Structural and Functional Insights into the Regulation of Helicobacter pylori Arginase Activity by an Evolutionary Nonconserved Motif

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• 作者：Abhishek Srivastava ; Shiv Kumar Meena ; Mashkoor Alam ; Shahid M. Nayeem ; Shashank Deep ; Apurba Kumar Sau
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：January 22, 2013
• 年：2013
• 卷：52
• 期：3
• 页码：508-519
• 全文大小：727K
• 年卷期：v.52,no.3(January 22, 2013)
• ISSN：1520-4995

文摘

Urea producing bimetallic arginases are essential for the synthesis of polyamine, DNA, and RNA. Despite conservation of the signature motifs in all arginases, a nonconserved ¹⁵³ESEEKAWQKLCSL¹⁶⁵ motif is found in the Helicobacter pylori enzyme, whose role is yet unknown. Using site-directed mutagenesis, kinetic assays, metal analyses, circular dichroism, heat-induced denaturation, molecular dynamics simulations and truncation studies, we report here the significance of this motif in catalytic function, metal retention, structural integrity, and stability of the protein. The enzyme did not exhibit detectable activity upon deletion of the motif as well as on individual mutation of Glu155 and Trp159 while Cys163Ala displayed significant decrease in the activity. Trp159Ala and Glu155Ala show severe loss of thermostability (14鈥?7掳) by a decrease in the 伪-helical structure. The role of Trp159 in stabilization of the structure with the surrounding aromatic residues is confirmed when Trp159Phe restored the structure and stability substantially compared to Trp159Ala. The simulation studies support the above results and show that the motif, which was previously solvent exposed, displays a loop-cum-small helix structure (Lys161鈥揅ys163) and is located near the active-site through a novel Trp159鈥揂sp126 interaction. This is consistent with the mutational analyses, where Trp159 and Asp126 are individually critical for retaining a bimetallic center and thereby for function. Furthermore, Cys163 of the helix is primarily important for dimerization, which is crucial for stimulation of the activity. Thus, these findings not only provide insights into the role of this motif but also offer a possibility to engineer it in human arginases for therapeutics against a number of carcinomas.