Oxidase or peptidase? A computational insight into a putative aflatoxin oxidase from Armillariella tabescens

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.     

Dipeptidyl peptidase III from rat liver cytosol: purification, molecular cloning and immunohistochemical localization

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of beta-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the cytosol of various rat tissues by immunohistochemical examination.     

Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance.The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of the enzyme's residual activity with fluorimetric detection.Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3523+/-567M(-1)min(-1). Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540M(-1)s(-1) for slower reacting sulfhydryl, and 21,855M(-1)s(-1) for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H(2)O(2) inactivated the rat DPP III. Human enzyme was resistant to H(2)O(2) attack and less affected by reduced glutathione (GSH) than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues.These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III.     

Novel amidino-substituted benzimidazoles: synthesis of compounds and inhibition of dipeptidyl peptidase III

Dipeptidyl peptidase III (DPP III), also known as enkephalinase B, is a zinc-hydrolase with an indicated role in the mammalian pain modulatory system. In order to find a potent antagonist of this enzyme, we synthesized and screened the effect of a small set of benzimidazole derivatives on its activity. To improve the inhibitory potential, a cyclobutane ring was introduced as rigid conformation support to the diamidino substituted dibenzimidazoles. Two such compounds (1' and 4') from the group of cyclobutane derivatives containing amidino-substituted benzimidazole moieties, obtained by photochemical cyclization in water and by respecting rules of the "green chemistry" approach, were found to be strong DPP III inhibitors, with IC(50) value below 5 microM. Compound 1' displayed time-dependent inhibition towards human DPP III, characterized by the second-order rate constant of 6924+/-549 M(-1)min(-1) (K(i)=0.20 microM). The peptide substrate valorphin protected the enzyme from inactivation by 1'.     

Reactive cysteine in the active-site motif of Bacteroides thetaiotaomicron dipeptidyl peptidase III is a regulatory residue for enzyme activity

Dipeptidyl peptidase III (DPP III), a member of the metallopeptidase family M49, was considered as an exclusively eukaryotic enzyme involved in intracellular peptide catabolism and pain modulation. In 2003, new data on genome sequences revealed the first prokaryotic orthologs, which showed low sequence similarity to eukaryotic ones and a cysteine (Cys) residue in the zinc-binding motif HEXXGH. Here we report the cloning and heterologous expression of DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The catalytic efficiency of bacterial DPP III for preferred synthetic substrate hydrolysis was very similar to that of the human host enzyme. Substitution of Cys450 from the active-site motif by serine did not substantially change the enzymatic activity. However, this residue was wholly responsible for the inactivation effect of sulfhydryl reagents. Molecular modeling indicated seven basic amino acid residues in the local environment of Cys450 as a possible cause for its high reactivity. Sequence analysis of 81 bacterial M49 peptidases showed conservation of the HECLGH motif in 68 primary structures with the majority of proteins lacking an active-site Cys originated from aerobic bacteria. Data obtained suggest that Cys450 of B. thetaiotaomicron DPP III is a regulatory residue for the enzyme activity.     

Human and rat dipeptidyl peptidase III: biochemical and mass spectrometric arguments for similarities and differences

Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82500+/-60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co2+, differing in their pH optima and kcat/Km. Zn2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III (Ki < 1 microM) and a preference for ahydrophobic residue at the P1' site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.     

Functional tyrosine residue in the active center of human dipeptidyl peptidase III

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.     

Flexibility of the coordination geometry around the cupric ions in Cu(II)-rat dipeptidyl peptidase III is important for the expression of enzyme activity

Dipeptidyl peptidase III (DPP III), the zinc peptidase, has a unique helix portion in the metal-binding motif (HELLGH). The enzyme activity of the cupric derivative of rat DPP III (Cu(II)-rat DPP III) for Lys-Ala-β-NA is about 30% of that of the wild-type enzyme. On the other hand, the enzyme activity of Cu(II)-rat del-DPP III, in which Leu453 is deleted from the metal-binding motif, possesses only 1-2% of the enzyme activity of rat del-DPP III. The EPR spectra of Cu(II)-rat DPP III in the presence of various concentrations of the substrate, Lys-Ala-β-NA, changed dramatically, showing formation of the enzyme-metal-substrate complex. The EPR spectra of Cu(II)-rat del-DPP III did not change in the presence of excess Lys-Ala-β-NA. The deletion of Leu453 from the HELLGH motif of rat DPP III leads to a complete loss of flexibility in the ligand geometry around the cupric ions. Under the formation of the enzyme-metal-substrate complex, Glu451 of Cu(II)-rat DPP III is sufficiently able to approach the water molecule via a very different orientation from that of the resting state; however, Glu451 of Cu(II)-rat del-DPP III is not able to access the water molecule.     

The isolation and some properties of dipeptidyl peptidases II and III from porcine spleen

1. Dipeptidyl peptidases (DPP) II and III from porcine spleen have been purified to homogeneity as assessed by disc gel electrophoresis, HPLC and chromatofocusing. 2. The enzyme are both inhibited by diisopropylfluorophosphate suggesting that the active site contains an essential serine residue, but they are also inhibited by a variety of other reagents. 3. The pI of DPP II is 4.8, that of DPP III, 4.0. 4. The former enzyme has a molecular weight of 97,000, the latter 66,000 and both are glycoproteins. 5. The enzymes are compared with those from other sources.     

