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Study of seven new guanidiniocarbonylpyrrole (GCP)–fluorophore conjugates interactions with dipeptidyl peptidase III (DPP III) showed that all compounds bind strongly (Ks ≈ µM) to enzyme active site, but with very different fluorimetric response (varying from quenching to strong increase), dependent on the fluorophore type and intramolecular pre-organisation of molecule. Positively charged lysine side chain improved significantly compound solubility but diminished fluorescence increase upon DPP III binding and completely abolished inhibitory effect on DPP III activity, whereas linker-neutral analogues showed stronger emission increase and were efficient enzyme inhibitors. By far the best fluorimetric response and inhibitive properties showed cyanine–GCP analogue, thus being promising lead compound for both enzyme sensing and bio-activity inhibiting (theragnostic) studies of DPP III in the future. Communicated by Ramaswamy H. Sarma

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.

Dipeptidil-peptidaze III (DPP III) su metalopeptidaze iz porodice M49 koje hidroliziraju dipeptide s N-kraja peptidnih supstrata veličine 3 do 10 aminokiselina. Fiziološka funkcija ove peptidaze nije još do kraja razjašnjena, ali pretpostavlja se da ljudska DPP III osim uloge u završnim koracima razgradnje peptida u stanici ima ulogu i u nekoliko patofizioloških procesa, uključujući razvitak raka. U cBioPortal bazi podataka nalaze se sekvencirani uzorci DNA izolirane iz ljudskih tumora te su nađene mutacije u kodirajućoj regiji gena za humanu DPP III. Odabrane su tri mutacije kako bi se ispitao učinak istih na biokemijska svojstva i aktivnost humane DPP III. Istraživanje se provodilo na mutantima humane DPP III: E451K, R638L i R638W koji su dobiveni ciljanom in vitro mutagenezom ekspresijskih plazmida. Nakon tog slijedila je ekspresija mutanata u bakteriji E. coli te njihova izolacija i pročišćavanje afinitetnom kromatografijom na Ni-NTA koloni. Mutanti su podvrgnuti raznim testovima aktivnosti i mjerenju kinetike te su analizirani nativnom elektroforezom i izoelektričnim fokusiranjem na gelu. Dobiveni parametri za mutante uspoređeni su s onima „divljeg“ tipa. Dipeptidyl peptidases III (DPP III) are members of the M49 metalopeptidase family which hydrolyze dipeptides from the N-termini of 3 to 10 aminoacids long peptides. Their physiological function is not yet fully understood, but it is assumed that the human DPP III, apart from the role in the final steps of peptide degradation in the cell, has the role in several pathophysiological processes, including the development of cancer. The cBioPortal database contains sequenced DNA samples isolated from human cancers and mutations in the coding region of the human DPP III gene are found. Three mutations were selected to examine their effect on the biochemical properties and the activity of human DPP III. The study was performed on human DPP III: E451K, R638L and R638W mutants obtained by site-directed in vitro mutagenesis of expression plasmids, followed by the expression of mutants in E. coli, and the isolation and purification by affinity chromatography on Ni-NTA column. The mutants were subjected to various activity assays, kinetics measurement, and they were also analysed by native and isoelectric focusing gel electrophoresis. The obtained mutant parameters were compared to those of the wild type.

