National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Departments of Chemical Sciences, Tata Institute of Fundamental Research, Dr. Homi Bhabha Road, Colaba, Mumbai 400005, India.
Biochem J. 2021 Feb 26;478(4):871-894. doi: 10.1042/BCJ20200978.
The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.
幽门螺杆菌双金属酶精氨酸酶对该细菌在人胃中的发病机制非常重要。尽管催化残基保守，但这种单一的色氨酸酶具有一个插入序列（-153ESEEKAWQKLCSL165-），对其功能至关重要。该序列包含关键残基，这些残基在其他幽门螺杆菌胃病原体的同源物中保守。然而，该模体在催化功能中的作用的基础尚不完全清楚。在这里，我们使用生化、生物物理和分子动力学模拟研究来确定该延伸中的Glu155 与 Lys57 和 Ser152 相互作用。这些相互作用对于通过位于 Glu155 附近的 Trp159 定位该模体至关重要（His122-Trp159-Tyr125 接触对于三级结构完整性至关重要）。Lys57 和 Ser152 单独或双突变为 Ala 会大大降低催化活性，其中 Lys57 到 Ala 的突变更为显著，表明它们对功能至关重要。我们的数据表明，Lys57-Glu155-Ser152 相互作用影响含有催化 His133 的环的定位，使得该 His 可以参与催化，从而提供了对该模体在催化功能中的作用的机制理解。Lys57 也仅存在于其他幽门螺杆菌胃病原体的精氨酸酶中。基于非保守模体，我们发现了一种新的分子，它可以特异性抑制这种酶。因此，本研究不仅为该模体在功能中的作用提供了分子基础，还为设计更有效的抑制剂提供了机会。
