Acylaminoacyl Peptidase Research Articles

Structural adaptations for carboxypeptidase activity in putative S9 acylaminoacyl peptidase from Bacillus subtilis

Prolyl oligopeptidase, or S9 (MEROPS) family enzymes are crucial drug targets due to their association with various diseases, neurological disorders, cell growth, and survival. These implications render them an exceptionally fascinating field of research. Despite sharing similar structural features, they exhibit diverse enzyme activities, including endopeptidase, dipeptidyl peptidase, and acylaminoacyl peptidase. Additionally, a few members of the S9 family demonstrate carboxypeptidase activity. A recent study showed that the S9 peptidase of Bacillus subtilis (S9bs) possesses the conserved sequence feature necessary for carboxypeptidase activity despite being annotated as an acylaminoacyl peptidase in the UniProt database. However, the mechanism of action and identity of S9bs as carboxypeptidase remain unclear. Consequently, we focused our studies on thoroughly investigating S9bs for its carboxypeptidase activity. In the present study, we report biochemical and biophysical analyses of S9bs, confirming its identity as a carboxypeptidase. Further, structural analysis reveals the molecular basis of S9bs' carboxypeptidase activity, highlighting the crucial structural elements like the “cavity loop” and the “two-arginine” residues essential for this activity. Additionally, our studies confirmed that S9bs forms a stable tetrameric assembly and established its quaternary molecular arrangement, which reveals the presence of an oligomeric pore. Altogether, these structural features play a are crucial role in substrate selection for S9 carboxypeptidases. Overall, our findings reveal a distinct carboxypeptidase within the S9 family and significantly enhance our understanding of these enzymes. Moreover, this study sheds light on the mechanisms underlying carboxypeptidase activity, offering valuable insights that could contribute to therapeutic and drug design.

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis

Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms.

Cryo-EM structure of acylpeptide hydrolase reveals substrate selection by multimerization and a multi-state serine-protease triad.

The first structure of tetrameric mammalian acylaminoacyl peptidase, an enzyme that functions as an upstream regulator of the proteasome through the removal of terminal N-acetylated residues from its protein substrates, was determined by cryo-EM and further elucidated by MD simulations. Self-association results in a toroid-shaped quaternary structure, guided by an amyloidogenic β-edge and unique inserts. With a Pro introduced into its central β-sheet, sufficient conformational freedom is awarded to the segment containing the catalytic Ser587 that the serine protease catalytic triad alternates between active and latent states. Active site flexibility suggests that the dual function of catalysis and substrate selection are fulfilled by a novel mechanism: substrate entrance is regulated by flexible loops creating a double-gated channel system, while binding of the substrate to the active site is required for stabilization of the catalytic apparatus – as a second filter before hydrolysis. The structure not only underlines that within the family of S9 proteases homo-multimerization acts as a crucial tool for substrate selection, but it will also allow drug design targeting of the ubiquitin-proteasome system.

A carbapenem antibiotic inhibiting a mammalian serine protease: structure of the acylaminoacyl peptidase-meropenem complex.

The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem antibiotic. AAP is a modulator of the ubiquitin-proteasome degradation system and the site of a drug-drug interaction between the widely used antipsychotic, valproate and carbapenems. The active form of pAAP - a toroidal tetramer - binds four meropenem molecules covalently linked to the catalytic Ser587 of the serine-protease triad, in an acyl-enzyme state. AAP is hindered from fully processing the antibiotic by the displacement and protonation of His707 of the catalytic triad. We show that AAP is made susceptible to the association by its unusually sheltered active pockets and flexible catalytic triads, while the carbapenems possess sufficiently small substituents on their β-lactam rings to fit into the shallow substrate-specificity pocket of the enzyme.