Dipeptidil-peptidaza III (DPP III) je široko rasprostranjena citosolna metalopeptidaza iz porodice M49, koja odcjepljuje dipeptide s N-kraja peptidnih supstrata. Većina istraživanja o DPP III napravljena je na ortolozima iz eukariota koja ukazuju na njenu opću funkciju u intracelularnom katabolizmu proteina, ulogu u regulaciji boli te odgovoru na oksidativni stres. U ovom radu je strukturno karakterizirana prva prokariotska DPP III, i to iz bakterije Bacteroides thetaiotaomicron, ljudskog simbionta koji nastanjuje tanko i debelo crijevo. Kinetičkom analizom utvrđeno je da je Arg2-2-naftilamid njezin najbolji sintetski supstrat. Elektroforetskim metodama je utvrđeno da pročišćeni proteinski uzorak sadrži tri forme DPP III. Zamjenom svih cisteinskih ostataka u serinske pripremljen je dovoljno homogen uzorak za kristalizaciju bakterijske DPP III. Riješene su strukture u otvorenoj i zatvorenoj konformaciji proteina. Pokazalo se da ovaj protein pripada istoj strukturnoj klasi kao i ljudska i kvaščeva DPP III. S obzirom na gotovo jednako aktivno mjesto može se pretpostaviti i isti mehanizam djelovanja. Dipeptidyl peptidase III (DPP III) is a widespread cytosolic metalopeptidase of the M49 family, which cleaves dipeptides from the N-terminus of its peptide substrates. Previous studies of the DPP III enzyme were focused on eukaryotic orthologs and indicate its general function in the intracellular protein catabolism, pain regulation as well as the response to oxidative stress. In this work, the first prokaryotic DPP III, that from the human gut symbiont bacterium Bacteroides thetaiotaomicron, was structurally characterized. Kinetic analysis showed that Arg2-2-naphthylamide is the preferred synthetic substrate. Electrophoretic methods revealed that the purified protein sample contains three forms. By replacing all cysteine residues with serines, homogeneity of the sample improved enough to enable successful crystallization of the bacterial DPP III. Structures in open and closed conformations were solved and they have the same protein fold as the human and yeast DPP III. Considering the fact that these three enzymes have almost identical active sites, the same mechanism of action can be assumed.

Nudix-dipeptidil-peptidaza III je biljni enzim s dvojnom hidrolaznom aktivnošću: dipeptidilpeptidaznom (DPP III) i fosfataznom (Nudix). Biokemijski su okarakterizirani homolozi iz mahovine (Physcomitrella patens) i uročnjaka (Arabidopsis thaliana). C-terminalna domena oba homologa predstavlja atipičnu DPP III s obzirom na aktivno mjesto (pentapeptid HEXXH umjesto heksapeptida HEXXGH u tipičnim DPP III), te je slabije aktivna prema sintetskim supstratima u usporedbi s tipičnim, do sada istraženim DPP III. N-terminalna domena je član nadporodice Nudix proteina. Utvrđeno je da Nudix domena ima fosfataznu aktivnost prema supstratima izopentenil-difosfatu i dimetilalil-difosfatu, prekursorima u sintezi izoprenoida. Funkcionalnim istraživanjima na mutantama mahovine P. patens koje ne eksprimiraju ovaj enzim pokazano je da on sudjeluje u metabolizmu biljnih hormona citokinina. Preliminarni rezultati analize ekspresije gena u mahovini u prisutnosti stresora sugeriraju da enzim Nudix- DPP III sudjeluje u odgovoru biljaka na stres (sušu, solni i oksidacijski stres). Zaključno, predložen je naziv izopentenil-difosfat-fosfohidrolaza za novopronađenu enzimsku aktivnost. Nudix-dipeptidyl peptidase III is a plant enzyme with dual hydrolase activity: dipeptidylpeptidase III (DPP III) and phosphatase (Nudix). Homologues from moss (Physcomitrella patens) and angiosperm (Arabidopsis thaliana) were biochemically characterized. C-terminal domain of both homologues represents an atypical DPP III considering the active site (pentapeptide HEXXH instead of hexapeptide HEXXGH in typical DPPs III), and it is less active towards synthetic supstrates than typical, formerly characterized DPPs III. N-terminal domain is a member of Nudix superfamily. It was established that it has phosphatase activity towards isopentenyl-diphosphate and dimethylallyl-diphosphate, precursors in isoprenoid sythesis. Functional research on moss P. patens knock-out mutants showed that this enzyme is involved in cytokinin metabolism. Preliminary analysis of gene expression in the presence of stressors suggests that Nudix-DPP III enzyme is involved in plant response to stress (drought, salt and oxidative stress). Finally, the name isopentenyl diphosphate phosphohydrolase is suggested for the newly identified enzyme activity.

The hydrogen deuterium exchange (HDX) mass spectrometry combined with molecular dynamics (MD) simulations was employed to investigate conformational dynamics and ligand binding within the M49 family (dipeptidyl peptidase III family). Six dipeptidyl peptidase III (DPP III) orthologues, human, yeast, three bacterial and one plant (moss) were studied. According to the results, all orthologues seem to be quite compact wherein DPP III from the thermophile Caldithrix abyssi seems to be the most compact. The protected regions are located within the two domains core and the overall flexibility profile consistent with semi-closed conformation as the dominant protein form in solution. Besides conservation of conformational dynamics within the M49 family, we also investigated the ligand, pentapeptide tynorphin, binding. By comparing HDX data obtained for unliganded protein with those obtained for its complex with tynorphin it was found that the ligand binding mode is conserved within the family. Tynorphin binds within inter-domain cleft, close to the lower domain β-core and induces its stabilization in all orthologues. Docking combined with MD simulations revealed details of the protein flexibility as well as of the enzyme– ligand interactions.

