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Identification of histidine residues that act as zinc ligands in beta-lactamase II by differential tritium exchange.

ABSTRACT: 1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.

SUBMITTER: Baldwin GS

PROVIDER: S-EPMC1186650 | biostudies-other | 1979 Jun

REPOSITORIES: biostudies-other

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Histidine residues of zinc ligands in beta-lactamase II.

Project description:1. The Zn(II)-requiring beta-lactamase from Bacillus cereus 569/H/9, which has two zinc-binding sites, was examined by 270 MHz 1H n.m.r. spectroscopy. Resonances were assigned to five histidine residues. 2. Resonances attributed to three of the histidine residues in the apoenzyme shift on the addition of one equivalent of Zn(II). 3. Although these three histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH range 6.0--9.0 in the mono-zinc enzyme. 4. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. 5. It is proposed that these three histidine residues act as zinc ligands at the tighter zinc-binding site. 6. Resonances attributed to a fourth histidine residue shift on addition of further zinc to the mono-zinc enzyme. It is proposed that this histidine residue acts as a Zn(II) ligand at the second zinc-binding site.

| S-EPMC1186089 | biostudies-other

Structure of bradavidin-C-terminal residues act as intrinsic ligands.

Project description:Bradavidin is a homotetrameric biotin-binding protein from Bradyrhizobium japonicum, a nitrogen fixing and root nodule-forming symbiotic bacterium of the soybean. Wild-type (wt) bradavidin has 138 amino acid residues, whereas the C-terminally truncated core-bradavidin has only 118 residues. We have solved the X-ray structure of wt bradavidin and found that the C-terminal amino acids of each subunit were uniquely bound to the biotin-binding pocket of an adjacent subunit. The biotin-binding pocket occupying peptide (SEKLSNTK) was named "Brad-tag" and it serves as an intrinsic stabilizing ligand in wt bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and a binding affinity of ?25 µM was measured. In order to study the potential of Brad-tag, a green fluorescent protein tagged with Brad-tag was prepared and successfully concentrated from a bacterial cell lysate using core-bradavidin-functionalized Sepharose resin.

| S-EPMC3344845 | biostudies-literature

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.

Project description:Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.Using His-HDX-MS, the pK(a) values and the half-lives (t(1/2)) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK(a) and t(1/2)) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a), t(1/2) or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a) and t(1/2) changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

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Identifying Zn-bound histidine residues in metalloproteins using hydrogen-deuterium exchange mass spectrometry.

Project description:In this work, we have developed a method that uses hydrogen-deuterium exchange (HDX) of C2-hydrogens of histidines coupled with mass spectrometry (MS) to identify Zn-bound histidines in metalloproteins. This method relies on differences in HDX reaction rates of Zn-bound and Zn-free His residues. Using several model peptides and proteins, we find that all Zn-bound His residues have substantially lower HDX reaction rates in the presence of the metal. The vast majority of non-Zn-binding His residues undergo no significant changes in HDX reaction rates when their reactivity is compared in the presence and absence of Zn. Using this new approach, we then determined the Zn binding site of ?-2-microglobulin, a protein associated with metal-induced amyloidosis. Together, these results suggest that HDX-MS of His C2-hydrogens is a promising new method for identifying Zn-bound histidines in metalloproteins.

| S-EPMC3912567 | biostudies-literature

Structure of the plasmid-mediated class C beta-lactamase ACT-1.

Project description:The crystallographic structure of ACT-1, which is the first plasmid-mediated AmpC-type beta-lactamase to have been completely analyzed in terms of nucleotide sequence and which has a high degree of sequence similarity to the chromosomal AmpC enzymes of Enterobacter cloacae and the plasmid-encoded MIR-1, has been solved at 2.4 A resolution. The overall structure of ACT-1 is similar to those of other class C beta-lactamases, such as the AmpC enzymes from E. cloacae P99 and Escherichia coli.

| S-EPMC2376412 | biostudies-literature

The structure of the dizinc subclass B2 metallo-beta-lactamase CphA reveals that the second inhibitory zinc ion binds in the histidine site.

