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Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism
Biochemistry ( IF 2.9 ) Pub Date : 2018-01-12 00:00:00 , DOI: 10.1021/acs.biochem.7b01151
Boguslaw Nocek 1 , Cory Reidl 2 , Anna Starus 2 , Tahirah Heath 2 , David Bienvenue 3, 4 , Jerzy Osipiuk 1 , Robert Jedrzejczak 1 , Andrzej Joachimiak 1 , Daniel P. Becker 2 , Richard C. Holz 4
Affiliation  

The X-ray crystal structure of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae (HiDapE) bound by the products of hydrolysis, succinic acid and l,l-DAP, was determined at 1.95 Å. Surprisingly, the structure bound to the products revealed that HiDapE undergoes a significant conformational change in which the catalytic domain rotates ∼50° and shifts ∼10.1 Å (as measured at the position of the Zn atoms) relative to the dimerization domain. This heretofore unobserved closed conformation revealed significant movements within the catalytic domain compared to that of wild-type HiDapE, which results in effectively closing off access to the dinuclear Zn(II) active site with the succinate carboxylate moiety bridging the dinculear Zn(II) cluster in a μ-1,3 fashion forming a bis(μ-carboxylato)dizinc(II) core with a Zn–Zn distance of 3.8 Å. Surprisingly, His194.B, which is located on the dimerization domain of the opposing chain ∼10.1 Å from the dinuclear Zn(II) active site, forms a hydrogen bond (2.9 Å) with the oxygen atom of succinic acid bound to Zn2, forming an oxyanion hole. As the closed structure forms upon substrate binding, the movement of His194.B by more than ∼10 Å is critical, based on site-directed mutagenesis data, for activation of the scissile carbonyl carbon of the substrate for nucleophilic attack by a hydroxide nucleophile. Employing the HiDapE product-bound structure as the starting point, a reverse engineering approach called product-based transition-state modeling provided structural models for each major catalytic step. These data provide insight into the catalytic reaction mechanism and also the future design of new, potent inhibitors of DapE enzymes.

中文翻译:

DapE酶中底物结合触发的主要构象变化的结构证据:对催化机制的影响。

的dapE的编码的X射线晶体结构Ñ -succinyl-从二氨基庚二酸desuccinylase流感嗜血杆菌您好通过水解,琥珀酸和的产物结合的DAPE)-DAP,在1.95埃确定。出乎意料的是,与产物结合的结构表明,Hi DapE经历了显着的构象变化,其中催化结构域相对于二聚结构域旋转了约50°,移位了约10.1Å(在Zn原子的位置测量)。迄今为止,这种未观察到的闭合构象揭示了与野生型Hi相比,在催化域内有明显的运动。DapE,其有效封闭了琥珀酸羧酸酯部分以μ-1,3方式桥接小分子Zn(II)簇的琥珀酸羧酸酯部分,从而形成了双(μ-羧基硅基)二嗪(II) )的Zn-Zn距离为3.8Å。令人惊讶的是,His194.B位于双链Zn(II)活性位点附近相对链的二聚化域〜10.1Å处,与琥珀酸的氧原子键合到Zn2上形成氢键(2.9Å),从而形成氧阴离子孔。由于封闭结构是在与底物结合时形成的,因此基于定点诱变数据,His194.B的移动超过约10Å是至关重要的,对于激活底物的易裂的羰基碳以通过氢氧化物亲核试剂进行亲核攻击是至关重要的。聘请DapE产品绑定结构作为起点,一种称为基于产品的过渡状态建模的逆向工程方法为每个主要的催化步骤提供了结构模型。这些数据提供了对催化反应机理的了解,也为新型,有效的DapE酶抑制剂的未来设计提供了依据。
更新日期:2018-01-12
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