National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, Delhi, India.
IUBMB Life. 2018 Jul;70(7):658-669. doi: 10.1002/iub.1754. Epub 2018 Apr 23.
Despite importance of arginine decarboxylase (ADC: EC 4.1.1.19) of Helicobacter pylori (H. pylori) 26695 pathogenic strain for acid adaptation in host, the enzyme has not yet been studied at a molecular level. Using combined approaches that include kinetic assays, site-directed mutagenesis, circular dichroism, heat-induced denaturation, analytical gel-filtration, and homology modeling, we report here a detailed investigation of H. pylori ADC. The pyridoxal 5'-phosphate (PLP)-dependent enzyme exhibits higher catalytic activity in the presence of Mg ions at pH ∼8.5. Unlike other bacterial ADCs, this homolog exists as a hexamer. The higher thermal stability (T ∼65.8 ± 0.2 °C) of the enzyme observed from the heat-induced circular dichroism measurements indicates its secondary structural stabilization in the presence of PLP. The kinetic parameters K and k of the enzyme are determined to be 3.4 ± 0.2 mM and 55.2 ± 1.0 min , respectively. We elucidate that Cys487, a conserved residue located at the active-site, is involved in the catalysis, whose pK value was estimated to be ∼7.2. The homology model of the protein show conserved α/β TIM barrel and β-sandwich domains, which are characteristic features of fold III decarboxylases. A lower sequence identity (∼21%) of this enzyme compared with its human counterpart has enabled us to screen putative inhibitors of H. pylori ADC. We found that α-difluoromethylarginine inhibits the activity of the H. pylori enzyme competitively with a K value ∼118 µM and thus it can serve as a basis to design inhibitors with higher efficacy against this ADC. © 2018 IUBMB Life, 70(7):658-669, 2018.
尽管幽门螺杆菌（H. pylori）26695 致病株的精氨酸脱羧酶（ADC：EC 4.1.1.19）对宿主的酸适应非常重要，但该酶尚未在分子水平上进行研究。本研究采用包括动力学测定、定点突变、圆二色性、热诱导变性、分析凝胶过滤和同源建模在内的综合方法，详细研究了 H. pylori ADC。依赖于吡哆醛 5'-磷酸（PLP）的酶在 pH∼8.5 时在存在镁离子的情况下表现出更高的催化活性。与其他细菌 ADC 不同，该同源物以六聚体形式存在。从热诱导圆二色性测量中观察到的酶较高的热稳定性（T∼65.8±0.2°C）表明其在存在 PLP 的情况下二级结构稳定。酶的动力学参数 K 和 k 分别确定为 3.4±0.2 mM 和 55.2±1.0 min 。我们阐明了位于活性部位的保守残基半胱氨酸 487 参与催化，其 pK 值估计约为 7.2。该蛋白的同源模型显示保守的α/β TIM 桶和β-三明治结构域，这是折叠 III 脱羧酶的特征。与人类对应物相比，该酶的序列同一性（约 21%）较低，这使我们能够筛选幽门螺杆菌 ADC 的潜在抑制剂。我们发现α-二氟甲基精氨酸以 K 值约 118 µM 竞争性抑制 H. pylori 酶的活性，因此它可以作为设计对该 ADC 具有更高疗效的抑制剂的基础。©2018 IUBMB Life，70（7）：658-669，2018。
