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

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.

The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability.

Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate.

Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III.

Human dipeptidyl peptidase III (hDPP III) is a member of the M49 metallopeptidase family, which is involved in intracellular protein catabolism and oxidative stress response. To investigate the structural basis of hDPP III preference for diarginyl arylamide, using site-directed mutagenesis, we altered its S2 subsite to mimic the counterpart in yeast enzyme. Kinetic studies revealed that the single mutant D496G lost selectivity due to the increase of the Km value. The D496G, but not S504G, showed significantly decreased binding of peptides with N-terminal arginine, and of tynorphin. The results obtained identify Asp496 as an important determinant of human DPP III substrate specificity.

Importance of the three basic residues in the vicinity of the zinc-binding motifs for the activity of the yeast dipeptidyl peptidase III.

Yeast dipeptidyl peptidase III (yDPP III) is a member of the metallopeptidase family M49 involved in intracellular protein catabolism. Elucidation of the yDPP III crystal structure has pinpointed the zinc-coordinating residues (two His from H(460)ELLGH(465) motif and the second Glu from E(516)ECRAE(521) motif), and several amino acid residues potentially important for catalytic activity whose roles have not been investigated. Here, three putative catalytic residues of the yDPP III, His578, Arg582 and Lys638 were substituted and the resultant single mutants characterized. The replacement of His578 with an asparagine significantly (122-fold) lowered the catalytic efficiency, kcat/Km, for Arg-Arg-2-naphthylamide (Arg2-2NA) hydrolysis, and affinity for hydroxamate inhibitor Tyr-Phe-NHOH (decline by 14-fold). The R582Q mutant exhibited an order of magnitude higher activity with all four dipeptide derivatives examined, compared to the wild type. The molecular dynamics simulations revealed the change in the H-bond networking in the R582Q variant active-site region. The mutation of Lys638, to Leu, slightly increased the specificity constant for Arg2-2NA hydrolysis. However, the affinity for Tyr-Phe-NHOH, and activity for the substrates with uncharged P2 side chains (Ala-Ala-, Ala-Arg- and Phe-Arg-2NA) were dramatically reduced, indicating the importance of the evolutionary conserved salt bridge Lys(638)-Glu(516) for the modulation of DPP III substrate specificity.

Hydrolysis of dipeptide derivatives reveals the diversity in the M49 family.

Dipeptidyl peptidase III, a metallopeptidase of the M49 family, was first identified (in the pituitary) by its specific cleavage of diarginyl arylamides, which have been used as preferred assay substrates until now. Here we examined the activity of the yeast and human dipeptidyl peptidase III in parallel. The human enzyme preferred Arg(2)-ß-naphthylamide and showed 620-fold higher k(cat)/K(m) for this substrate. In contrast, the yeast enzyme did not display a preference for any of the X-Arg-ß-naphthylamide analyzed. The replacement of Gly(505) with Asp, resulted in a less active, but more selective, yeast enzyme form. These results indicate diversity in cleavage specificity in the M49 family.

Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III.

Dipeptidyl peptidases III (DPPs III) form a distinct metallopeptidase family characterized by the unique HEXXGH motif. High susceptibility to inactivation by organomercurials suggests the presence of a reactive cysteine residue(s) in, or close to, their active site. Yeast DPP III contains five Cys, none of which is absolutely conserved within the family. In order to identify reactive residue(s), site-directed mutagenesis on yeast His(6)-tagged DPP III was employed to substitute specifically all five cysteine residues to serine. The variant enzymes thus obtained were enzymatically active and showed an overall structure not greatly affected by the mutations as judged by circular dichroism. Analysis by native and SDS-PAGE under non-reducing conditions revealed the existence of a monomeric and dimeric form in all DPP III proteins except in the C130S, implying that dimerization of yeast DPP III is mediated by the surface-exposed cysteine 130. The investigation of the effect of thiol reagent 4,4'-dithiodipyridine (DTDP) on all five Cys to Ser single protein variants showed that Cys639 and Cys518 are more reactive than the remainder. Only the C639S mutant protein displayed the remarkable resistance against p-hydroxy-mercuribenzoate (pHMB) indicating that modification of Cys639 is responsible for the fast inactivation of yeast DPP III by this sulfhydryl reagent.

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.

The first structure of dipeptidyl-peptidase III provides insight into the catalytic mechanism and mode of substrate binding.

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-A 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.