Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. DeltaN54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191) Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.     

Equine peptidases: Correspondence with human peptidases and polymorphism for erythrocyte peptidase a

Equine erythrocyte peptidases were compared to the six human erythrocyte peptidases, A, B, C, D, E, and F, regarding substrate specificity, relative activity, and electrophoretic mobility. Five equine erythrocyte peptidases appeared homologous to human peptidases A, B, D, E, and F. In contrast to human, equine peptidase C was absent in red cells, although it was weakly active in white cells. On the other hand, an equine peptidase, probably homologous to human peptidase S, was weakly active in red cells as well as present in white cells. Polymorphism for equine erythrocyte peptidase A is reported.     

The 1.8 A crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species

Proteins from organisms living in extreme conditions are of particular interest because of their potential for being templates for redesign of enzymes both in biotechnological and other industries. The crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species has been solved to 1.8 A. The structure has been compared with the structures of proteinase K from Tritirachium album Limber and Vibrio sp. PA44 in order to reveal structural explanations for differences in biophysical properties. The Serratia peptidase shares around 40 and 64% identity with the Tritirachium and Vibrio peptidases, respectively. The fold of the three enzymes is essentially identical, with minor exceptions in surface loops. One calcium binding site is found in the Serratia peptidase, in contrast to the Tritirachium and Vibrio peptidases which have two and three, respectively. A disulfide bridge close to the S2 site in the Serratia and Vibrio peptidases, an extensive hydrogen bond network in a tight loop close to the substrate binding site in the Serratia peptidase and different amino acid sequences in the S4 sites are expected to cause different substrate specificity in the three enzymes. The more negative surface potential of the Serratia peptidase, along with a disulfide bridge close to the S2 binding site of a substrate, is also expected to contribute to the overall lower binding affinity observed for the Serratia peptidase. Clear electron density for a tripeptide, probably a proteolysis product, was found in the S' sites of the substrate binding cleft.     

Activation of the 20S Proteasome of Xenopus Oocytes by SDS: Evidence for the Substrate-Induced Conformational Change Characteristic of Trypsin-Like Peptidase

The 20S proteasome of eukaryotic cells has at least three distinct peptidase activities (trypsin-like, chymotrypsin-like and peptidylglutamylpeptide (PGP) hydrolase activities). These peptidases are latent and require appropriate activators. SDS has been widely used as an activator of these peptidases, but the mechanism of its activation remains unresolved. In this study, we investigated the kinetics of the SDS-activated hydrolysis of the above three types of peptidase of the 20S proteasome purified from Xenopus oocytes. When the reaction was started by simultaneous adding both SDS and substrate, maximal rates of hydrolysis were reached after appreciable lag phases with the trypsin-type substrate [t-butyloxycarbonylLeu-Arg-Arg-4-methylcoumaryl-7-amide (Boc-LRR-MCA)], but no such lag phases were observed with the chymotrypsin-type and PGP hydrolase-type substrates [succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (Suc-LLVY-MCA), and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Cbz-LLE-2NA), respectively]. Similarly, changes in the hydrolysis rate to a reduced level upon dilution of SDS occurred after an appreciable lag phase again in the trypsin-like peptidase, but not in the other types. The lag phase characteristic of the trypsin-like peptidase was dependent on the substrate concentration. Thus, the lag phase was less discernible at very low concentrations of the substrate (e.g. at concentrations in the order of 1/100 of the Km value), but became more conspicuous with the increases in the substrate concentration. This lag phase also vanished upon preincubation of the activator (SDS) for a short period of 5 sec. These results suggest that the formation of the enzyme-substrate complex in the trypsin-like reaction induces a conformational change in the enzyme which makes the SDS activator site(s) in an occluded form, reducing the rates of SDS binding and dissociation.     

Factors governing nonoverlapping substrate specificity by mitochondrial inner membrane peptidase

At least three peptidases are involved in cleaving presequences from imported mitochondrial proteins. One of the peptidase, the inner membrane peptidase, has two catalytic subunits, Imp1p and Imp2p, which are structurally related but functionally distinct in the yeast Saccharomyces cerevisiae. Whereas both subunits are members of the type I signal peptidase family, they exhibit nonoverlapping substrate specificities. A clue to the substrate specificity mechanism has come from our discovery of the importance not only of the -1 and -3 residues in the signal peptides cleaved by Imp1p and Imp2p but also the +1 cargo residues attached to the signal peptides. We specifically find that Imp1p prefers substrates having a negatively charged residue (Asp or Glu) at the +1 position, whereas Imp2p prefers substrates having the Met residue at the +1 position. We further suggest that the conformation of the cargo is important for substrate recognition by Imp2p. A role for the cargo in presequence recognition distinguishes Imp1p and Imp2p from other type I signal peptidases.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.     

