当前位置：
X-MOL 学术
›
Biochemistry
›
论文详情
Our official English website, www.x-mol.net, welcomes your
feedback! (Note: you will need to create a separate account there.)
Cofactor Complexes of DesD, a Model Enzyme in the Virulence-related NIS Synthetase Family.
Biochemistry ( IF 2.9 ) Pub Date : 2020-09-04 , DOI: 10.1021/acs.biochem.9b00899 Katherine M Hoffmann 1 , Eliana S Goncuian 1 , Kimya L Karimi 1 , Caroline R Amendola 2 , Yasi Mojab 1 , Kaitlin M Wood 2 , Gregory A Prussia 2 , Jay Nix 3 , Margaret Yamamoto 2 , Kiera Lathan 1 , Iris W Orion 2
Biochemistry ( IF 2.9 ) Pub Date : 2020-09-04 , DOI: 10.1021/acs.biochem.9b00899 Katherine M Hoffmann 1 , Eliana S Goncuian 1 , Kimya L Karimi 1 , Caroline R Amendola 2 , Yasi Mojab 1 , Kaitlin M Wood 2 , Gregory A Prussia 2 , Jay Nix 3 , Margaret Yamamoto 2 , Kiera Lathan 1 , Iris W Orion 2
Affiliation
|
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.
中文翻译：
DesD的辅因子复合物，毒力相关的NIS合成酶家族中的模型酶。
由于其在异羟肟酸酯和羧酸盐“隐身”铁载体的合成中起关键作用，因此未被充分研究的非核糖体肽合成酶非依赖性铁载体（NIS）合成酶家族与细菌中的毒力越来越相关。我们已经确定了模型的家庭成员，可持续发展教育，从天蓝色链霉菌，使用野生型的组合和Arg306Gln变体在结构表征APO，辅因子产物AMP结合和辅因子反应物ATP结合的复合物。该家族中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基于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DesD的先前估算值高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，并证实了其不可检测的活性。这些是DesD首次报道的结构，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测定法。
更新日期：2020-09-22
中文翻译：
DesD的辅因子复合物，毒力相关的NIS合成酶家族中的模型酶。
由于其在异羟肟酸酯和羧酸盐“隐身”铁载体的合成中起关键作用，因此未被充分研究的非核糖体肽合成酶非依赖性铁载体（NIS）合成酶家族与细菌中的毒力越来越相关。我们已经确定了模型的家庭成员，可持续发展教育，从天蓝色链霉菌，使用野生型的组合和Arg306Gln变体在结构表征APO，辅因子产物AMP结合和辅因子反应物ATP结合的复合物。该家族中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基于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DesD的先前估算值高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，并证实了其不可检测的活性。这些是DesD首次报道的结构，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测定法。
京公网安备 11010802027423号