Peptidase inhibitors in the MEROPS database

The MEROPS website (http://merops.sanger.ac.uk) includes information on peptidase inhibitors as well as on peptidases and their substrates. Displays have been put in place to link peptidases and inhibitors together. The classification of protein peptidase inhibitors is continually being revised, and currently inhibitors are grouped into 67 families based on comparisons of protein sequences. These families can be further grouped into 38 clans based on comparisons of tertiary structure. Small molecule inhibitors are important reagents for peptidase characterization and, with the increasing importance of peptidases as drug targets, they are also important to the pharmaceutical industry. Small molecule inhibitors are now included in MEROPS and over 160 summaries have been written.     

Hierarchical classification of glycoside hydrolases

This review deals with structural and functional features of glycoside hydrolases, a widespread group of enzymes present in almost all living organisms. Their catalytic domains are grouped into 120 amino acid sequence-based families in the international classification of the carbohydrate-active enzymes (CAZy database). At a higher hierarchical level some of these families are combined in 14 clans. Enzymes of the same clan have common evolutionary origin of their genes and share the most important functional characteristics such as composition of the active center, anomeric configuration of cleaved glycosidic bonds, and molecular mechanism of the catalyzed reaction (either inverting, or retaining). There are now extensive data in the literature concerning the relationship between glycoside hydrolase families belonging to different clans and/or included in none of them, as well as information on phylogenetic protein relationship within particular families. Summarizing these data allows us to propose a multilevel hierarchical classification of glycoside hydrolases and their homologs. It is shown that almost the whole variety of the enzyme catalytic domains can be brought into six main folds, large groups of proteins having the same three-dimensional structure and the supposed common evolutionary origin.     

'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.     

Carboxylic ester hydrolases: Classification and database derived from their primary,secondary, and tertiary structures

We classified the carboxylic ester hydrolases (CEHs) into families and clans by use of multiple sequence alignments, secondary structure analysis, and tertiary structure superpositions. Our work for the first time has fully established their systematic structural classification. Family members have similar primary, secondary, and tertiary structures, and their active sites and reaction mechanisms are conserved. Families may be gathered into clans by their having similar secondary and tertiary structures, even though primary structures of members of different families are not similar. CEHs were gathered from public databases by use of Basic Local Alignment Search Tool (BLAST) and divided into 91 families, with 36 families being grouped into five clans. Members of one clan have standard α/β‐hydrolase folds, while those of other two clans have similar folds but with different sequences of their β‐strands. The other two clans have members with six‐bladed β‐propeller and three‐α‐helix bundle tertiary structures. Those families not in clans have a large variety of structures or have no members with known structures. At the time of writing, the 91 families contained 321,830 primary structures and 1378 tertiary structures. From these data, we constructed an accessible database: CASTLE (CArboxylic eSTer hydroLasEs, http://www.castle.cbe.iastate.edu ).     

The structure and mechanism of action of cellulolytic enzymes

The modern structural classification of polysaccharases comprising cellulase–hemicellulase enzyme systems is dis cussed. Their catalytic domains are currently grouped into 15 of more than 80 known glycosyl hydrolase families, whereas substrate binding domains fall into 13 families. The structures of catalytic and substrate binding domains, as well as linker sequences, are briefly considered. A hypothetical mechanism of concerted action of catalytic and substrate binding domains of cellobiohydrolases on the surface of highly ordered cellulose is suggested.     

