A novel Porphyromonas gingivalis enzyme: An atypical dipeptidyl peptidase III with an ARM repeat domain

Abstract

Porphyromonas gingivalis, an asaccharolytic Gram-negative oral anaerobe, is a major pathogen associated with adult periodontitis, a chronic infective disease that a significant percentage of the human population suffers from. It preferentially utilizes dipeptides as its carbon source, suggesting the importance of dipeptidyl peptidase (DPP) types of enzyme for its growth. Until now DPP IV, DPP5, 7 and 11 have been extensively investigated. Here, we report the characterization of DPP III using molecular biology, biochemical, biophysical and computational chemistry methods. In addition to the expected evolutionarily conserved regions of all DPP III family members, PgDPP III possesses a C-terminal extension containing an Armadillo (ARM) type fold similar to the AlkD family of bacterial DNA glycosylases, implicating it in alkylation repair functions. However, complementation assays in a DNA repair-deficient Escherichia coli strain indicated the absence of alkylation repair function for PgDPP III. Biochemical analyses of recombinant PgDPP III revealed activity similar to that of DPP III from Bacteroides thetaiotaomicron, and in the range between activities of human and yeast counterparts. However, the catalytic efficiency of the separately expressed DPP III domain is ~1000-fold weaker. The structure and dynamics of the ligand-free enzyme and its complex with two different diarginyl arylamide substrates was investigated using small angle X-ray scattering, homology modeling, MD simulations and hydrogen/deuterium exchange (HDX). The correlation between the experimental HDX and MD data improved with simulation time, suggesting that the DPP III domain adopts a semi-closed or closed form in solution, similar to that reported for human DPP III. The obtained results reveal an atypical DPP III with increased structural complexity: its superhelical C-terminal domain contributes to peptidase activity and influences DPP III interdomain dynamics. Overall, this research reveals multifunctionality of PgDPP III and opens direction for future research of DPP III family proteins.

Conservation of the conformational dynamics and ligand binding within M49 enzyme family

“… 12 In the case of Pg DPP III this resulted in concurrent movements of both the lower DPP III domain and ARM fragment in the direction of the upper DPP III domain as it was discussed in our recent publication. 33 This could serve as an explanation for the measured HDX decrease in the regions distant from the tynorphin binding site.…”

“…Detailed expression protocols were described elsewhere. 8,12,25,26,33 Protein purity was conrmed by SDS-PAGE according to Laemmli. 34 Protein concentrations were determined by the Bradford method.…”

“…On the other hand, its peptidase activity is comparable to that of BtDPP III. 33 Weak changes of the HDX proles upon the enzyme complexation with tynorphin were detected in the DPP III part of the protein region represented by peptides p18, p37, p39, and p45, ( 161 IIKASSVNF 169 , 355 LTIAGDSYPATPIG 365 ,…”

Dipeptidyl‐peptidases: Key enzymes producing entry forms of extracellular proteins in asaccharolytic periodontopathic bacterium Porphyromonas gingivalis

“…P. gingivalis DPP3 is composed of 906 and 886 amino acid residues in the strains ATCC 33277 and W83, respectively, which are substantially larger than 675 residues of Bacteroides thetiotaomicron DPP3 and 737 residues of human DPP3 due to the C‐terminally tagged sequence. The additional sequence is predicted at high confidence to have α‐α superhelix fold, belonging to Armadillo‐type fold family similar to the AlkD family of bacterial glycosylases (Hromić‐Jahjefendić et al., 2017). The genes encoding DPP3 with AlkD domain are specifically found within the genus Porphyromonas .…”

Metabolic cooperativity between Porphyromonas gingivalis and Treponema denticola

“…Following the initial proteolytic degradation of complex proteins by gingipains or other proteases into oligopeptides, numerous endopeptidases work in concert for downstream digestion to produce tri- and dipeptides for P. gingivalis internalization and utilization [ 7 , 9 , 11 ]. Peptidases of P. gingivalis that have been characterized include: dipeptidyl peptidase (DPP) III [ 12 ], DPPIV [ 13 ], DPP5 [ 14 ], DPP7 [ 15 ], DPP11 [ 16 ], prolyl tripeptidyl peptidase A (PtpA) [ 17 ], as well as acylpeptidyl oligopeptidase (AOP) [ 18 ] and carboxypeptidase (CPG70) [ 19 , 20 ].…”