Outer-Sphere Tyrosine 159 within the 3-Mercaptopropionic Acid Dioxygenase S-H-Y Motif Gates Substrate-Coordination Denticity at the Non-Heme Iron Active Site.
Outer-Sphere Tyrosine 159 within the 3-Mercaptopropionic Acid Dioxygenase S-H-Y Motif Gates Substrate-Coordination Denticity at the Non-Heme Iron Active Site.
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DOI：
10.1021/acs.biochem.9b00674
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发表时间：
2019-12-24
期刊：
影响因子：
2.9
通讯作者：
Pierce, Brad S.
中科院分区：
文献类型：
--
作者：
Sardar, Sinjinee;Weitz, Andrew;Hendrich, Michael P.;Pierce, Brad S.
巯基双加氧酶是非血红素单核铁酶，其催化游离巯基（-SH）的O2-依赖性氧化以产生相应的亚磺酸（-SO2-）。无论是哪一种结构域，这类酶的活性位点通常由两个主要特征组成：（1）由三个蛋白质衍生的组氨酸配位的单核亚铁和（2）与铁位点（丝氨酸-组氨酸-酪氨酸）空间相邻的外铁配位球氨基酸（丝氨酸-组氨酸-酪氨酸）的保守序列。在这里，我们利用一个混杂的3-巯基丙酸双加氧酶克隆棕色固氮菌（Av MDO），以探讨保守的S-H-Y基序的功能。该酶与3-巯基丙酸（3 mpa）、L-半胱氨酸（cys）以及其他几种含巯基的底物具有活性，因此使其成为研究高度保守的S-H-Y基序（H157和Y159）内的残基对底物特异性和反应性的影响的理想系统。通过pH依赖性稳态动力学确定这些残基的值，并通过与H157 N和Y159 F变体的比较验证其归属。互补电子顺磁共振和穆斯堡尔研究表明，在酶促Fe位点内连接H157-Y159和Fe结合配体的氢键网络。至关重要的是，这些实验表明，羟基的Y159氢键铁结合的NO，并通过扩展，铁结合的氧在天然催化。这种相互作用改变了NO的结合亲和力和菱形的3 mpa结合的铁-亚硝基网站。此外，铁的协调cys切换从硫醇盐只有双齿（硫醇盐/胺）的Y159 F的变体，表明扰动内的S-H-Y质子中继网络也影响cys铁结合denticity。
Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2–). Regardless of the phylogenic domain, the active site for this enzyme class is typically comprised of two major features: (1) a mononuclear ferrous iron coordinated by three protein-derived histidines and (2) a conserved sequence of outer Fe-coordination-sphere amino acids (Ser-His-Tyr) spatially adjacent to the iron site (∼3 Å). Here, we utilize a promiscuous 3-mercaptopropionic acid dioxygenase cloned from Azotobacter vinelandii (Av MDO) to explore the function of the conserved S-H-Y motif. This enzyme exhibits activity with 3-mercaptopropionic acid (3mpa), L-cysteine (cys), as well as several other thiol-bearing substrates, thus making it an ideal system to study the influence of residues within the highly conserved S-H-Y motif (H157 and Y159) on substrate specificity and reactivity. The values for these residues were determined by pH-dependent steady-state kinetics, and their assignments verified by comparison to H157N and Y159F variants. Complementary electron paramagnetic resonance and Mössbauer studies demonstrate a network of hydrogen bonds connecting H157–Y159 and Fe-bound ligands within the enzymatic Fe site. Crucially, these experiments suggest that the hydroxyl group of Y159 hydrogen bonds to Fe-bound NO and, by extension, Fe-bound oxygen during native catalysis. This interaction alters both the NO binding affinity and rhombicity of the 3mpa-bound iron–nitrosyl site. In addition, Fe coordination of cys is switched from thiolate only to bidentate (thiolate/amine) for the Y159F variant, indicating that perturbations within the S-H-Y proton relay network also influence cys Fe binding denticity.
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影响因子：
15
作者：
Fielding, Andrew J.;Lipscomb, John D.;Que, Lawrence, Jr.
通讯作者：
Que, Lawrence, Jr.
影响因子：
2.9
作者：
Diebold, Adrienne R.;Neidig, Michael L.;Moran, Graham R.;Straganz, Grit D.;Solomon, Edward I.
通讯作者：
Solomon, Edward I.
影响因子：
4.1
作者：
GEORGE, P;HANANIA, G
通讯作者：
HANANIA, G
影响因子：
2.9
作者：
Crowell, Joshua K.;Li, Wei;Pierce, Brad S.
通讯作者：
Pierce, Brad S.
影响因子：
4.8
作者：
Dominy, John E.;Hwang, Jesse;Stipanuk, Martha H.
通讯作者：
Stipanuk, Martha H.