Thioesterases: A new perspective based on their primary and tertiary structures

Thioesterases (TEs) are classified into EC 3.1.2.1 through EC 3.1.2.27 based on their activities on different substrates, with many remaining unclassified (EC 3.1.2.–). Analysis of primary and tertiary structures of known TEs casts a new light on this enzyme group. We used strong primary sequence conservation based on experimentally proved proteins as the main criterion, followed by verification with tertiary structure superpositions, mechanisms, and catalytic residue positions, to accurately define TE families. At present, TEs fall into 23 families almost completely unrelated to each other by primary structure. It is assumed that all members of the same family have essentially the same tertiary structure; however, TEs in different families can have markedly different folds and mechanisms. Conversely, the latter sometimes have very similar tertiary structures and catalytic mechanisms despite being only slightly or not at all related by primary structure, indicating that they have common distant ancestors and can be grouped into clans. At present, four clans encompass 12 TE families. The new constantly updated ThYme (Thioester‐active enzYmes) database contains TE primary and tertiary structures, classified into families and clans that are different from those currently found in the literature or in other databases. We review all types of TEs, including those cleaving CoA, ACP, glutathione, and other protein molecules, and we discuss their structures, functions, and mechanisms.     

Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families.

Several types of domain occur in beta-1, 4-glycanases. The best characterized of these are the catalytic domains and the cellulose-binding domains. The domains may be joined by linker sequences rich in proline or hydroxyamino acids or both. Some of the enzymes contain repeated sequences up to 150 amino acids in length. The enzymes can be grouped into families on the basis of sequence similarities between the catalytic domains. There are sequence similarities between the cellulose-binding domains, of which two types have been identified, and also between some domains of unknown function. The beta-1, 4-glycanases appear to have arisen by the shuffling of a relatively small number of progenitor sequences.     

The MEROPS batch BLAST: a tool to detect peptidases and their non-peptidase homologues in a genome

Many of the 181 families of peptidases contain homologues that are known to have functions other than peptide bond hydrolysis. Distinguishing an active peptidase from a homologue that is not a peptidase requires specialist knowledge of the important active site residues, because replacement or lack of one of these catalytic residues is an important clue that the homologue in question is unlikely to hydrolyse peptide bonds. Now that the rate at which proteins are characterized is outstripped by the rate that genome sequences are determined, many genes are being incorrectly annotated because only sequence similarity is taken into consideration. We present a tool called the MEROPS batch BLAST which not only performs a comparison against the MEROPS sequence collection, but also does a pair-wise alignment with the closest homologue detected and calculates the position of the active site residues. A non-peptidase homologue can be distinguished by the absence or unacceptable replacement of any of these residues. An analysis of peptidase homologues in the genome of the bacterium Erythrobacter litoralis is presented as an example.     

The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.     

Architecture and function of metallopeptidase catalytic domains

The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single‐step reaction involving a solvent molecule, a general base/acid, and a mono‐ or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal‐binding motif (HEXXH), which includes two metal‐binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ～130–270‐residue catalytic domains, which are usually preceded by N‐terminal pro‐segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C‐terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N‐terminal subdomain spanning a five‐stranded β‐sheet, a backing helix, and an active‐site helix. The latter contains most of the metal‐binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C‐terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met‐turn—and a C‐terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs.     

Cytochrome P450 diversity in the tree of life

Sequencing in all areas of the tree of life has produced > 300,000 cytochrome P450 (CYP) sequences that have been mined and collected. Nomenclature has been assigned to > 41,000 CYP sequences and the majority of the remainder has been sorted by BLAST searches into clans, families and subfamilies in preparation for naming. The P450 sequence space is being systematically explored and filled in. Well-studied groups like vertebrates are covered in greater depth while new insights are being added into uncharted territories like horseshoe crab (Limulus polyphemus), tardigrades (Hypsibius dujardini), velvet worm (Euperipatoides_rowelli), and basal land plants like hornworts, liverworts and mosses. CYPs from the fungi, one of the most diverse groups, are being explored and organized as nearly 800 fungal species are now sequenced. The CYP clan structure in fungi is emerging with 805 CYP families sorting into 32 CYP clans. > 3000 bacterial sequences are named, mostly from terrestrial or freshwater sources. Of 18,379 bacterial sequences downloaded from the CYPED database, all are > 43% identical to named CYPs. Therefore, they fit in the 602 named P450 prokaryotic families. Diversity in this group is becoming saturated, however 25% of 3305 seawater bacterial P450s did not match known P450 families, indicating marine bacterial CYPs are not as well sampled as land/freshwater based bacterial CYPs. Future sequencing plans of the Genome 10 K project, i5k and GIGA (Global Invertebrate Genomics Alliance) are expected to produce more than one million cytochrome P450 sequences by 2020. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

