Carboxyl Peptidase Research Articles

Hyper-Responsive Chemiluminescent Probe Reveals Distinct PYRase Activity in Pseudomonas aeruginosa.

Pyrrolidone carboxyl peptidase, commonly known as PYRase, is an exopeptidase that catalytically cleaves an N-terminal pyroglutamic acid from peptides or proteins. The diverse functions of PYRases in bacterial enzymology have prompted the development of various bacterial diagnostic techniques. However, the specific physiological role and activity of this enzyme across the bacterial kingdom remain unclear. Here, we present a functional phenoxy-1,2-dioxetane chemiluminescent probe (PyrCL) that can selectively detect PYRase activity in both Gram-positive and Gram-negative bacteria. The probe activation mechanism is based on the cleavage of a pyroglutamyl substrate, followed by a release of the phenoxy-dioxetane luminophore, which then undergoes efficient chemiexcitation to emit a green photon. Probe PyrCL exhibits an effective turn-on response with superior detection capability in terms of response time and sensitivity compared to existing fluorescence probes. The superior detection sensitivity of the chemiluminescent probe enables us to reveal previously undetected PYRase activity in Streptococcus mutans. Furthermore, it enables the discrimination of Pseudomonas aeruginosa from other Gram-negative bacteria in the tested panel, based on their distinct PYRase activity. We expect that probe PyrCL will have great value for PYRase-based bacteria diagnosis with use in basic research and clinical applications.

Crystal structure analysis of pyrrolidone carboxyl peptidase from Thermus thermophilus

Pyrrolidone carboxyl peptidase (PCP) hydrolytically removes the L-pyroglutamic acid from the amino terminal region of pyroglutamyl proteins or peptides. So far, only a limited number of structures of PCP have been solved. Here we report the crystal structure of pyrrolidone carboxyl peptidase from Thermus thermophilus (TtPCP) which has been solved using the molecular replacement method and refined at 1.9 Å resolution. TtPCP follows the α/β/α architecture in which the central β-sheets are surrounded by α-helices on both sides. The inter subunit contact between two monomers consists of two short antiparallel β-strands and part of a long protrusion loop. By comparing the TtPCP with its structural homologs, we identified the putative catalytic triad residues as Glu76, Cys139 and His160. A unique disulfide link found in some homologs of TtPCP, formed between two monomers that provide thermal stability to the protein, is not observed in TtPCP. Hence, being a thermophilic protein, the putative thermal stability of TtPCP could be due to more intra and inter-molecular hydrogen bonds, hydrophobic and ion pair interactions when compared with its mesophilic counterpart. The structural details of TtPCP will be helpful to understand the basis of the intrinsic stability of thermophilic proteins. Also, it could be useful for protein engineering.

Isolation of an anti-entomopathogenic fungal protein secreted from Pseudomonas aeruginosa BGf-2: An intestinal bacteriam of Blattella germanica (L.)

A bacterial strain (BGf-2) with anti-Beauveria bassiana activity was obtained from the feces of Blattella germanica (L.) and identified as Pseudomonas aeruginosa based on biochemical tests and 16S rRNA sequence analysis. An antifungal protein (A0A0H2ZK06) was purified with Sephadex G-100 column and DEAE-sepharose Fast Flowanion exchange from sterile BGf-2 fermentation liquid. Based on MALDI-TOF MS analysis and protein model building, A0A0H2ZK06 showed homology with Pyrrolidone carboxyl peptidases (pcps). Fermentation liquid and antifungal proteins not only reduced the B. bassiana conidial germination rate but also inhibited hyphal growth. A per os test showed that the mortality of cockroaches decreased after treatment with BGf-2 suspension compared with control. We hypothesized that gut microbes with antifungal activity might play an important role in protect cockroaches from pathogenic fungi.

Angiotensin-converting enzyme affects the presentation of MHC class II antigens

Antigen processing and presentation through the MHC class II pathway is critical for activating T helper cells. Angiotensin-converting enzyme (ACE) is a carboxyl peptidase expressed by antigen-presenting cells. By analysis of ACE null (knockout), wild-type, and ACE-overexpressing (ACE10) mice and the antigen-presenting cells derived from these mice, we found that ACE has a physiological role in the processing of peptides for MHC class II presentation. The efficiency of presenting MHC class II epitopes from ovalbumin (OVA) and hen egg lysosome is markedly affected by cellular ACE levels. Mice overexpressing ACE in myeloid cells have a much more vigorous CD4+ T-cell and antibody response when immunized with OVA. ACE is present in the endosomal pathway where MHC class II peptide processing and loading occur. The efficiency of MHC class II antigen presentation can be altered by ACE overexpression or ACE pharmacological inhibition. Thus, ACE is a dynamic participant in processing MHC class II peptides. Manipulation of ACE expression by antigen-presenting cells may prove to be a novel strategy to alter the immune response.

