The "Gln-Type" Thiol Dioxygenase from Azotobacter vinelandii Is a 3-Mercaptopropionic Acid Dioxygenase
The "Gln-Type" Thiol Dioxygenase from Azotobacter vinelandii Is a 3-Mercaptopropionic Acid Dioxygenase
复制标题
DOI：
10.1021/acs.biochem.5b00636
复制
发表时间：
2015-12-29
期刊：
影响因子：
2.9
通讯作者：
Crowell, Joshua K.
中科院分区：
文献类型：
--
作者：
Pierce, Brad S.;Subedi, Bishnu P.;Crowell, Joshua K.
半胱氨酸双加氧酶（CDO）是一种非血红素铁酶，催化l -半胱氨酸o -2依赖性氧化生成半胱氨酸磺酸。基于保守活性位点残基的特性，细菌cdo被细分为“arg型”或“gln型”。迄今为止，“gln型”酶在很大程度上仍未被表征。最近注意到，“gln型”酶与Variovorax paradoxus中发现的另一种硫醇双加氧酶[3-巯基丙酸双加氧酶（MDO）]更为同源，这表明“gln型”亚类酶实际上是MDO。本研究纯化了一种推测为“gln型”硫醇双加氧酶，并对其进行了表征。采用三种底物[3-巯基丙酸（3mpa）、l -半胱氨酸（cys）和半胱胺（ca）]进行稳态测定。尽管最大速度相当，但“gln型”Av酶的特异性为3mpa (k(cat)/ k - m = 72000 M-1 s(-1))，比cys （110 M-1 s(-1)）和ca （11 M-1 s(-1)）高出近2个数量级。支持x波段电子顺磁共振（EPR）研究使用一氧化氮（NO）作为O-2结合的替代物，以确认在NO之前有选择性地添加底物。在3mpa结合酶的溶液中添加NO，可以定量地得到一个具有EPR特征的单核（S = 3/2） {FeNO}(7)位点的亚硝基铁（Av - ES-NO）。相反，在用cys和ca制备的Av - ES-NO样品中观察到两种不同的底物结合构象，表明酶活性位点内的异质结合。通过分析EPR模拟来建立每个底物的相对结合亲和力（3map > cys > ca）。动力学和光谱结果都表明3mpa是该酶的首选底物。
Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O-2-dependent oxidation of L-cysteine to produce cysteinesulfinic acid. Bacterial CDOs have been subdivided as either "Arg-type" or "Gln-type" on the basis of the identity of conserved active site residues. To date, "Gln-type" enzymes remain largely uncharacterized. It was recently noted that the "Gln-type" enzymes are more homologous with another thiol dioxygenase [3-mercaptopropionate dioxygenase (MDO)] identified in Variovorax paradoxus, suggesting that enzymes of the "Gln-type" subclass are in fact MDOs. In this work, a putative "Gln-type" thiol dioxygenase from Azotobacter vinelandii (Av) was purified to homogeneity and characterized. Steady-state assays were performed using three substrates [3-mercaptopropionic acid (3mpa), L-cysteine (cys), and cysteamine (ca)]. Despite comparable maximal velocities, the "Gln-type" Av enzyme exhibited a specificity for 3mpa (k(cat)/K-M = 72000 M-1 s(-1)) nearly 2 orders of magnitude greater than those for cys (110 M-1 s(-1)) and ca (11 M-1 s(-1)). Supporting X-band electron paramagnetic resonance (EPR) studies were performed using nitric oxide (NO) as a surrogate for O-2 binding to confirm obligate-ordered addition of substrate prior to NO. Stoichimetric addition of NO to solutions of 3mpa-bound enzyme quantitatively yields an iron-nitrosyl species (Av ES-NO) with EPR features consistent with a mononuclear (S = 3/2) {FeNO}(7) site. Conversely, two distinct substrate-bound conformations were observed in Av ES-NO samples prepared with cys and ca, suggesting heterogeneous binding within the enzymatic active site. Analytical EPR simulations are provided to establish the relative binding affinity for each substrate (3map > cys > ca). Both kinetic and spectroscopic results presented here are consistent with 3mpa being the preferred substrate for this enzyme.