Project description:Bacteria can defend themselves against beta-lactam antibiotics through the expression of class B beta-lactamases, which cleave the beta-lactam amide bond and render the molecule harmless. There are three subclasses of class B beta-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-beta-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

| S-EPMC2764157 | biostudies-literature

Identification of histidine-122alpha in human haemoglobin as one of the unknown alkaline Bohr groups by hydrogen--tritium exchange.

Project description:Human carbonmonoxy- and deoxy-haemoglobins were incubated at 37 degrees C in 3H2O at various pH values to measure the pH-dependent hydrogen--tritium exchange at the C-2 position of the imidazole ring of histidine-122alpha. To obtain the pseudo-first-order rate constants for the exchange, k, the two peptides containing histidine-122alpha were isolated and the amounts of tritium incorporated were determined. The rate constants gave pK values for the histidine of 6.1 in carbonmonoxyhaemoglobin and 6.6 in deoxyhaemoglobin, showing that it contributes about 20% to the total alkaline Bohr effect and about 10% at pH7.4.

| S-EPMC1185820 | biostudies-other

Impact of derepressed AmpC beta-lactamase ACT-9 on the clinical efficacy of ertapenem.

Project description:An in vivo development of Pantoea agglomerans mutants (isolates PA2 to PA4) with reduced ertapenem susceptibility from that of isolate PA1 was associated with an inadequate clinical response to ertapenem therapy. All four isolates harbored the bla(ACT-9) AmpC ?-lactamase gene. However, a loss-of-function mutation in the ampD gene in PA2 to PA4, but not PA1, led to derepressed ACT-9. The reduced ertapenem susceptibility caused by derepressed ACT-9 was confirmed with an ampD knockout mutant of PA1.

| S-EPMC3165326 | biostudies-literature

The exchange of histidine C-2 protons in superoxide dismutases. A novel method for assigning histidine-metal ligands in proteins.

Project description:The rates of exchange of the C-2 protons of histidine residues in copper-zinc superoxide dismutase are substantially decreased by metal ion binding. This observation was used to distinguish between ligand and non ligand histidine residues in bovine and yeast copper-zinc superoxide dismutases; the effect was shown to depend only on metal ion co-ordination and not as a consequence of concomitant changes in protein structure. Selective deuteration of the zinc-only proteins at pH (uncorrected pH-meter reading) 8.2 and 50 degrees C resulted in the distinction between copper and zinc ligand resonances in the 1H n.m.r. spectrum of the enzymes. This method is proposed as a generally applicable technique for identifying histidine residues as ligands in metalloproteins.

| S-EPMC1161480 | biostudies-other

A Cephalosporin Prochelator Inhibits New Delhi Metallo-β-lactamase 1 without Removing Zinc.

Project description:Antibacterial drug resistance is a rapidly growing clinical threat, partially due to expression of β-lactamase enzymes, which confer resistance to bacteria by hydrolyzing and inactivating β-lactam antibiotics. The increasing prevalence of metallo-β-lactamases poses a unique challenge, as currently available β-lactamase inhibitors target the active site of serine β-lactamases but are ineffective against the zinc-containing active sites of metallo-β-lactamases. There is an urgent need for metallo-β-lactamase inhibitors and antibiotics that circumvent resistance mediated by metallo-β-lactamases in order to extend the utility of existing β-lactam antibiotics for treating infection. Here we investigated the antibacterial chelator-releasing prodrug PcephPT (2-((((6R,7R)-2-carboxy-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methyl)thio) pyridine 1-oxide) as an inhibitor of New Delhi metallo-β-lactamase 1 (NDM-1). PcephPT is an experimental compound that we have previously shown inhibits growth of β-lactamase-expressing E. coli using a mechanism that is dependent on both copper availability and β-lactamase expression. Here, we found that PcephPT, in addition to being a copper-dependent antibacterial compound, inhibits hydrolysis activity of purified NDM-1with an IC50 of 7.6 μM without removing zinc from the active site and restores activity of the carbapenem antibiotic meropenem against NDM-1-producing E. coli. This work demonstrates that targeting a metal-binding pharmacophore to β-lactamase-producing bacteria is a promising strategy for inhibition of both bacterial growth and metallo-β-lactamases.

| S-EPMC7266096 | biostudies-literature

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