结构和机械重要性提出了显着的见解，并说明了构象抽样在酶催化中的作用。

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