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

Abstract

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.

S51 family peptidases provide resistance to peptidyl-nucleotide antibiotic McC

“…PCC6803 (28) belong to clades I and IV, respectively (Figure 4A). Several proteins that do not possess peptidase activity and have no established physiological function belong to clade II (Figure 4A) (29). MccG Nva , together with PepE Bs from Bacillus subtilis (29) belongs to clade V. PepE Bs was previously shown to hydrolyze the model substrate of aspartyl dipeptidases, L-aspartic acid α-(p-nitroanilide) (Asp-pNA), in vitro (29), however, its natural substrates are unknown.…”

“…cells from the McC action. To confirm that recombinant PepE is an active aspartyl dipeptidase we have synthesized its substrate Asp-pNA(29). Incubation of Asp-pNA with recombinant PepE led to hydrolysis of the compound at the carboxamide bond and formation of fluorogenic p-nitroaniline (FigureS4).Thus, recombinant PepE Eco is active but does not confer resistance to McC-like antibiotics.…”

“…substrate of aspartyl dipeptidases, L-aspartic acid α-(p-nitroanilide) (Asp-pNA), in vitro(29), however, its natural substrates are unknown.To check if the ability to confer resistance to McC-like compounds is unique to MccG Nva , we tested MccG Mab from the M. abscessus mcc BGC and several MccG Nva homologs from clade V encoded by stand-alone genes. The latter included MccG-like proteins from Artrobacter sp.…”

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

“…PepEse enzyme is dimeric and the active site is located at the dimeric interface 4 . The crystal structures of other homologs of PepExl has been determined from Deinococcus radiodurans (PDB code: 6IRU) by Yadav et al, 5 and from Listeria monocytogens (PDB code: 3L4E) by structural genomics consortium. The third residue of the catalytic triad, glutamate, is replaced by Asp and Asn in later two structures, respectively.…”

“…The third residue of the catalytic triad, glutamate, is replaced by Asp and Asn in later two structures, respectively. Moreover, none of these two proteins have been found to possess any peptidase activity 5 . PepExl is eukaryotic member of S51 peptidase family whose physiological role has been assigned but the three‐dimensional structure is not yet known 1 .…”