Enzymatic Preparation of Bioactive Peptides Exhibiting ACE Inhibitory Activity from Soybean and Velvet Bean: A Systematic Review.

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.

Exopeptidase Assisted N- and C-Terminal Proteome Sequencing.

Due to mechanisms such as proteolytic processing or alternative translation starts, in vivo proteoforms do not necessarily correspond directly to those encoded in the genome. Therefore, the knowledge of protein termini is an indispensable prerequisite to understand protein functions. So far, sequencing of protein N- and C-termini has been limited to single purified protein species, while the proteome-wide identification of N- and C-termini relies on the generation of single, terminal proteotypic peptides followed by chemical enrichment or depletion strategies to facilitate their detection via mass spectrometry (MS). To overcome the numerous limitations in such approaches, we present an alternative concept that readily enables unbiased ladder sequencing of protein N- and C-termini. The approach combines exopeptidase digestions of the proteome with two-dimensional chromatographic separation and tandem-MS. We demonstrate the potential of the methodology by analyzing the N- and C-terminome of S. cerevisiae, identifying 2190 N-termini and 1562 C-termini. In conclusion, the presented method largely expands the proteomics toolbox enabling N- and C-terminal sequential characterization of entire proteomes.

Ac-LysargiNase Complements Trypsin for the Identification of Ubiquitinated Sites.

Mass spectrometry (MS)-based identification of ubiquitinated sites requires trypsin digestion prior to MS analysis, and a signature peptide was produced with a diglycine residue attached to the ubiquitinated lysine (K-ε-GG peptide). However, the missed cleavage of modified lysines by trypsin results in modified peptides with increased length and charge, whose detection by MS analysis is suppressed by the vast majority of internally unmodified peptides. LysargiNase, the mirrored trypsin, is reported to cleave before lysine and arginine residues and to be favorable for the identification of methylation and phosphorylation, but its digestive characteristics related to ubiquitination are unclear. Herein, we tested the capacity of the in-house developed acetylated LysargiNase (Ac-LysargiNase) with high activity and stability, for cleaving ubiquitinated sites in both the seven types of ubiquitin chains and their corresponding K-ε-GG peptides. Interestingly, Ac-LysargiNase could efficiently cleave the K63-linked chain but had little effect on the other types of chains. Additionally, Ac-LysargiNase had higher exopeptidase activity than trypsin. Utilizing these features of the paired mirror proteases, a workflow of trypsin and Ac-LysargiNase tandem digestion was developed for the identification of ubiquitinated proteins. Through this method, the charge states and ionization capacity of the unmodified peptides were efficiently reduced, and the identification of modified sites was consequently increased by 30% to 50%. Strikingly, approximately 15% of the modified sites were cleaved by Ac-LysargiNase, resulting in shorter K-ε-GG peptides for better identification. The enzyme Ac-LysargiNase is expected to serve as an option for increasing the efficiency of modified site identification in ubiquitome research.

Broad-spectrum quorum sensing and biofilm inhibition by green tea against gram-negative pathogenic bacteria: Deciphering the role of phytocompounds through molecular modelling.

