Tight binding of heme to Staphylococcus aureus IsdG and IsdI precludes design of a competitive inhibitor.
Tight binding of heme to Staphylococcus aureus IsdG and IsdI precludes design of a competitive inhibitor.
复制标题
DOI：
10.1039/c7mt00035a
复制
发表时间：
2017-05-24
期刊：
影响因子：
--
通讯作者：
Liptak MD
中科院分区：
文献类型：
--
作者：
Conger MA;Pokhrel D;Liptak MD
文献中报道的IsdG和IsdI的血红素解离的微摩尔平衡常数令人质疑这些酶是否真的是金黄色葡萄球菌铁调节表面决定簇系统的成员，该系统在感染期间从宿主获取血红素铁。为了解决这个问题，使用三种方法重新评估了IsdG和IsdI的血红素解离常数。使用基于无假设模型分析的紫外 - 可见吸收检测法以及一种新开发的荧光检测法测量血红素解离平衡常数。使用脱辅基肌红蛋白竞争实验估计血红素解离速率常数。对紫外 - 可见吸收数据的分析揭示了先前测量中的一个关键缺陷；在紫外 - 可见吸收光谱所需的微摩尔浓度下，99.9%的血红素与蛋白质结合，这使得精确测量平衡常数几乎不可能。然而，可以对更稀的样品测量荧光，对这些数据的分析得出IsdG和IsdI的解离平衡常数分别为1.4 ± 0.6纳摩尔和12.9 ± 1.3纳摩尔。对脱辅基肌红蛋白竞争实验获得的动力学数据的分析估计IsdG的血红素解离速率常数为0.022 ± 0.002秒⁻¹，IsdI为0.092 ± 0.008秒⁻¹。基于这些数据以及关于IsdG和IsdI翻译后调控的已知信息，有人提出只有IsdG是血红素铁获取途径的成员，IsdI调节血红素内稳态。此外，纳摩尔解离常数意味着血红素被IsdG紧密结合，表明对该蛋白质的竞争性抑制将很困难。相反，基于对酶机制的详细了解的非竞争性抑制是一种更有前景的抗生素开发策略。
一种新的基于荧光的检测方法表明，金黄色葡萄球菌IsdG和IsdI对血红素的亲和力比先前认识到的高2 - 3个数量级。
The micromolar equilibrium constants for heme dissociation from IsdG and IsdI reported in the literature call into question whether these enzymes are actually members of the iron-regulated surface determinant system of Staphylococcus aureus, which harvests heme iron from a host during infection. In order to address this question, the heme dissociation constants for IsdG and IsdI were reevaluated using three approaches. The heme dissociation equilibrium constants were measured using a UV/Vis absorption-detected assay analyzed with an assumption-free model, and using a newly developed fluorescence-detected assay. The heme dissociation rate constants were estimated using apomyoglobin competition assays. Analyses of the UV/Vis absorption data revealed a critical flaw in the previous measurements; heme is 99.9% protein-bound at the micromolar concentrations needed for UV/Vis absorption spectroscopy, which renders accurate equilibrium constant measurement nearly impossible. However, fluorescence can be measured for more dilute samples, and analyses of these data resulted in dissociation equilibrium constants of 1.4 ± 0.6 nM and 12.9 ± 1.3 nM for IsdG and IsdI, respectively. Analyses of the kinetic data obtained from apomyoglobin competition assays estimated heme dissociation rate constants of 0.022 ± 0.002 s-1 for IsdG and 0.092 ± 0.008 s-1 for IsdI. Based upon these data, and what is known regarding the post-translational regulation of IsdG and IsdI, it is proposed that only IsdG is a member of the heme iron acquisition pathway and IsdI regulates heme homeostasis. Furthermore, the nanomolar dissociation constants mean that heme is bound tightly by IsdG and indicates that competitive inhibition of this protein will be difficult. Instead, uncompetitive inhibition based upon a detailed understanding of enzyme mechanism is a more promising antibiotic development strategy. A new fluorescence-based assay reveals that the affinities of Staphylococcus aureus IsdG and IsdI for heme are 2-3 orders of magnitude greater than previously realized.
登录
查看更多内容
影响因子：
2.9
作者：
HERNANDEZ, G;WILKS, A;LAMAR, GN
通讯作者：
LAMAR, GN
影响因子：
3.6
作者：
Reniere, Michelle L.;Skaar, Eric P.
通讯作者：
Skaar, Eric P.
影响因子：
2.9
作者：
BERRY, EA;TRUMPOWER, BL
通讯作者：
TRUMPOWER, BL
影响因子：
2.9
作者：
Koga, Shinya;Yoshihara, Shun;Sakamoto, Hiroshi
通讯作者：
Sakamoto, Hiroshi
影响因子：
4.8
作者：
Carter, Eric L.;Gupta, Nirupama;Ragsdale, Stephen W.
通讯作者：
Ragsdale, Stephen W.