Helical Shape of Helicobacter pylori Requires an Atypical Glutamine as a Zinc Ligand in the Carboxypeptidase Csd4

Chan, Anson; Blair, Kris M.; Liu, Yanjie; Frirdich, Emilisa; Gaynor, Erin C.; Tanner, Martin E.; Salama, Nina R.; Murphy, M.E.P.

doi:10.1074/jbc.m114.624734

Cited by 17 publications

(36 citation statements)

References 43 publications

(44 reference statements)

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“…It should be noted that in the structure of the bound tripeptide substrate, the carbonyl of the iso-Glu side chain amide (the site of hydrolysis) is not coordinated to the zinc atom (Zn-O distance = 3.8 Å, Figure 2A). 16,17 This implies that the mechanism proposed by Christianson and Lipscomb is operative, in which the metal acts to acidify the nucleophilic water and stabilize the tetrahedral intermediate, but does not directly activate the carbonyl of the substrate by electrophilic catalysis (Figure 1B). 19,20 As expected, the key acid/base catalyst Glu222 that serves to deprotonate the nucleophilic water molecule is hydrogen bonded to a phosphinic acid oxygen of inhibitor 1 .…”

Section: Resultsmentioning

confidence: 96%

“…16,29 For this assay, His-tagged diaminopimelate dehydrogenase (DAPDH) from Corynebacterium glutamicum was overproduced in Escherichia coli and purified by immobilized metal affinity chromatography for use as a coupling enzyme. 16 NADH formed from the oxidation of meso -Dap by DAPDH was monitored by UV spectroscopy.…”

Section: Resultsmentioning

confidence: 99%

“…16 These crystals were then sequentially soaked with solutions of ZnCl 2 , inhibitor 1 , and cryoprotectant. A dataset to 1.9 Å resolution was collected and the structure factors were directly refined against the apo-Csd4 crystal structure.…”

Section: Resultsmentioning

confidence: 99%

“…Csd4 has been found to be responsible for hydrolyzing the bond between meso -Dap and iso - d -Glu in the trimming of a peptidoglycan tripeptide to a dipeptide. 11,16,17 As the dipeptide no longer bears a meso -Dap residue, it can no longer participate in a crosslinking event. Deletion of csd4 does not impair growth rate, but results in a straight rod phenotype.…”

Section: Introductionmentioning

confidence: 99%

“…16,17 The Csd4 enzyme bears an N-terminal domain with a typical carboxypeptidase fold as well as two additional domains of unknown function. The enzyme has been shown to accept peptidoglycan fragments (MurNAc-GlcNAc tripeptide and N -acetylated tripeptide) as alternate substrates (where the tripeptide is l -Ala- iso - d -Glu- meso -Dap).…”

Section: Introductionmentioning

confidence: 99%

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A Bacterial Cell Shape-Determining Inhibitor

et al. 2016

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Helicobacter pylori and Campylobacter jejuni are human pathogens and causative agents of gastric ulcers/cancer and gastroenteritis, respectively. Recent studies have uncovered a series of proteases that are responsible for maintaining the helical shape of these organisms. The H. pylori metalloprotease Csd4, and its C. jejuni homologue Pgp1, cleave the amide bond between meso-diaminopimelate and iso-d-glutamic acid in truncated peptidoglycan side chains. Deletion of either csd4 or pgp1 results in bacteria with a straight rod phenotype, a reduced ability to move in viscous media, and reduced pathogenicity. In this work, a phosphinic acid-based pseudodipeptide inhibitor was designed to act as a tetrahedral intermediate analog against the Csd4 enzyme. The phosphinic acid was shown to inhibit the cleavage of the alternate substrate, Ac-l-Ala-iso-d-Glu-meso-Dap with a Ki value of 1.5 µM. Structural analysis of the Csd4-inhibitor complex shows that the phosphinic acid displaces the zinc-bound water and chelates the metal in a bidentate fashion. The phosphinate oxygens also interact with the key acid/base residue, Glu222, and the oxyanion-stabilizing residue, Arg86. The results are consistent with the "promoted-water pathway" mechanism for carboxypeptidase A catalysis. Studies on cultured bacteria showed that the inhibitor causes significant cell straightening when incubated with H. pylori at millimolar concentrations. A diminished, yet observable, effect on the morphology of C. jejuni was also apparent. Cell straightening was more pronounced with an acapsular C. jejuni mutant strain compared to the wild type, suggesting that the capsule impaired inhibitor accessibility. These studies demonstrate that a highly polar compound is capable of crossing the outer membrane, inhibiting cell shape determinant proteases and altering cell shape, thereby affecting the pathogenic attributes of these organisms. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials against these human pathogens.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Bacterial Cell Shape-Determining Inhibitor

et al. 2016

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Peptides Containing meso‐Oxa‐Diaminopimelic Acid as Substrates for the Cell‐Shape‐Determining Proteases Csd6 and Pgp2

et al. 2019

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Molecular Container and Metal Ion Sensor Chiral Cavitands

2020

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Chiral macrocyclic cavitands prepared by a template free, high dilution methodology have been discovered to function as molecular containers accommodating small organic molecules and host transition metal ions with specific sensing ability while having an outstanding capability to sense Zn2+ with switch‐on fluorescence and also act as a selective colorimetric sensor for Cu2+. The cavitand acts as a unique two‐directional molecular tweezers for the guest molecules.

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Helical Shape of Helicobacter pylori Requires an Atypical Glutamine as a Zinc Ligand in the Carboxypeptidase Csd4

Cited by 17 publications

References 43 publications

A Bacterial Cell Shape-Determining Inhibitor

A Bacterial Cell Shape-Determining Inhibitor

Peptides Containing meso‐Oxa‐Diaminopimelic Acid as Substrates for the Cell‐Shape‐Determining Proteases Csd6 and Pgp2

Molecular Container and Metal Ion Sensor Chiral Cavitands

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