Structure and function studies on enzymes with a catalytic carboxyl group(s): from ribonuclease T1 to carboxyl peptidases.

A group of enzymes, mostly hydrolases or certain transferases, utilize one or a few side-chain carboxyl groups of Asp and/or Glu as part of the catalytic machinery at their active sites. This review follows mainly the trail of studies performed by the author and his colleagues on the structure and function of such enzymes, starting from ribonuclease T1, then extending to three major types of carboxyl peptidases including aspartic peptidases, glutamic peptidases and serine-carboxyl peptidases.

Structural Characterization of a Trapped Folding Intermediate of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.

New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases

Peptidases or proteinases are now classified into seven families based on the nature of the catalytic residues [MEROPS-the peptidase database (http://merops.sanger.ac.uk/)]. They are aspartic- (first described in 1993), cysteine- (1993), serine- (1993) metallo- (1993), threonine- (1997), glutamic- (2004) and asparagine-peptidase (2010). By using an S-PI (pepstatin Ac) as a probe, a new subfamily of serine peptidase, serine-carboxyl peptidase (sedolisin) was discovered in 2001. In addition, the sixth family of peptidase, glutamic peptidase (eqolisin) was also discovered in 2004. The former peptidase is widely distributed in nature from archea to mammals, including humans. One of these enzymes is related to a human fatal hereditable disease, Batten disease. In contrast, the distribution of the latter peptidases is limited, with most of them found in human or plant pathogenic fungi. One such enzyme was isolated from a fungal infection in an HIV-infected patient. In this review, the background of the findings, and crystal structures, catalytic mechanisms, substrates specificities and distribution of the new peptidase families are described.

Understanding the Mechanism of Deacylation Reaction Catalyzed by the Serine Carboxyl Peptidase Kumamolisin-As: Insights from QM/MM Free Energy Simulations

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics and free energy simulations are performed to study the process of the deacylation reaction catalyzed by kumamolisin-As, a serine-carboxyl peptidase, and to elucidate the catalytic mechanism. The results given here suggest that Asp-164 acts as a general acid/base catalyst not only for the acylation reaction but also for the deacylation reaction. It is shown that the electrostatic oxyanion hole interactions may be less effective in transition state stabilization for the kumamolisin-As catalyzed reaction compared to the general acid/base mechanism involving the proton transfer from or to Asp-164. The dynamic substrate-assisted catalysis (DSAC) involving His at the P1 site of the substrate is found to be less important for the deacylation reaction than for the acylation reaction in the kumamolisin-As catalyzed reaction. The proton transfer processes during the enzyme-catalyzed process are examined and their role in the catalysis is discussed.

1P-041 モルテングロビュール段階で見出されるPCP(ピロリドンカルボキシルペプチダーゼ)の逐次折れたたみ経路とそれに続く天然構造への転移(蛋白質-物性(安定性,折れたたみなど),第47回日本生物物理学会年会)

1P-041 モルテングロビュール段階で見出されるPCP(ピロリドンカルボキシルペプチダーゼ)の逐次折れたたみ経路とそれに続く天然構造への転移(蛋白質-物性(安定性,折れたたみなど),第47回日本生物物理学会年会)

The confirmation of the denatured structure of pyrrolidone carboxyl peptidase under nondenaturing conditions: Difference in helix propensity of two synthetic peptides with single amino acid substitution

In the denatured state (D(1) state) of cystein-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus, a hyperthermophile under nondenaturing conditions, a fairly stable alpha-helix (alpha6-helix) has been determined from H/D exchange-NMR experiments. On the other hand, the alpha6-helix region of the proline-mutant at position 199 (A199P) was unstructured in the D(1) state unlike that of the wild-type PCP-0SH, although the folded conformations of both proteins were almost identical to each other. This finding has been deduced from the information regarding the remaining amide hydrogens in the HSQC spectra after H/D exchanges in the D(1) state. To confirm this inference, we examined the helical propensities of two synthetic peptides from their NMR structural analysis in the presence of trifluoroethanol (TFE). One is an 18-residue peptide called the wild-type H6-peptide corresponding to the alpha6-helix (from Ser188 to Glu205) of the wild-type PCP-0SH, and the other is the mutant H6-peptide corresponding to the alpha6-helix region of A199P. The NOE-contact information obtained from the 2D-(1)H-NOESY spectra measured for both peptides in the presence of 30% TFE clearly demonstrated that the wild-type H6-peptide had a high helical propensity, but the mutant H6-peptide was almost totally unstructured. The TFE-induced helical propensities for these peptide fragments confirmed the conclusions deduced from the H/D exchange data measured in the D(1) states of two proteins.

3P-003 モルテングロビュール状の変性状態においてピロリドンカルボキシルペプチダーゼ(PCP)の折りたたみ反応は順次進行していく(蛋白質・物性(3),第46回日本生物物理学会年会)

3P-003 モルテングロビュール状の変性状態においてピロリドンカルボキシルペプチダーゼ(PCP)の折りたたみ反応は順次進行していく(蛋白質・物性(3),第46回日本生物物理学会年会)

Characterization of the Denatured Structure of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile under Nondenaturing Conditions: Role of the C-Terminal α-Helix of the Protein in Folding and Stability,

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.

