Dipeptidyl peptidase III (DPP III) from the human gut symbiont Bacteroides thetaiotaomicron (Bt) is the first identified prokaryotic DPP III orthologue. It has low sequence similarity to the thoroughly studied human DPP III, and differently from eukaryotic orthologues it has a cysteine (Cys450) residue in the zinc-binding motif HEXXGH (HECLGH). The recently determined crystal structure of BtDPP III showed that its 3D structure, similar to the structure of the human DPP III, consists of two domains with a wide cleft in between. Although such a striking similarity of the 3D structures of orthologues with low sequence similarity is not surprising, it is no guarantee for similarity of their dynamic properties and the catalytic performance. Here, we report the results of the molecular modelling study of BtDPP III, wild type and its C450S mutant, as well as their complexes with characteristic DPP III substrates Arg-Arg-2-naphthylamide (RRNA) and Lys-Ala-2-naphtylamide (KANA). During several hundred nanoseconds of all-atom MD simulations of the wild type protein, the long range conformational changes, which can be described as protein 'closing', have been traced. We have determined a similar conformational change for the human orthologue as well. However, the amplitude of the change is lower for BtDPP III than for the human DPP III. The MD simulations have been performed using ff03, ff12SB and ff14SB force fields wherein the results of the last two better fit to the experimental results. The hydrogen bond analysis indicates reasons for higher substrate specificity of BtDPP III towards RRNA than KANA as well as for the decrease of the RRNA hydrolysis rate induced by the Cys450 to Ser mutation. The obtained results are in line with the experimental data.

Dipeptidyl peptidase III (DPP III) isolated from the thermophilic bacteria Caldithrix abyssi (Ca) is a two-domain zinc exopeptidase, a member of the M49 family. Like other DPPs III, it cleaves dipeptides from the N-terminus of its substrates but differently from human, yeast and Bacteroides thetaiotaomicron (mesophile) orthologs, it has the pentapeptide zinc binding motif (HEISH) in the active site instead of the hexapeptide (HEXXGH). The aim of our study was to investigate structure, dynamics and activity of CaDPP III, as well as to find possible differences with already characterized DPPs III from mesophiles, especially B. thetaiotaomicron. The enzyme structure was determined by X-ray diffraction, while stability and flexibility were investigated using MD simulations. Using molecular modeling approach we determined the way of ligands binding into the enzyme active site and identified the possible reasons for the decreased substrate specificity compared to other DPPs III. The obtained results gave us possible explanation for higher stability, as well as higher temperature optimum of CaDPP III. The structural features explaining its altered substrate specificity are also given. The possible structural and catalytic significance of the HEISH motive, unique to CaDPP III, was studied computationally, comparing the results of long MD simulations of the wild type enzyme with those obtained for the HEISGH mutant. This study presents the first structural and biochemical characterization of DPP III from a thermophile.

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.

Aflatoxin oxidase (AFO), an enzyme isolated from Armillariella tabescens, has been reported to degrade aflatoxin B1 (AFB1). However, recent studies reported sequence and structure similarities with the dipeptidyl peptidase III (DPP III) family of enzymes and confirmed peptidase activity toward DPP III substrates. In light of these investigations, an extensive computational study was performed in order to improve understanding of the AFO functions. Steered MD simulations revealed long‐range domain motions described as protein opening, characteristic for DPPs III and necessary for substrate binding. Newly identified open and partially open forms of the enzyme closely resemble those of the human DPP III orthologue. Docking of a synthetic DPP III substrate Arg2‐2‐naphthylamide revealed a binding mode similar to the one found in crystal structures of human DPP III complexes with peptides with the S1 and S2 subsites’ amino acid residues conserved. On the other hand, no energetically favorable AFB1 binding mode was detected, suggesting that aflatoxins are not good substrates of AFO. High plasticity of the zinc ion coordination sphere within the active site, consistent with that of up to date studied DPPs III, was observed as well. A detailed electrostatic analysis of the active site revealed a predominance of negatively charged regions, unsuitable for the binding of the neutral AFB1. The present study is in line with the most recent experimental study on this enzyme, both suggesting that AFO is a typical member of the DPP III family.

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