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.

A novel enzyme with dual activity - an isopentenyl diphosphate (IPP) hydrolase from the Nudix family fused to a dipeptidyl peptidase III (DPP III) domain has been discovered in Physcomitrella patens and Arabidopsis thaliana and biochemically characterized.1 Heterologously expressed Physcomitrella patens and Arabidopsis thaliana Nudix-DPP III (PpND and AtND) proteins showed peptidase activity against the preferred artificial substrate of DPPs III. The Nudix domain of these proteins was presumed to be a Nudix hydrolase (phosphatase), since it contains a functional Nudix box motif. Based on sequence similarity with isopentenyl diphosphate isomerase (also a Nudix fold protein), we recognized IPP as a possible substrate. Indeed, in a screen of 73 potential Nudix hydrolase substrates, both PpND and AtND showed preference for IPP, producing isopentenyl phosphate (IP) as a final product. With both phosphatase and peptidase activity confirmed by enzyme assays, we used site- directed mutagenesis to investigate structure- activity relationships. In PpND, Glu92 and Glu592 were identified as putative catalytic residues. Point mutations of each Glu92 and Glu592 to Ala caused an absence of phosphatase and peptidase activity, respectively. Our results confirm the presence of two separate active sites, although both domains are needed for proper folding and activity of this enzyme. Separate domains produced as recombinant proteins were either insoluble or unstable. The exclusive presence of this fusion protein in plants is still unclear and may have an adaptive role for life on land. The physiological role of the phosphatase might be in the regulation of the pool of IPP, the isoprenoid building block, in plant cell cytoplasm, while DPP III is supposed to be involved in protein catabolism. Functional role of this dual enzyme is being investigated on Physcomitrella patens mutants. From a biotechnological point of view, this enzyme might be useful in microbial isopentenol biofuel production. References 1 Karačić, Z. et al. (2016), A novel plant enzyme with dual activity: an atypical Nudix hydrolase and a dipeptidyl peptidase III, Biological Chemistry (in print), DOI: 10.1515/hsz-2016-0141

Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-Å ; crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.

The activity of pork muscle dipeptidyl peptidases I, II, III, and IV was followed during the processing of Serrano dry-cured ham. The effects of NaCl, pH, and temperature on these enzymes have also been studied for a better understanding of their importance in ham processing. The lysosomal enzymes DPP I and DPP II showed maximal activity at pH around 5.5–6.0, i.e., close to the pH in ham. At low temperatures, only DPP I and DPP IV showed relevant activity. NaCl considerably inhibited DPP activities except DPP I which was only slightly affected. Dipeptidyl peptidases remained active during the whole process except DPP II whose activity disappeared after 240 days of dry-curing.

Dipeptidyl peptidase III (DPP III) is a widely distributed cytosolic zinc peptidase from the M49 family, which hydrolyses dipeptides from the N-termini of its peptide substrates. Until now, most studies on DPP III were done on orthologs from eukaryotic organisms, showing that DPP III participates in the intracellular protein catabolism and oxidative stress response. The knowledge about prokaryotic DPP III is very scarce, particularly data on its catalytic mechanism, physiological function and structural characteristics are lacking. We have been studying two bacterial orthologs: DPP III from the human gut symbiont Bacteroides thetaiotaomicron (Bt) and from Caldithrix abyssi (Ca), which inhabits hydrothermal vents. Both proteins were produced in Escherichia coli and then purified. Diverse crystallization screens were applied and wild-type CaDPP III crystals were obtained. Crystallization experiments with wild-type BtDPP III failed probably due to charge heterogeneity. In order to overcome this problem, specific amino acid replacements (all cysteines were replaced by serines) were introduced and crystals of the variant protein were grown. Datasets for both DPP III proteins were collected at BESY II, Germany. Up to date, all efforts to solve the structures of the two proteins using molecular replacement method and anomalous dispersion of the zinc ions, were unsuccessful. Therefore, selenomethionine labeled BtDPP III, non-cysteine variant, was prepared and the crystals were obtained in the same crystallization condition as the non-labelled protein. Diffraction data up to 1.8 Å resolution were collected at Elettra Trieste, Italy, and the first prokaryotic DPP III structure was solved using single-wavelength anomalous dispersion of the selenium atoms. BtDPP III crystallized in space group P3121 with unit cell dimensions a=103.5 Å and c=141.6 Å and one molecule per asymmetric unit. The overall fold is typical for a member of the M49 family with two domains separated by a wide cleft. The upper domain is mostly helical with a tetracoordinated zinc ion, while the lower domain contains mixed secondary structure elements with a five-stranded β-barrel core. The main structural difference between BtDPP III and eukaryotic DPP III is the absence of a 30 amino acid loop in upper domain. In human DPP III this loop contains the ETGE motif which is supposed to be responsible for binding KEAP1, a repressor protein from the Keap1-Nrf2 signaling pathway.

