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

Abstract

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.

The immunomodulatory dCache chemoreceptor TlpA ofHelicobacter pyloribinds multiple attractant and antagonistic ligands via distinct sites

“…The three molecules that elicited a chemotaxis response, arginine, fumarate, and cysteine, have been shown to have important biological roles in H. pylori biology. Arginine is an essential amino acid for H. pylori under in vitro growth conditions (Nedenskov, 1994), and H. pylori uses arginine to promote acid tolerance and dampen host immune responses (Alam et al, 2018;Gobert and Wilson, 2016;Mcgee et al, 1999;Valenzuela et al, 2014). Fumarate is predicted to be an alternative terminal electron acceptor for growth under anaerobic respiration (Mendz et al, 1995), and the associated enzyme, fumarate reductase, is essential for H. pylori colonization in vivo (Ge et al, 2000).…”

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

“…4 In H. pylori, the enzyme arginase, which is encoded by the rocF gene plays an important role in acid resistance. [5][6][7] In general, arginase (EC 3.5.3.1) uses divalent metal ions to catalyse the conversion of L-arginine to urea and L-ornithine. This protein is found in both prokaryotes and eukaryotes.…”

Hydroxylamine Analogue of Agmatine: Magic Bullet for Arginine Decarboxylase

“…The spectrum of the ADC inhibitors is not as extensive as in the case of ODC. The most well-known of these molecules is D,L-α-(difluoromethyl)arginine (DFMA) [26], which has been successfully used to inhibit ADC from different microorganisms and plants [27][28][29][30].…”