Structural and Functional Insights into the Regulation ofHelicobacter pyloriArginase Activity by an Evolutionary Nonconserved Motif

Abstract

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 (14–17°) by a decrease in the α-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–Cys163) and is located near the active-site through a novel Trp159–Asp126 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.

Synthesis and Characterization of 4‐, 5‐, and 6‐Coordinate Tris(1‐ethyl‐4‐isopropylimidazolyl‐κN)phosphine Cobalt(II) Complexes

“…DFT studies were performed to help understand the basis for these changes. 3 ]-(OTf) 2 (4) were synthesized by using similar methods but by altering either the cobalt salt or the solvent to achieve the respective complexes. For example, 1 was obtained by treating cobalt(II) triflate with T1Et4iPrIP (1 equiv.)…”

Illuminating the structure–function landscape of an evolutionary nonconserved motif in the arginases of Helicobacter gastric pathogens

“…62,67 However, due to the absence of the analogous 13-residue insertion sequence in other homologs, in the model structure this stretch was found as a loop and present on the surface of the protein. 60 This also could not explain the possible role of the motif in structure or function.…”

“…Srivastava et al performed detailed sequence analysis and showed that this stretch is not present in the other homologs. 60 To obtain insight into this motif's role in structure and function, they performed molecular dynamics (MD) simulations studies using the model structure of H. pylori apoarginase for 450 ns. During the simulations, this motif was found to move towards the active site and forms a loop-cum-helix structure.…”

“…24,62,64 A notable difference was also observed in the H. pylori enzyme, where a 13-residue long nonconserved motif ( 153 ESEEKAWQKLCSL 165 ) was present in the middle of the protein sequence (Figure 2). 5,30,60,65,66 This review delivers a structural basis into the role of the nonconserved motif of this enzyme in function and the knowledge gained could be utilised to design small molecule inhibitors specific to the arginases of H. pylori and other Helicobacter gastric pathogens.…”

Biochemical and biophysical studies of Helicobacter pylori arginine decarboxylase, an enzyme important for acid adaptation in host

“…The bacterium has a unique mechanism to survive in the acidic environment of the stomach lumen (pH ∼1–2) and colonizes near the mucosal layer, where the pH is around 5–7 . The acid protection is conferred by H. pylori arginase (RocF), a well‐studied enzyme that utilizes L‐arginine to generate urea and L‐ornithine Further, the degradation of urea to ammonia by urease elevates the pH of the microenvironment , which helps in the survival of the bacterium. Like other arginine decarboxylases (ADCs), the H. pylori enzyme has been shown to catalyze the conversion of L‐arginine to agmatine and CO 2 .…”