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.
Highlights
Porphyromonas gingivalis is a major pathogen that can be found in the oral cavity of mammals and represents a major cause of chronic periodontitis [1] which affects a significant percentage of the human population [2]
Dipeptidyl peptidases of P. gingivalis have been the subject of extensive research because these exopeptidases liberate dipeptides from the amino-end of their natural substrates, and P. gingivalis is known to utilize dipeptides preferentially, instead of free amino acids, as the source of energy [28]
Investigations with gene-disrupted mutants of P. gingivalis ATCC 33277 indicated that dipeptidyl peptidase (DPP) and gingipains cooperatively liberate dipeptides from nutrient oligopeptides [67]
Summary
Porphyromonas gingivalis is a major pathogen that can be found in the oral cavity of mammals and represents a major cause of chronic periodontitis [1] which affects a significant percentage of the human population [2]. This Gram-negative oral anaerobe colonizes the subgingival region by adherence to adsorbed salivary molecules, matrix proteins, epithelial cells and bacteria that are already established as a biofilm on tooth and epithelial surfaces [3,4]. Periodontal disease represents a risk factor for non-Hodgkin lymphoma [11]. There is a demonstrated link between diagnosed periodontitis and the risk of ischemic stroke [12]
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