'Species' of peptidases

A good system for the naming and classification of peptidases can contribute much to the study of these enzymes. Having already described the building of families and clans in the MEROPS system, we here focus on the lowest level in the hierarchy, in which the huge number of individual peptidase proteins are assigned to a lesser number of what we term 'species' of peptidases. Just over 2000 peptidase species are recognised today, but we estimate that 25 000 will one day be known. Each species is built around a peptidase protein that has been adequately characterised. The cluster of peptidase proteins that represent the single species is then assembled primarily by analysis of a sequence 'tree' for the family. Each peptidase species is given a systematic identifier and a summary page of data regarding it is assembled. Because the characterisation of new peptidases lags far behind the sequencing, the majority of peptidase proteins are so far known only as amino acid sequences and cannot yet be assigned to species. We suggest that new forms of analysis of the sequences of the unassigned peptidases may give early indications of how they will cluster into the new species of the future.     

Multiple intracellular peptidases in Neurospora crassa.

Neurospora crassa possesses multiple intracellular peptidases which display overlapping substrate specificities. They were readily detected by an in situ staining procedure for peptidases separated in polyacrylamide gels, within which the auxilliary enzyme, l-amino acid oxidase, was immobilized. Eleven different intracellular peptidases were identified by electrophoretic separation and verified by their individual patterns of substrate specificities. Most peptide substrates tested were hydrolyzed by several different peptidases. The multiple intracellular peptidases may play overlapping roles in several basic cell processes which involve peptidase activity. The amount of peptidase activity for leucylglycine present in crude extracts of cells grown under widely different conditions was relatively constant, suggesting that this enzyme may be constitutive, although alterations in the amounts of individual peptidase isozymes may occur. A single enzyme, designated peptidase II, was partially purified and obtained free from the other peptidase species. Peptidase II was found to be an aminopeptidase with activity toward many peptides of varied composition and size. It was more active with tripeptides than homologous dipeptides and showed strong activity toward methionine-containing peptides. This enzyme, with a molecular weight of about 37,000, was thermolabile at 65 degrees C and was strongly inhibited by p-hydroxymercuribenzoate, Zn(2+), Co(2+), and Mn(2+), but was insensitive to the serine protease inhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses an essential sulfhydryl group and may be a metalloenzyme.     

Type I signal peptidases of Gram-positive bacteria

Proteins that are exported from the cytoplasm to the periplasm and outer membrane of Gram-negative bacteria, or the cell wall and growth medium of Gram-positive bacteria, are generally synthesized as precursors with a cleavable signal peptide. During or shortly after pre-protein translocation across the cytoplasmic membrane, the signal peptide is removed by signal peptidases. Importantly, pre-protein processing by signal peptidases is essential for bacterial growth and viability. This review is focused on the signal peptidases of Gram-positive bacteria, Bacillus and Streptomyces species in particular. Evolutionary concepts, current knowledge of the catalytic mechanism, substrate specificity requirements and structural aspects are addressed. As major insights in signal peptidase function and structure have been obtained from studies on the signal peptidase LepB of Escherichia coli, similarities and differences between this enzyme and known Gram-positive signal peptidases are highlighted. Notably, while the incentive for previous research on Gram-positive signal peptidases was largely based on their role in the biotechnologically important process of protein secretion, present-day interest in these essential enzymes is primarily derived from the idea that they may serve as targets for novel anti-microbials.     

The chemistry and enzymology of the type I signal peptidases

The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membrane-bound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes. These peptidases are termed type I signal (or leader) peptidases. They share the unusual feature of being resistant to the general inhibitors of the four well-characterized peptidase classes. The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family. This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases.     