The activity and stability of a cold-active acylaminoacyl peptidase rely on its dimerization by domain swapping

The study of enzymes from extremophiles arouses interest in Protein Science because of the amazing solutions these proteins adopt to cope with extreme conditions. Recently solved, the structure of the psychrophilic acyl aminoacyl peptidase from Sporosarcina psychrophila (SpAAP) pinpoints a mechanism of dimerization unusual for this class of enzymes. The quaternary structure of SpAAP relies on a domain-swapping mechanism involving the N-terminal A1 helix. The A1 helix is conserved among homologous mesophilic and psychrophilic proteins and its deletion causes the formation of a monomeric enzyme, which is inactive and prone to aggregate. Here, we investigate the dimerization mechanism of SpAAP through the analysis of chimeric heterodimers where a protomer lacking the A1 helix combines with a protomer carrying the inactivated catalytic site. Our results indicate that the two active sites are independent, and that a single A1 helix is sufficient to partially recover the quaternary structure and the activity of chimeric heterodimers. Since catalytically competent protomers are unstable and inactive unless they dimerize, SpAAP reveals as an “obligomer” for both structural and functional reasons.

Carboxypeptidase in prolyl oligopeptidase family: Unique enzyme activation and substrate-screening mechanisms

Serine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders. Proper annotation and knowledge of substrate specificity mechanisms in this family are highly valuable. Although endopeptidase, dipeptidyl peptidase, tripeptidyl peptidase, and acylaminoacyl peptidase activities have been reported previously, here we report the first instance of carboxypeptidase activity in a POP family member. We determined the crystal structures of this carboxypeptidase, an S9C subfamily member from Deinococcus radiodurans, in its active and inactive states at 2.3-Å resolution, providing an unprecedented view of assembly and disassembly of the active site mediated by an arginine residue. We observed that this residue is poised to bind substrate in the active structure and disrupts the catalytic triad in the inactive structure. The assembly of the active site is accompanied by the ordering of gating loops, which reduces the effective size of the oligomeric pore. This prevents the entry of larger peptides and constitutes a novel mechanism for substrate screening. Furthermore, we observed structural adaptations that enable its carboxypeptidase activity, with a unique loop and two arginine residues in the active site cavity orienting the peptide substrate for catalysis. Using these structural features, we identified homologs of this enzyme in the POP family and confirmed the presence of carboxypeptidase activity in one of them. In conclusion, we have identified a new type within POP enzymes that exhibits not only unique activity but also a novel substrate-screening mechanism.

Understanding the interactions of different substrates with wild-type and mutant acylaminoacyl peptidase using molecular dynamics simulations

Acylaminoacylpeptidase (AAP) belongs to peptidase protein family, which can degrade amyloid β-peptide forms in the brains of patients, and hence leads to Alzheimer’s disease. And so, AAP is considered to be a novel target in the design of drugs against Alzheimer’s disease. In this investigation, six molecular dynamics simulations were used to find that the interaction between the wild-type and R526V AAP with two different substrates (p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide). Our results were as follows: firstly, Ac-Leu-p-nitroanilide bound to R526V AAP to form a more disordered loop (residues 552–562) in the α/β-hydrolase fold like of AAP, which caused an open and inactive AAP domain form, secondly, binding p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide to AAP can decrease the flexibility of residues 225–250, 260–270, and 425–450, in which the ordered secondary structures may contain the suitable geometrical structure and so it is useful to serine attack. Our theoretical results showed that the binding of the two substrates can induce specific conformational changes responsible for the diverse AAP catalytic specificity. These theoretical substrate-induced structural diversities can help explain the abilities of AAPs to recognize and hydrolyze extremely different substrates.

A bacterial acyl aminoacyl peptidase couples flexibility and stability as a result of cold adaptation.

Structural data are available in the Protein Data Bank under the accession number 5L8S.

Solving the structure of a mammalian acylaminoacyl-peptidase

Solving the structure of a mammalian acylaminoacyl-peptidase

A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides

Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm(-1) s(-1), optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (β-propeller domain); and residues Ala(496)-Phe(736) (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides.