The emerging prevalence of multidrug-resistance in Gram-negative pathogens, due to conventional antimicrobial therapeutics, has led the researchers to emphasize on development of alternative novel strategies to suppress the bacterial virulence and pathogenicity through inhibition of quorum sensing (QS) and biofilms. QS is a bacterial communication system to produce density-dependent response via chemical signalling that controls pathogenesis and biofilms formation. Leaves of green tea are used worldwide as beverage which is also known for its broad-spectrum therapeutic efficacy. In this work, we have identified and characterized the most bioactive faction of green tea extract and evaluated the anti-QS and antibiofilm activity of green tea ethyl acetate fraction (GTEF) i.e. most active fraction, on three different Gram-negative bacterial pathogens. GTEF inhibited the violacein production by >75% in C. violaceum 12472. Many virulence factors of P. aeruginosa PAO1 viz. pyocyanin, pyoverdin, exoprotease, elastase, rhamnolipid production, and swimming motility were remarkably reduced in presence of sub-MICs of GTEF. Moreover, prodigiosin, protease activity, cell surface hydrophobicity, and swimming of S. marcescens MTCC 97 were also decreased significantly by the supplementation of GTEF in culture media. GTEF exhibited broad-spectrum antibiofilm action with >80% reduction in biofilm formation of test pathogens. In silico studies gave a mechanistic insight of action of GTEF. Molecular modelling revealed that phytoconstituents detected by GC/MS exhibited affinity (in order of 104 M-1) towards AHL synthases (LasI and EsaI). The molecular binding between phytocompounds and receptor proteins (LasR, RhlR, and PqsR) of QS circuit was also energetically favourable (ΔG°≥ 5.0 kcal mol-1) and supported by hydrogen bonds and hydrophobic interactions. These compounds were found to be docked in ligand binding domain of CviR and occupied same cavity as that of its antagonist. Squalene and thunbergol interacted with LasA at tartaric acid binding pocket and the complex was strengthened with binding energy -5.9 kcal mol-1. Moreover, interaction of thunbergol with biofilm-associated proteins viz. PilT and PilY1, might be disabling the pilus assembly and consequently inhibiting biofilm formation. In vivo validation of results suggested the protective role GTEF against QS-mediated pathogenicity and it might become a novel non-antibiotic QS inhibitor to control bacterial infection.

Establishment of an HPLC-based method to identify key proteases of proteins in vitro.

Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detection in vitro substantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability.

Aeromonas hydrophila biofilm, exoprotease, and quorum sensing responses to co-cultivation with diverse foodborne pathogens and food spoilage bacteria on crab surfaces.

The effects of dual species interactions on biofilm formation by Aeromonas hydrophila in the presence of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pectobacterium carotovorum, Salmonella Typhimurium, and Listeria monocytogenes were examined. High-performance liquid chromatography and liquid-chromatography-mass spectrometry were performed to identify N-acyl homoserine lactone (AHL) molecules secreted by monocultures and dual cultures grown in crab broth. Field emission scanning electron microscopy was performed to observe attachment and biofilm formation. P. aeruginosa and P. fluorescens inhibited biofilm formation by A. hydrophila on the crab surface, without affecting their own biofilm-forming abilities. Dual biofilms of S. Typhimurium, L. monocytogenes, or P. carotovorum did not affect A. hydrophila biofilm formation. Exoprotease, AHL, and AI-2 levels were significantly reduced in dual cultures of P. aeruginosa and P. fluorescens with A. hydrophila, supporting the relationship between quorum sensing and biofilm formation. Dual-species biofilms were studied in their natural environment and in the laboratory.

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) (ß-propeller domain); and residues Ala(496)-Phe(736) (α/ß-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.

Antimicrobial peptides are degraded by the cytosolic proteases of human erythrocytes.

Well-studied and promising antimicrobial peptides (AMPs), with potent bactericidal activity, in vitro, have yet to have a significant impact in human medicine beyond topical applications. We previously showed that interactions of AMPs with concentrated human erythrocytes inhibit many of them, and suggested that screens and assays should be done in their presence to mimic host cell inhibition. Here, we use AMPs to characterize the activity of proteases that are associated with human erythrocytes. The representative AMPs, ARVA and indolicidin, are degraded significantly during incubation with dilute, washed erythrocytes and yield a variety of degradation products, suggesting significant exopeptidase activity. Comparison of these fragments with those obtained from incubation with serum shows that the proteolytic activity associated with cells yields unique products that are not explained by residual serum proteases. By separately testing the membrane and cytosolic fractions, we show that erythrocyte proteolytic activity is found only in the cytosol. Finally, we incubated a diverse cross-section of natural and synthetic linear AMPs with human erythrocyte cytosolic extracts and observed degradation of all of them. These results show that, in addition to cell binding, proteolysis can also contribute significantly to host cell inhibition of AMPs in vitro and possibly also in vivo.

Stationary-phase induction of vvpS expression by three transcription factors: repression by LeuO and activation by SmcR and CRP.