Dipeptidyl peptidase III (EC 3.4.14.4) was purified from porcine skeletal muscle by ammonium sulfate fractionation and by chromatography and rechromatography on a Biosept-DEAE HPLC column. The enzy...

Human dipeptidyl peptidase III (DPP III) is a two domain metallo-peptidase from the M49 family. The wide interdomain cleft and broad substrate specificity suggest that this enzyme could experience significant conformational change. Long (100 ns) molecular dynamics (MD) simulations of DPP III revealed large range conformational changes of the protein, suggesting the pre-existing equilibrium model for a substrate binding. The binding free energy calculations revealed tighter binding of the preferred synthetic substrate Arg-Arg-2-naphtylamide to the "closed" than to the "open" DPP III conformation. Our assumption that Asp372 plays a crucial role in the large scale interdomain closure was proved by the MD simulations of the Asp372Ala variant. During the same simulation time, the variant remained more "open" than the wild type protein. Apparently, Ala was not as efficient as Asp in establishing the interdomain interactions. According to the MM-PBSA calculations, the electrostatic component of the free energy of solvation turned out to be higher for the "closed" protein than for its less compact form. However, the gain in entropy due to water released from the interdomain cleft nicely balanced this negative effect.

Human dipeptidyl peptidase III (DPP III) is a member of the zinc-metallopeptidase family M49 with a role in intracellular protein catabolism. Ligand-free crystal structure of yeast and human orthologues provided insight into the enzyme's active center and enabled some assumptions about the enzyme - substrate interactions. The molecular modeling performed for human and yeast DPP III indicated involvement of Asn406 and Asn415, respectively in ligand binding. To investigate further the role of this asparagine residue, conserved in all eukaryotic M49 peptidases, a site-directed mutagenesis of human DPP III was carried out. Replacement of Asn406 with Gln, but not with Ser, resulted in a large decrease in the enzyme's binding affinity for competitive peptide inhibitor and in catalytic efficiency. Molecular dynamics simulations performed for enzyme-substrate complexes, revealed changed coordination of the active- site zinc ion and the orientation of the catalytically important His568, in N406Q, but not in the N406S complexes.(doi: 10.5562/cca1808)

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.

The soluble fraction of a cell extract of Saccharomyces cerevisiae was found to contain hydrolytic activity toward arginyl-arginyl-beta-naphthylamide (Arg-Arg-beta NA). Most of the contaminating Arg-beta NA hydrolysis activity was removed by diethylaminoethyl (DEAE)-cellulose column chromatography and the enzyme was partially purified. It is very similar in various properties to mammalian dipeptidyl aminopeptidase III (DAP III), although it differs slightly in some respects.

A partial purification of dipeptidyl peptidase III has been achieved from human cataractous lens. The specific activity was increased 45.5-fold over that of the original aqueous extract. The exopeptidase exhibited a marked preference for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 8.8 and 37 degrees. The Km for this substrate was estimated to be 6.061 X 10(-3). Lens DPP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, puromycin and DFP. The preparation contained leucyl aminopeptidase and a neutral endopeptidase as contaminating proteases.

A new neutral endopeptidase having the properties of prolyl endopeptidase was detected in bovine lenses. The enzyme hydrolyzed the prolyl bond in the newly-developed fluorogenic substrate, t-butyloxycarbonyl-Arg-Pro-2-NNap, optimally at pH 8 and 37 degrees. The Km value was estimated to be 0.033 mM. An approximately 4-fold purification was achieved. DFP completely inhibited the hydrolysis of Boc-Arg-Pro-2-NNap by the endopeptidase.