Exploring the different ligand escape pathways in acylaminoacyl peptidase by random acceleration and steered molecular dynamics simulations

Acylaminoacyl peptidase (APH, EC 3.4.19.1) is a novel class of serine-type protease belonging to the prolyl oligopeptidase (POP) family.

Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction

Acylaminoacyl peptidase has been named acyl-peptide releasing enzyme( AARE),catalyses the removal of an N-acylated amino acid from N-α-acylpeptides. In this research,a novel acyl-peptide releasing enzyme gene( BSU32230) from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BLP DE3 codon plus to produce acyl-peptide releasing enzyme. The recombinant enzyme was purified in two steps:ammonium sulfate precipitation and Ni2+-column affinity chromatography. The optimum temperature and p H of enzyme were 50 ℃ and 8. 0,respectively. The half-life of recombinant enzyme at 40 ℃ was 29 h,the enzyme was stable at p H range from 4 to 10. This report demonstrated the acylaminoacyl peptidase from Bacillus subtilis peptidase can catalyze aldol reaction and showed high enantioselectivity. The reaction provided optically active secondary alcohol with satisfying enantioselectivity( 84. 6% e.e.).

Catalytically distinct states captured in a crystal lattice: the substrate-bound and scavenger states of acylaminoacyl peptidase and their implications for functionality

Acylaminoacyl peptidase (AAP) is an oligopeptidase that only cleaves short peptides or protein segments. In the case of AAP from Aeropyrum pernix (ApAAP), previous studies have led to a model in which the clamshell-like opening and closing of the enzyme provides the means of substrate-size selection. The closed form of the enzyme is catalytically active, while opening deactivates the catalytic triad. The crystallographic results presented here show that the open form of ApAAP is indeed functionally disabled. The obtained crystal structures also reveal that the closed form is penetrable to small ligands: inhibitor added to the pre-formed crystal was able to reach the active site of the rigidified protein, which is only possible through the narrow channel of the propeller domain. Molecular-dynamics simulations investigating the structure of the complexes formed with longer peptide substrates showed that their binding within the large crevice of the closed form of ApAAP leaves the enzyme structure unperturbed; however, their accessing the binding site seems more probable when assisted by opening of the enzyme. Thus, the open form of ApAAP corresponds to a scavenger of possible substrates, the actual cleavage of which only takes place if the enzyme is able to re-close.

Gene cloning and biochemical characterization of eryngase, a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases.

Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH2 as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH2 by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH2. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH2 substrate.

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

Protein structure and dynamics are crucial for protein function. Thus, the study of conformational properties can be very informative for characterizing new proteins and to rationalize how residue substitutions at specific protein sites affect its dynamics, activity and thermal stability. Here, we investigate the structure and dynamics of the recently isolated cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila (SpAAP) by the integration of simulations, circular dichroism, mass spectrometry and other experimental data. Our study notes traits of cold-adaptation, such as lysine-to-arginine substitutions and a lack of disulphide bridges. Cold-adapted enzymes are generally characterized by a higher number of glycine residues with respect to their warm-adapted counterparts. Conversely, the SpAAP glycine content is lower than that in the warm-adapted variants. Nevertheless, glycine residues are strategically located in proximity to the functional sites in SpAAP, such as the active site and the linker between the two domains.. In particular, G457 reduces the steric hindrance around the nucleophile elbow. Our results suggest a local weakening of the intramolecular interactions in the cold-adapted enzyme. This study offers a basis for the experimental mutagenesis of SpAAP and related enzymes. The approaches employed in this study may also provide a more general framework to characterize new protein structures in the absence of X-ray or NMR data.