Catalytic Role of Proton Transfers in the Formation of a Tetrahedral Adduct in a Serine Carboxyl Peptidase

Quantum mechanical/molecular mechanical molecular dynamics and 2D free energy simulations are performed to study the formation of a tetrahedral adduct by an inhibitor N-acetyl-isoleucyl-prolyl-phenylalaninal (AcIPF) in a serine-carboxyl peptidase (kumamolisin-As) and elucidate the role of proton transfers during the nucleophilic attack by the Ser278 catalytic residue. It is shown that although the serine-carboxyl peptidases have a fold resembling that of subtilisin, the proton transfer processes during the nucleophilic attack by the Ser residue are likely to be more complex for these enzymes compared to the case in classical serine proteases. The computer simulations demonstrate that both general base and acid catalysts are required for the formation and stabilization of the tetrahedral adduct. The 2D free energy maps further demonstrate that the proton transfer from Ser278 to Glu78 (the general base catalyst) is synchronous with the nucleophilic attack, whereas the proton transfer from Asp164 (the general acid catalyst) to the inhibitor is not. The dynamics of the protons at the active site in different stages of the nucleophilic attack as well as the motions of the corresponding functional groups are also studied. It is found that the side chain of Glu78 is generally rather flexible, consistent with its possible multifunctional role during catalysis. The effects of proton shuffling from Asp82 to Glu78 and from Glu32 to Asp82 are examined, and the results indicate that such proton shuffling may not play an important role in the stabilization of the tetrahedral intermediate analogue.

Completely Buried, Non-Ion-Paired Glutamic Acid Contributes Favorably to the Conformational Stability of Pyrrolidone Carboxyl Peptidases from Hyperthermophiles,

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.

Pyroglutamic acid and iron regulate the expression of the pcp gene in Pseudomonas fluorescens MFO

Pyrrolidone carboxyl peptidase (Pcp) is an aminopeptidase (EC 3.4.11.8) able to specifically remove the l-pyroglutamyl residue from the amino-terminus of polypeptides. Since nothing was known concerning the regulation and function of Pcps, a mutant of a milk-isolated strain lacking Pcp activity (Pseudomonas fluorescens MB1), was constructed by homologous recombination using a transcriptional fusion between pcp and a reporter gene (uidA). The wild-type and mutant strains were grown in synthetic media and in milk to investigate the environmental effects on pcp transcription. The expression of pcp and of the transcriptional fusion pcp::uidA was not sensitive to environmental conditions like temperature, osmolarity or nitrogen and phosphate starvation but was induced by the product of the enzymatic activity, pyroglutamic acid (pGlu). The expression of the native gene and the fusion in inducing conditions was also controlled by the iron concentration. The identification in the pcp promoter sequence of putative ferric uptake regulator (Fur) binding sites suggests a transcriptional regulation in a Fur-dependent fashion. Two other putative regulatory stretches, corresponding to inverted repeated sequences with perfect and imperfect symmetry, were also identified. pGlu and iron are therefore at least two of the transcriptional effectors of pcp expression.

1P118 H/D exchange characterization of the C-terminal α-helix in the denatured state of pyrrolidone carboxyl peptidase from a hyperthermophile(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P118 H/D exchange characterization of the C-terminal α-helix in the denatured state of pyrrolidone carboxyl peptidase from a hyperthermophile(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P123 TFE-induced conformations of a synthesized oligopeptide originating from the C-terminal α-helix of pyrrolidone carboxyl peptidase(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P123 TFE-induced conformations of a synthesized oligopeptide originating from the C-terminal α-helix of pyrrolidone carboxyl peptidase(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

A General Acid−Base Mechanism for the Stabilization of a Tetrahedral Adduct in a Serine−Carboxyl Peptidase: A Computational Study

The QM/MM MD and free energy simulations show that serine-carboxyl peptidases (sedolisins) may stabilize the tetrahedral intermediates and tetrahedral adducts primarily through a general acid-base mechanism involving Asp (Asp164 for kumamolisin-As) rather than the oxyanion-hole interactions as in the cases of serine proteases.

Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-peptidase I in Vitro and in Vivo

Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal exopeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t((1/2)) of mature enzyme at pH 7.4 increased by approximately 8-fold in the presence of heparin) and in vivo ( approximately 2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.

2P072 NMRによる超好熱菌由来のPyrrolidone carboxyl peptidaseのリフォールディング中間体に関する研究(蛋白質 C) 物性(安定性、折れたたみなど)))

2P072 NMRによる超好熱菌由来のPyrrolidone carboxyl peptidaseのリフォールディング中間体に関する研究(蛋白質 C) 物性(安定性、折れたたみなど)))