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

A dipeptidyl peptidase (DPP) was purified to homogeneity using lys-ala-beta-naphthylamide, the standard substrate for DPP II. The enzyme is a monomer with a Mr of 70kDa, pl 5.2, and Km 5.0 microM. Its terminal amino acid sequence was XXLLYAIQKRLF and was not identical to that of any known protein. Although initially considered to be a DPP II, the enzyme differed in some properties from classical DPP IIs. It had a pH optimum of 7.9, was not active on X-pro-naphthylamides, the usual substrates of mammalian DPP II, but was active on arg-arg- and asp-arg-naphthylamides, substrates acted on by the DPP III class of enzymes. This enzyme therefore combines properties typical of both DPP II and III and differs from all previously described DPPs. Activity on lys-ala-beta-naphthylamide was most abundant during aggregation and its activity is consistent with processing specific peptides during development.     

Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster.

A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723-residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was functionally expressed in Schneider S2 cells. Immunocytochemical studies using anti-(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands (82 and 86 kDa) were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites, PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) at the Tyr-Leu bond (Km approximately 4 micro m). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC50 = 0.62 +/- 0.15 micro m). These results constitute the first characterization of an evolutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation.     

Absolutely conserved tryptophan in M49 family of peptidases contributes to catalysis and binding of competitive inhibitors

The role of the unique fully conserved tryptophan in metallopeptidase family M49 (dipeptidyl peptidase III family) was investigated by site-directed mutagenesis on human dipeptidyl peptidase III (DPP III) where Trp300 was subjected to two substitutions (W300F and W300L). The mutant enzymes showed thermal stability equal to the wild-type DPP III. Conservative substitution of the Trp300 with phenylalanine decreased enzyme activity 2-4 fold, but did not significantly change the K_m values for two dipeptidyl 2-naphthylamide substrates. However, the K_m for the W300L mutant was elevated 5-fold and the k_cat value was reduced 16-fold with Arg-Arg-2-naphthylamide. Both substitutions had a negative effect on the binding of two competitive inhibitors designed to interact with S1 and S2 subsites.These results indicate the importance of the aromatic nature of W300 in DPP III ligand binding and catalysis, and contribution of this residue in maintaining the functional integrity of this enzyme’s S2 subsite.     

Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity

Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.     

Dentin phosphoprotein gene locus is not associated with dentinogenesis imperfecta types II and III.

Dentinogenesis imperfecta (DGI) is an autosomal dominant inherited dental disease which affects dentin production and mineralization. Genetic linkage studies have been performed on several multigeneration informative kindreds. These studies determined linkage between DGI type II and III and group-specific component (vitamin D-binding protein). This gene locus has been localized to the long arm of human chromosome 4 in the region 4q11-q21. Although this disease has been mapped to chromosome 4, the defective gene product is yet to be determined. Biochemical studies have suggested abnormal levels of dentin phosphoprotein (DPP) associated with DGI type II. This highly acidic protein is the major noncollagenous component of dentin, being solely expressed by the ectomesenchymal derived odontoblast cells of the tooth. The purpose of the present study was to establish whether DPP is associated with DGI types II and III, by using molecular biology techniques. The strategy was to use a synthetic degenerative DPP oligonucleotide probe to map this sequence to the long arm of human chromosome 4, 4q13-q21, by using somatic cell hybrids. Our results indicated that DPP is not localized to any region of human chromosome 4, thus suggesting that the DPP gene is not directly associated with DGI type II or DGI type III. Our data do not exclude the possibility that other proteins associated with DPP posttranslational modifications might be responsible for this genetic disease.     

Dipeptidyl peptidase 4 – An important digestive peptidase in Tenebrio molitor larvae

Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0–9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor.     

In rat dipeptidyl peptidase III,His is essential for catalysis,and Glu or Glu stabilizes the coordination bond between His or His and zinc ion

Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, “HELLGH,” as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al. (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K_m values or in the substrate specificity. However, the k_cat values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His⁵⁶⁸ residue of rat DPP III is essential for enzyme catalysis. The k_cat values of the mutants E507A and E512A were 2.38 and 3.88 s^− 1 toward Arg-Arg-NA, and 0.097 and 0.59 s⁻¹ toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 × 10³ to 2 × 10⁴. The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu⁵⁰⁷ and Glu⁵¹² form the hydrogen bonds to the nitrogen atoms of His⁴⁵⁵ and His⁴⁵⁰. All of these results showed that Glu⁵⁰⁷ or Glu⁵¹² stabilizes the coordination bond between the zinc ion and His⁴⁵⁵ or His⁴⁵⁰.     

Characterization and purification of adenosine deaminase 1 from human and chicken liver

Adenosine deaminase 1 (ADA1) was purified from human and chicken liver. The purified enzyme had a molecular weight of approximately 42,000 Da on SDS-PAGE. In humans, ADA1 was mainly purified concomitant with ADA-binding protein, dipeptidyl peptidase IV (DPP IV)/CD26; however, in chickens, only ADA1 without DPP IV was purified. Both human and chicken ADA1s showed similar properties on substrate specificities, sensitivities on inhibitors, and pH profile. However, they had different affinities with adenosine-Sepharose and IgG anti-ADA1-Sepharose. Human ADA1 was not adsorbed in adenosine-Sepharose column, but chicken ADA1 was adsorbed. As for IgG anti-ADA1-Sepharose column, the results were converse. Furthermore, human ADA1 could bind to DPP IV whereas chicken ADA1 could not.