An exoprotease of Vibrio vulnificus, VvpS, exhibits an autolytic function during the stationary phase. To understand how vvpS expression is controlled, the regulators involved in vvpS transcription and their regulatory mechanisms were investigated. LeuO was isolated in a ligand-fishing experiment, and experiments using a leuO-deletion mutant revealed that LeuO represses vvpS transcription. LeuO bound the extended region including LeuO-binding site (LBS)-I and LBS-II. Further screening of additional regulators revealed that SmcR and cyclic adenosine monophosphate-receptor protein (CRP) play activating roles in vvpS transcription. SmcR and CRP bound the regions overlapping LBS-I and -II, respectively. In addition, the LeuO occupancy of LBS-I and LBS-II was competitively exchanged by SmcR and CRP, respectively. To examine the mechanism of stationary-phase induction of vvpS expression, in vivo levels of three transcription factors were monitored. Cellular level of LeuO was maximal at exponential phase, while those of SmcR and CRP were maximal at stationary phase and relatively constant after the early-exponential phase, respectively. Thus, vvpS transcription was not induced during the exponential phase by high cellular content of LeuO. When entering the stationary phase, however, LeuO content was significantly reduced and repression by LeuO was relieved through simultaneous binding of SmcR and CRP to LBS-I and -II, respectively.

Bacillus subtilis SalA is a phosphorylation-dependent transcription regulator that represses scoC and activates the production of the exoprotease AprE.

Bacillus subtilis Mrp family protein SalA has been shown to indirectly promote the production of the exoprotease AprE by inhibiting the expression of scoC, which codes for a repressor of aprE. The exact mechanism by which SalA influences scoC expression has not been clarified previously. We demonstrate that SalA possesses a DNA-binding domain (residues 1-60), which binds to the promoter region of scoC. The binding of SalA to its target DNA depends on the presence of ATP and is stimulated by phosphorylation of SalA at tyrosine 327. The B. subtilis protein-tyrosine kinase PtkA interacts specifically with the C-terminal domain of SalA in vivo and in vitro and is responsible for activating its DNA binding via phosphorylation of tyrosine 327. In vivo, a mutant mimicking phosphorylation of SalA (SalA Y327E) exhibited a strong repression of scoC and consequently overproduction of AprE. By contrast, the non-phosphorylatable SalA Y327F and the ΔptkA exhibited the opposite effect, stronger expression of scoC and lower production of the exoprotease. Interestingly, both SalA and PtkA contain the same ATP-binding Walker domain and have thus presumably arisen from the common ancestral protein. Their regulatory interplay seems to be conserved in other bacteria.

A highly sensitive and simply operated protease sensor toward point-of-care testing.

Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.

Global identification of peptidase specificity by multiplex substrate profiling.

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases.

VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling iron-homeostasis. Fur repressed the transcription of vvhBA operon via binding to the promoter region. However, haemolysin content and haemolytic activity were lowered in cell-free supernatant of fur mutant. This discrepancy between the levels of vvhA transcript and VvhA protein in fur mutant was caused by exoproteolytic activities of the elastase VvpE and another metalloprotease VvpM, which were also regulated by Fur. vvpE gene expression was repressed by Fur via binding to the Fur-box homologous region. Regulation of VvpM expression by Fur did not occur at the level of vvpM transcription. In vitro proteolysis assays showed that both proteases efficiently degraded VvhA. In addition, the extracellular levels of VvhA were higher in culture supernatants of vvpE or vvpM mutants than in the wild type. Thus this study demonstrates that Fur regulates haemolysin production at the transcription level of the vvhBA operon and at the post-translation level by regulating the expressions of two VvhA-degrading exoproteases, VvpE and VvpM.

The prevalence and origin of exoprotease-producing cells in the Bacillus subtilis biofilm.

Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialized cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease-producing cells increases as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in exoprotease activity over time. We go on to demonstrate that exoprotease-producing cells arise from more than one cell type, namely matrix-producing and non-matrix-producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialized cell types that are derived during B. subtilis biofilm formation and furthermore the data highlight the plasticity in the origin of differentiated cells.

The exo-proteome and exo-metabolome of Nostoc punctiforme (Cyanobacteria) in the presence and absence of nitrate.

