Identification of the Catalytic Triad of Family S46 Exopeptidases, Closely Related to Clan PA Endopeptidases

Abstract

The exopeptidases of family S46 are exceptional, as the closest homologs of these enzymes are the endopeptidases of clan PA. The three-dimensional structure of S46 enzymes is unknown and only one of the catalytic residues, the serine, has been identified. The catalytic histidine and aspartate residues are not experimentally identified. Here we present phylogenetic and experimental data that identify all residues of the catalytic triad of S46 peptidase, dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24. Phylogenetic comparison with the protein and S46 peptidases, revealed His-86, Ser-657, and five aspartate residues as possible catalytic residues. Mutation studies identified the catalytic triad of DAP BII as His-86, Asp-224, and Ser-657, while secondary structure analysis predicted an extended alpha-helical domain in between Asp-224 and Ser-657. This domain is unique for family S46 exopeptidases and its absence from the endopeptidases of clan PA might be key to their different hydrolysis activities.

Identification of a new subtype of dipeptidyl peptidase 11 and a third group of the S46-family members specifically present in the genus Bacteroides

“…putrefaciens [20], P. mexicana [18], and Capnocytophaga canimorsus [29]. At present, though their homologues are widely distributed among eubacteria, most S46-family members are tentatively classified by their sequence similarity to authentic S46 members of P. gingivalis, and their enzymatic characteristics remain to be elucidated.…”

“…Currently, the S46 family is composed of three members, DPP7 (S46.001), DPP11 (S46.002), characterized in P. gingivalis (PgDPP7, PgDPP11, respectively), and DAPBII from Pseudoxanthomonas mexicana (S46.003) [18,19]. Since the biochemical property and substrate preference of DAPBII are nearly identical to those of PgDPP7, the S46 family should be composed solely of two members, i.e., DPP7 preferentially liberates dipeptides with both P1 and P2 hydrophobic residues, and DPP11 is specific for P1 Asp and Glu.…”

S46 Peptidases are the First Exopeptidases to be Members of Clan PA

“…DAP BII sequentially releases Asp-Arg, Val-Tyr, Ile-His, Pro-Phe, and His-Leu from the N terminus of angiotensin I to degrade it completely into dipeptides10. The S1 site of DAP BII has a tendency to prefer a large hydrophobic side chain11, although strict specificity is not observed.…”

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl peptidase 11 fromPorphyromonas gingivalis

“…Initially, we tried to solve the structure of PgDPP11 by molecular-replacement techniques. Structures of DAP BII complexed with or without a peptide (Sakamoto, Suzuki, Iizuka, Tateoka, Roppongi, Okada et al, 2014), belonging to the S46 peptidase family (Suzuki et al, 2014) and having 29% sequence identity with PgDPP11, were used as search models. Since these attempts failed, a search for heavyatom derivatives intended for phasing by the multiple/single isomorphous replacement method and the preparation of selenomethionine-substituted PgDPP11 using LeMaster medium (Hendrickson et al, 1990) and E. coli B834(DE3) cells for phasing by Se-MAD/SAD methods are under way.…”

“…The predicted amino-acid sequences of DPP7s and DPP11s from Porphyromonas species revealed that these enzymes are $80 kDa serine proteases and are classified into clan PA family S46 in the MEROPS database (Rawlings et al, 2012). The S46 enzymes are widely distributed in anaerobic Gram-negative species in the genera Bacteroides, Parabacteroides and Porphyromonas, but are not found in mammals (Ohara-Nemoto et al, 2011;Suzuki et al, 2014). In order to further characterize the properties of the S46 enzymes, we initiated structural studies of PgDPP11.…”