Crystal Structures of Trypanosoma brucei Oligopeptidase B Broaden the Paradigm of Catalytic Regulation in Prolyl Oligopeptidase Family Enzymes

Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

OsPOP5, A Prolyl Oligopeptidase Family Gene from Rice Confers Abiotic Stress Tolerance in Escherichia coli

The prolyl oligopeptidase family, which is a group of serine peptidases, can hydrolyze peptides smaller than 30 residues. The prolyl oligopeptidase family in plants includes four members, which are prolyl oligopeptidase (POP, EC3.4.21.26), dipeptidyl peptidase IV (DPPIV, EC3.4.14.5), oligopeptidase B (OPB, EC3.4.21.83), and acylaminoacyl peptidase (ACPH, EC3.4.19.1). POP is found in human and rat, and plays important roles in multiple biological processes, such as protein secretion, maturation and degradation of peptide hormones, and neuropathies, signal transduction and memory and learning. However, the function of POP is unclear in plants. In order to study POP function in plants, we cloned the cDNA of the OsPOP5 gene from rice by nested-PCR. Sequence analysis showed that the cDNA encodes a protein of 596 amino acid residues with Mw ≈ 67.29 kD. In order to analyze the protein function under different abiotic stresses, OsPOP5 was expressed in Escherichia coli. OsPOP5 protein enhanced the tolerance of E. coli to high salinity, high temperature and simulated drought. The results indicate that OsPOP5 is a stress-related gene in rice and it may play an important role in plant tolerance to abiotic stress.

Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from Sporosarcina psychrophila

Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution.

Coupled motions during dynamics reveal a tunnel toward the active site regulated by the N-terminal α-helix in an acylaminoacyl peptidase

Acylaminoacyl peptidase (AAP) subfamily belongs to the prolyl oligopeptidase (POP) family of serine-proteases. There is a great interest in the definition of molecular mechanisms related to the activity and substrate recognition of these complex multi-domain enzymes. The active site relies at the interface between the C-terminal catalytic domain and the β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In AAP, the N-terminal extension is characterized by a structurally conserved α1-helix, which is known to affect thermal stability and thermal dependence of the catalytic activity. In the present contribution, results from hundreds nanosecond all-atom molecular dynamics simulations, along with analyses of the networks of cross-correlated motions of a member of the AAP subfamily are discussed. The MD investigation identifies a tunnel that from the surrounding of the N-terminal α1-helix bring to the catalytic site. This cavity seems to be regulated by conformational changes of the α1-helix itself during the dynamics. The evidence here provided can be a useful guide for a better understanding of the mechanistic aspects related to AAP activity, but also for drug design purposes.

Mechanisms of Intramolecular Communication in a Hyperthermophilic Acylaminoacyl Peptidase: A Molecular Dynamics Investigation

Protein dynamics and the underlying networks of intramolecular interactions and communicating residues within the three-dimensional (3D) structure are known to influence protein function and stability, as well as to modulate conformational changes and allostery. Acylaminoacyl peptidase (AAP) subfamily of enzymes belongs to a unique class of serine proteases, the prolyl oligopeptidase (POP) family, which has not been thoroughly investigated yet. POPs have a characteristic multidomain three-dimensional architecture with the active site at the interface of the C-terminal catalytic domain and a β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In the present contribution, protein dynamics signatures of a hyperthermophilic acylaminoacyl peptidase (AAP) of the prolyl oligopeptidase (POP) family, as well as of a deletion variant and alanine mutants (I12A, V13A, V16A, L19A, I20A) are reported. In particular, we aimed at identifying crucial residues for long range communications to the catalytic site or promoting the conformational changes to switch from closed to open ApAAP conformations. Our investigation shows that the N-terminal α1-helix mediates structural intramolecular communication to the catalytic site, concurring to the maintenance of a proper functional architecture of the catalytic triad. Main determinants of the effects induced by α1-helix are a subset of hydrophobic residues (V16, L19 and I20). Moreover, a subset of residues characterized by relevant interaction networks or coupled motions have been identified, which are likely to modulate the conformational properties at the interdomain interface.