Structure of Asp‐bound peptidase E from <i>Salmonella enterica</i>: Active site at dimer interface illuminates Asp recognition

Yadav, Pooja; Goyal, Venuka Durani; Gaur, Neeraj; Kumar, Ashwani; Gokhale, Sadashiv M.; Makde, Ravindra D.

doi:10.1002/1873-3468.13247

Cited by 7 publications

(19 citation statements)

References 27 publications

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“…Hence, the lack of activity toward these substrates could be due to other reasons. Recently, PepEse has been reported as a dimer, not monomer, and its dimer interface has been shown to shield the active site and is thought to be important for activity . We probed the oligomeric state of PepElm by size exclusion chromatography and found that the enzyme is monomeric (Figure S4).…”

Section: Resultsmentioning

confidence: 97%

“…However, the dimeric structure of PepEse was solved recently and its dimer orientation does not use the extension of any regular secondary structure. Moreover, the active site of PepEse was found to be well shielded by the dimer interface and loop A plays an important role in both dimer formation and active site shielding (Figure D) . The N‐terminal extension of loop A houses the His157 of the catalytic triad.…”

Section: Resultsmentioning

confidence: 99%

“…Percent relative activity of PepE homologs is shown in the bar graph on the right considering PepEse activity as 100%. Superscript a in PepEse represents previously characterized peptidase E . N.A.…”

Section: Resultsmentioning

confidence: 99%

“…Activity toward X‐β‐naphthylamide substrates (X‐βNA; where X is any amino acid) was tested as described by Jamdar . Activity toward other peptide substrates were tested using a modified Ninhydrin assay as described by Yadav et al Activity toward chromogenic peptides (with p‐nitroanilide, pNA) and chromogenic esters (with p‐nitrophenyl, pnp) were tested by incubating with substrates at assay conditions for 10 minutes and measuring absorbance at 410 nm or 405 nm, respectively. PepEdr was found to be active toward ester substrates.…”

Section: Methodsmentioning

confidence: 99%

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Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability

et al. 2019

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Peptidase E (PepE) is a nonclassical serine peptidase with a Ser‐His‐Glu catalytic triad. It is specific for dipeptides with an N‐terminal aspartate residue (Asp‐X dipeptidase activity). Its homolog from Listeria monocytogenes (PepElm) has a Ser‐His‐Asn “catalytic triad.” Based on sequence alignment we predicted that the PepE homolog from Deinococcus radiodurans (PepEdr) would have a Ser‐His‐Asp “catalytic triad.” We confirmed this by solving the crystal structure of PepEdr to 2.7 Å resolution. We show that PepElm and PepEdr lack the Asp‐X dipeptidase activity. Our analyses suggest that absence of P1 pocket in the active site could be the main reason for this lack of typical activity. Sequence and structural data reveal that the PepE homologs can be divided into long and short PepEs based on presence or absence of a C‐terminal tail which adopts a β‐hairpin conformation in the canonical PepE from Salmonella enterica. A long PepE from Bacillus subtilis with Ser‐His‐Asp catalytic triad exhibits Asp‐X dipeptidase activity. Whereas the three long PepEs enzymatically characterized till date have been found to possess the Asp‐X dipeptidase activity, the three enzymatically characterized short PepEs lack this activity irrespective of the nature of their catalytic triads. This study illuminates the structural and functional heterogeneity in the S51 family and also provides structural basis for the functional variability among PepE homologs.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability

et al. 2019

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“…Crystallization was carried out by under oil microbatch method [20] as per previously published protocol [21]. Crystallization was carried out by under oil microbatch method [20] as per previously published protocol [21].…”

Section: Protein Crystallization and Structure Determinationmentioning

confidence: 99%

Crystal structures and biochemical analyses of intermediate cleavage peptidase: role of dynamics in enzymatic function

et al. 2019

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Intermediate cleavage peptidase (Icp55) processes a subset of mitochondrial matrix proteins by removing a bulky residue at their N termini, leaving behind smaller N‐terminal residues (icp activity). This contributes towards the stability of the mitochondrial proteome. We report crystal structures of yeast Icp55 including one bound to the apstatin inhibitor. Apart from icp activity, the enzyme was found to exhibit Xaa‐Pro aminopeptidase activity in vitro. Structural and biochemical data suggest that the enzyme exists in a rapid equilibrium between monomer and dimer. Furthermore, the dimer, and not the monomer, was found to be the active species with loop dynamics at the dimer interface playing an important role in activity. Based on the new evidence, we propose a model for binding and processing of cellular targets by Icp55. Database The atomic coordinates and structure factors for the structures of Icp55 (code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6A9T, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6A9U, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6A9V) have been deposited in the Protein Data Bank (PDB) (http://www.pdb.org/).

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Crystal structure of aspartyl dipeptidase from Xenopus laevis revealed ligand binding induced loop ordering and catalytic triad assembly

et al. 2021

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Gene encoding aspartyl dipeptidase from Xenopus levies (PepExl) is upregulated by thyroid hormone and is proposed to play a significant role in resorption of tadpole tail during metamorphosis. However, the importance of peptidase activity for the resorption of the tail remain elusive. Here we report the crystal structures of first eukaryotic S51 peptidase, PepExl, in its ligand‐free and Asp‐bound states at 1.4 and 1.8 Å resolutions, respectively. The active site is located at dimeric interface and the catalytic triad is found to be dissembled in ligand‐free and assembled in Asp‐bound state. Structural comparison and molecular dynamic simulations of ligand‐free and Asp‐bound states shows that distinct loop (loop‐A) plays an important role in active site shielding, substrate binding and enzyme activation. This study illuminates the Asp‐X dipeptide binding in PepExl is associated with ordering of the loop‐A and assembly of residues of catalytic triad in active conformation for enzymatic activity.

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Structure of Asp‐bound peptidase E from Salmonella enterica: Active site at dimer interface illuminates Asp recognition

Cited by 7 publications

References 27 publications

Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability

Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability

Crystal structures and biochemical analyses of intermediate cleavage peptidase: role of dynamics in enzymatic function

Crystal structure of aspartyl dipeptidase from Xenopus laevis revealed ligand binding induced loop ordering and catalytic triad assembly

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