How to use the MEROPS database and website to help understand peptidase specificity

The MEROPS website ( https://www.ebi.ac.uk/merops ) and database was established in 1996 to present the classification and nomenclature of proteolytic enzymes. This was expanded to include a classification of protein inhibitors of proteolytic enzymes in 2004. Each peptidase or inhibitor is assigned to a distinct identifier, based on its biochemical and biological properties, and homologous sequences are assembled into a family. Families in which the proteins share similar tertiary structures are assembled into a clan. The MEROPS classification is thus a hierarchy with at least three levels (protein‐species, family, and clan) showing the evolutionary relationship. Several other data collections have been assembled, which are accessed from all levels in the hierarchy. These include, sequence homologs, selective bibliographies, substrate cleavage sites, peptidase–inhibitor interactions, alignments, and phylogenetic trees. The substrate cleavage collection has been assembled from the literature and includes physiological, pathological, and nonphysiological cleavages in proteins, peptides, and synthetic substrates. In this article, we make recommendations about how best to analyze these data and show analyses to indicate peptidase binding site preferences and exclusions. We also identify peptidases where co‐operative binding occurs between adjacent binding sites.     

The identification of residues that control signal peptidase cleavage fidelity and substrate specificity

Signal peptidase, which removes signal peptides from preproteins, has a substrate specificity for small uncharged residues at -1 (P1) and small or larger aliphatic residues at the -3 (P3) position. Structures of the catalytic domain with a 5S-penem inhibitor and a lipopeptide inhibitor reveal candidate residues that make up the S1 and S3 pockets that bind the P1 and P3 specificity residues of the preprotein substrate. We have used site-directed mutagenesis, mass spectrometric analysis, and in vivo and in vitro activity assays as well as molecular modeling to examine the importance of the substrate pocket residues. Generally, we find that the S1 and S3 binding sites can tolerate changes that are expected to increase or decrease the size of the pocket without large effects on activity. One residue that contributes to the high fidelity of cleavage of signal peptidase is the Ile-144 residue. Changes of the Ile-144 residue to cysteine result in cleavage at multiple sites, as determined by mass spectrometry and Edman sequencing analysis. In addition, we find that signal peptidase is able to cleave after phenylalanine at the -1 residue in a double mutant in which both Ile-86 and Ile-144 were changed to an alanine. Also, alteration of the Ile-144 and Ile-86 residues to the corresponding residues found in the homologous Imp1 protease changes the specificity to promote cleavage following a -1 Asn residue. This work shows that Ile-144 and Ile-86 contribute to the signal peptidase substrate specificity and that Ile-144 is important for the accuracy of the cleavage reaction.     

Crystal structure of scytalidoglutamic peptidase with its first potent inhibitor provides insights into substrate specificity and catalysis

Scytalidoglutamic peptidase (SGP) from Scytalidium lignicolum is the founding member of the newly discovered\ family of peptidases, G1, so far found exclusively in fungi. The crystal structure of SGP revealed a previously undescribed fold for peptidases and a unique catalytic dyad of residues Gln53 and Glu136. Surprisingly, the beta-sandwich structure of SGP is strikingly similar to members of the carbohydrate-binding concanavalin A-like lectins/glucanases superfamily. By analogy with the active sites of aspartic peptidases, a mechanism employing nucleophillic attack by a water molecule activated by the general base functionality of Glu136 has been proposed. Here, we report the first crystal structures of SGP in complex with two transition state peptide analogs designed to mimic the tetrahedral intermediate of the proteolytic reaction. Of these two analogs, the one containing a central S-hydroxyl group is a potent sub-nanomolar inhibitor of SGP. The inhibitor binds non-covalently to the concave surface of the upper beta-sheet and enables delineation of the S4 to S3' substrate specificity pockets of the enzyme. Structural differences in these pockets account for the unique substrate preferences of SGP among peptidases having an acidic pH optimum. Inhibitor binding is accompanied by a structuring of the region comprising residues Tyr71-Gly80 from being mostly disordered in the apoenzyme and leading to positioning of crucial active site residues for establishing enzyme-inhibitor contacts. In addition, conformational rearrangements are seen in a disulfide bridged surface loop (Cys141-Cys148), which moves inwards, partially closing the open substrate binding cleft of the native enzyme. The non-hydrolysable scissile bond analog of the inhibitor is located in the active site forming close contacts with Gln53 and Glu136. The nucleophilic water molecule is displaced and a unique mode of binding is observed with the S-OH of the inhibitor occupying the oxyanion binding site of the proposed tetrahedral intermediate. Details of the enzyme-inhibitor interactions and mechanistic interpretations are discussed.     

