Hpn-like (Hpnl) protein, encoded by the hpnl gene in Helicobacter pylori and featuring a histidine-rich and two glutamine-rich motifs, can render nickel tolerance to H. pylori when the external nickel level reaches toxic limits. We found that the recombinant Hpnl exists as an oligomer in the native state and binds to two molar equivalents of nickel ions per monomer with a dissociation constant of 3.8 microM. Nickel could be released from Hpnl either at acidic pH (pH(1/2) 4.6) or in the presence of chelate ligands, such as EDTA (t(1/2) = 220, 355, and 716 min at pH 6.0, 7.0, and 7.5, respectively). Our combined spectroscopic data show that nickel ion coordinates to a nitrogen of a histidine residue possibly with a coordination number of four (square-planar geometry) or five. The growth of Escherichia coli cells with or without the hpnl gene implied a protective role of Hpnl under higher concentrations of external nickel ions. Hpnl may serve a role in binding/storage or detoxification of excess nickel ions.
Bismuth compounds are widely used for the treatment of peptic ulcers and Helicobacter pylori infections. It has been suggested that enzyme inhibition plays an important role in the antibacterial activity of bismuth towards this bacterium. Urease, an enzyme that converts urea into ammonia and carbonic acid, is crucial for colonization of the acidic environment of the stomach by H. pylori. Here, we show that three bismuth complexes exhibit distinct mechanisms of urease inhibition, with some differences dependent on the source of the enzyme. Bi(EDTA) and Bi(Cys)(3) are competitive inhibitors of jack bean urease with K(i) values of 1.74 +/- 0.14 and 1.84 +/- 0.15 mM, while the anti-ulcer drug, ranitidine bismuth citrate (RBC) is a non-competitive inhibitor with a K (i) value of 1.17 +/- 0.09 mM. A (13)C NMR study showed that Bi(Cys)(3) reacts with jack bean urease during a 30 min incubation, releasing free cysteines from the metal complex. Upon incubation with Bi(EDTA) and RBC, the number of accessible cysteine residues in the homohexameric plant enzyme decreased by 5.80 +/- 0.17 and 11.94 +/- 0.13, respectively, after 3 h of reaction with dithiobis(2-nitrobenzoic acid). Kinetic analysis showed that Bi(EDTA) is both a competitive inhibitor and a time-dependent inactivator of the recombinant Klebsiella aerogenes urease. The active C319A mutant of the bacterial enzyme displays a significantly reduced sensitivity toward inactivation by Bi(EDTA) compared with the wild-type enzyme, consistent with binding of Bi(3+) to the active site cysteine (Cys(319)) as the mechanism of enzyme inactivation.
Enzyme-mediated damage repair or mitigation, while common for nucleic acids, is rare for proteins. Examples of protein damage are elimination of phosphorylated Ser/Thr to dehydroalanine/dehydrobutyrine (Dha/ Dhb) in pathogenesis and aging. Bacterial LanC enzymes use Dha/Dhb to form carbon-sulfur linkages in antimicrobial peptides, but the functions of eukaryotic LanC-like (LanCL) counterparts are unknown. We show that LanCLs catalyze the addition of glutathione to Dha/Dhb in proteins, driving irreversible C-glutathionylation. Chemo-enzymatic methods were developed to site-selectively incorporate Dha/Dhb at phospho-regulated sites in kinases. In human MAPK-MEK1, such ''elimination damage'' generated aberrantly activated kinases, which were deactivated by LanCL-mediated C-glutathionylation. Surveys of endogenous proteins bearing damage from elimination (the eliminylome) also suggest it is a source of electrophilic reactivity. LanCLs thus remove these reactive electrophiles and their potentially dysregulatory effects from the proteome. As knockout of LanCL in mice can result in premature death, repair of this kind of protein damage appears important physiologically. ll
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.