Cofactor Complexes of DesD, a Model Enzyme in the Virulence-related NIS Synthetase Family
Cofactor Complexes of DesD, a Model Enzyme in the Virulence-related NIS Synthetase Family
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DOI：
10.1021/acs.biochem.9b00899
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发表时间：
2020-09-22
期刊：
影响因子：
2.9
通讯作者：
Orion, Iris W.
中科院分区：
文献类型：
--
作者：
Hoffmann, Katherine M.;Goncuian, Eliana S.;Orion, Iris W.
由于非核糖体-肽-合成酶独立的铁载体（NIS）合成酶家族在合成羟酸盐和羧酸盐“隐形”铁载体中起关键作用，因此越来越多地与细菌物种的毒力相关。我们已经从链霉菌中鉴定出一个模型家族成员DesD，并在载脂蛋白、辅因子产物amp结合和辅因子反应物atp结合的复合物中使用野生型和Arg306Gln变体的组合进行结构表征。该家族的动力学受到溶解度和报告基因测定的限制，因此我们开发了一种利用单次注射等温滴定-量热法的无标记动力学测定。我们报告的二阶速率常数比以前的估计高50倍。我们的Arg306Gln DesD变体也在相同的缓冲液和底物条件下进行了测试，证实了其不可检测的活性。这些是首次报道的DesD结构，它们描述了关键的辅因子协调。这也是第一个明确确定NIS合成酶动力学的无标记分析。
The understudied nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase family has been increasingly associated with virulence in bacterial species due to its key role in the synthesis of hydroxamate and carboxylate "stealth" siderophores. We have identified a model family member, DesD, from Streptomyces coelicolor, to structurally characterize using a combination of a wild-type and a Arg306Gln variant in apo, cofactor product AMP-bound, and cofactor reactant ATP-bound complexes. The kinetics in the family has been limited by solubility and reporter assays, so we have developed a label-free kinetics assay utilizing a single-injection isothermal-titration-calorimetry-based method. We report second-order rate constants that are 50 times higher than the previous estimations for DesD. Our Arg306Gln DesD variant was also tested under identical buffer and substrate conditions, and its undetectable activity was confirmed. These are the first reported structures for DesD, and they describe the critical cofactor coordination. This is also the first label-free assay to unambiguously determine the kinetics for an NIS synthetase.