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.     

Open relationships in the castles of clay: high diversity and low host specificity of Termitomyces fungi associated with fungus-growing termites in Africa

In the African and Asian tropics, termites of the subfamily Macrotermitinae play a major role in the decomposition of dead plant material. Their ecological success lies in the obligate mutualism of the termites with fungi of the genus Termitomyces. Before the advent of molecular studies, the interaction with these fungi was poorly understood. Here, we combined available ITS sequence data from West, Central, and South Africa with data of 39 new samples from East Africa to achieve the most comprehensive view of the diversity and host specificity of Termitomyces symbionts across Africa to date. A high amount of sequence divergence in the ITS sequences was found; 11 different Termitomyces lineages in East Africa and >30 lineages across Africa were identified, and the expected diversity is estimated to be about 41 lineages. The fungal lineages belong to four major clades, each almost exclusively associated with one termite host genus. Analysis of molecular variance revealed that 40% of the ITS sequence variation occurred between host genera, indicating close co-evolution at this level. However, within host genera, fungal lineages and haplotypes were frequently shared among host species and sampling localities, except for fungal symbionts of Odontotermes. Horizontal transmission of fungal symbionts may facilitate the transfer of haplotypes and species among hosts. However, at present, we have little understanding of the maintenance of specificity at the genus level. Possible explanations range from substrate specificity of fungi to an active selection of fungi by termites.     

Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid

Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (beta subunits) from several vertebrates (47-52% and 50-60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa.     

Extracellular peptidases of imaginal discs of Drosophila melanogaster

The imaginal discs of Drosophila melanogaster give rise to the adult epidermis during metamorphosis. During this developmental period several peptidase genes are expressed in disc cells, but there is a paucity of biochemical information regarding substrate specificity. We have used peptides and peptidyl 7-amino-4-methylcoumarin (AMC) substrates to detect several peptidases either positioned on the surface of wing discs or secreted by the imaginal cells. Using [Leu(5)]enkephalin as a substrate, a captopril sensitive dipeptidyl carboxypeptidase (angiotensin I-converting enzyme) and an amastatin-sensitive aminopeptidase were detected as prominent activities associated with intact discs. The formation of [Leu(5)]enkephalin-derived Phe was attributed to the concerted action of the D. melanogaster angiotensin I-converting enzyme (Ance) and a dipeptidase. The disc Ance also showed endopeptidic activity towards locust tachykinin-1 (LomTK-I) by cleaving the Gly-Val peptide bond, but this enzyme was not the sole endopeptidase activity associated with discs. Complete inhibition of the endopeptidic hydrolysis of the LomTK-1 by a disc homogenate required a combination of captopril and the neprilysin inhibitor, phosphoramidon, providing biochemical evidence for a neprilysin-like peptidase, in addition to Ance, in imaginal discs of D. melanogaster. Peptidyl AMC substrates for furin, prohormone convertase and tryptase provided evidence for trypsin-like serine endopeptidases in addition to the metalloendopeptidases. We conclude that imaginal discs are endowed with a variety of peptidases from different families that together are capable of hydrolyzing a broad range of peptides and proteins. Some of these peptidases might be responsible for the metabolic activation/inactivation of signaling peptides, as well as being involved in the production of dipeptides and free amino acids required for protein synthesis and osmotic balance during adult morphogenesis.     

Type I signal peptidase: an overview

The signal hypothesis suggests that proteins contain information within their amino acid sequences for protein targeting to the membrane. These distinct targeting sequences are cleaved by specific enzymes known as signal peptidases. There are various type of signal peptidases known such as type I, type II, and type IV. Type I signal peptidases are indispensable enzymes, which catalyze the cleavage of the amino-terminal signal-peptide sequences from preproteins, which are translocated across biological membranes. These enzymes belong to a novel group of serine proteases, which generally utilize a Ser-Lys or Ser-His catalytic dyad instead of the prototypical Ser-His-Asp triad. Despite having no distinct consensus sequence other than a commonly found 'Ala-X-Ala' motif preceding the cleavage site, signal sequences are recognized by type I signal peptidase with high fidelity. Type I signal peptidases have been found in bacteria, archaea, fungi, plants, and animals. In this review, I present an overview of bacterial type I signal peptidases and describe some of their properties in detail.