The ability of nitrogen-fixing filamentous Cyanobacteria to adapt to multiple environments comes in part from assessing and responding to external stimuli, an event that is initiated in the extracellular milieu. While it is known that these organisms produce numerous extracellular substances, little work has been done to characterize both the metabolites and proteins present under standard laboratory growth conditions. We have assessed the extracellular milieu of Nostoc punctiforme when grown in liquid culture in the presence and absence of a nitrogen source (nitrate). The extracellular proteins identified were enriched in integrin ß-propellor domains and calcium-binding sites with sequences unique to N. punctiforme, supporting a role for extracellular proteins in modulating species-specific recognition and behavior processes. Extracellular proteases are present and active under both conditions, with the cells grown with nitrate having a higher activity when normalized to chlorophyll levels. The released metabolites are enriched in peptidoglycan-derived tetrasaccharides, with higher levels in nitrate-free media.

Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways.

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.

Identification, immunolocalization, and characterization analyses of an exopeptidase of papain superfamily, (cathepsin C) from Clonorchis sinensis.

Cathepsin C is an important exopeptidase of papain superfamily and plays a number of great important roles during the parasitic life cycle. The amino acid sequence of cathepsin C from Clonorchis sinensis (C. sinensis) showed 54, 53, and 49% identities to that of Schistosoma japonicum, Schistosoma mansoni, and Homo sapiens, respectively. Phylogenetic analysis utilizing the sequences of papain superfamily of C. sinensis demonstrated that cathepsin C and cathepsin Bs came from a common ancestry. Cathepsin C of C. sinensis (Cscathepsin C) was identified as an excretory/secretory product by Western blot analysis. The results of transcriptional level and translational level of Cscathepsin C at metacercaria stage were higher than that at adult worms. Immunolocalization analysis indicated that Cscathepsin C was specifically distributed in the suckers (oral sucker and ventral sucker), eggs, vitellarium, intestines, and testis of adult worms. In the metacercaria, it was mainly detected on the cyst wall and excretory bladder. Combining with the results mentioned above, it implies that Cscathepsin C may be an essential proteolytic enzyme for proteins digestion of hosts, nutrition assimilation, and immune invasion of C. sinensis. Furthermore, it may be a potential diagnostic antigen and drug target against C. sinensis infection.

Potential elevation of exopeptidase activity of Glu-specific endopeptidase I/GluV8 mediated by hydrophobic P1'-position amino acid residue.

We recently reported that the activities of dipeptidyl-peptidase (DPP)7 and DPP11, S46-family exopeptidases were significantly elevated by the presence of prime-side amino acid residues of substrates caused by an increase in kcat [Ohara-Nemoto Y. et al., J Biol Chem 298(3):101585. doi: 10.1016/j.jbc.2022]. In the present study, the effects of prime-side residues on Glu-specific endopeptidase I/GluV8 from Staphylococcus aureus were investigated using a two-step cleavage method with tetrapeptidyl-methycoumaryl-7-amide (MCA) carrying P2- to P2'-position residues coupled with DPP11 as the second enzyme. GluV8 showed maximal activity toward benzyloxycarbonyl (Z)-LLE-MCA, while the effects of hydrolysis of substrates one residue shorter, such as acetyl (Ac)-Val-Glu- and Leu-Glu-MCA, were negligible. Nevertheless, activity towards Ac-VE-|-ID-MCA, a substrate carrying P1' and P2' residues, emerged and reached a level 44 % of that for Z-LLE-MCA. Among 11 Ac-HAXD-MCA (X is a varied amino acid), the highest level of activity enhancement was achieved with P1'-Leu and Ile, followed by Phe, Val, Ser, Tyr, and Ala, while Gly and Lys showed scant effects. This activation order was in parallel with the hydrophobicity indexes of these amino acids. The prime-side residues increased kcat/KM primarily through a maximum 500-fold elevation of kcat as well as S46-family exopeptidases. The MEROPS substrate database also indicates a close relationship between activity and hydrophobicity of the P1' residues in 93 N-terminal-truncated substrates, though no correlation was observed among all 4328 GluV8 entities examined. Taken together, these results are the first to demonstrate N-terminal exopeptidase activity of GluV8, considered to be prompted by hydrophobic P1' amino acid residues.

Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics-assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28-proline-specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino- and carboxyl-terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.