The MEROPS database as a protease information system

Peptidases (often termed proteases) are of great relevance to biology, medicine, and biotechnology. This practical importance creates a need for an integrated source of information about peptidases. In the MEROPS database (www.merops.ac.uk), peptidases are classified by structural similarities in the parts of the molecules responsible for their enzymatic activity. They are grouped into families on the basis of amino acid sequence homology, and the families are assembled into clans in light of evidence that they share common ancestry. The evidence for clan-level relationships usually comes from similarities in tertiary structure, but we suggest that secondary structure profiles may also be useful in the future. The classification forms a framework around which a wealth of supplementary information about the peptidases is organized. This includes images of three-dimensional structures, alignments of matching human and mouse ESTs, comments on biomedical relevance, human and other gene symbols, and literature references linked to PubMed. For each family, there is an amino acid sequence alignment and a dendrogram. There is a list of all peptidases known from each of over 1000 species, together with summary data for the distributions of the families and clans throughout the major groups of organisms. A set of online searches provides access to information about the location of peptidases on human chromosomes and peptidase substrate specificity.     

Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides.

Endoglucanase B (CenB) from the bacterium Cellulomonas fimi is divided into five discrete domains by linker sequences rich in proline and hydroxyamino acids (A. Meinke, C. Braun, N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, J. Bacteriol. 173:308-314, 1991). The catalytic domain of 608 amino acids is at the N terminus. The sequence of the first 477 amino acids in the catalytic domain is related to the sequences of cellulases in family E, which includes procaryotic and eucaryotic enzymes. The sequence of the last 131 amino acids of the catalytic domain is related to sequences present in a number of cellulases from different families. The catalytic domain alone can bind to cellulose, and this binding is mediated at least in part by the C-terminal 131 amino acids. Deletion of these 131 amino acids reduces but does not eliminate activity. The catalytic domain is followed by three domains which are repeats of a 98-amino-acid sequence. The repeats are approximately 50% identical to two repeats of 95 amino acids in a chitinase from Bacillus circulans which are related to fibronectin type III repeats (T. Watanabe, K. Suzuki, K. Oyanagi, K. Ohnishi, and H. Tanaka, J. Biol. Chem. 265:15659-15665, 1990). The C-terminal domain of 101 amino acids is related to sequences, present in a number of bacterial cellulases and xylanases from different families, which form cellulose-binding domains (CBDs). It functions as a CBD when fused to a heterologous polypeptide. Cells of Escherichia coli expressing the wild-type cenB gene accumulate both native CenB and a stable proteolytic fragment of 41 kDa comprising the three repeats and the C-terminal CBD. The 41-kDa polypeptide binds to cellulose but lacks enzymatic activity.     

Evolutionary lines of cysteine peptidases

The proteolytic enzymes that depend upon a cysteine residue for activity have come from at least seven different evolutionary origins, each of which has produced a group of cysteine peptidases with distinctive structures and properties. We show here that the characteristic molecular topologies of the peptidases in each evolutionary line can be seen not only in their three-dimensional structures, but commonly also in the two-dimensional structures. Clan CA contains the families of papain (C1), calpain (C2), streptopain (C10) and the ubiquitin-specific peptidases (C12, C19), as well as many families of viral cysteine endopeptidases. Clan CD contains the families of clostripain (C11), gingipain R (C25), legumain (C13), caspase-1 (C14) and separin (C50). These enzymes have specificities dominated by the interactions of the S1 subsite. Clan CE contains the families of adenain (C5) from adenoviruses, the eukaryotic Ulp1 protease (C48) and the bacterial YopJ proteases (C55). Clan CF contains only pyroglutamyl peptidase I (C15). The picornains (C3) in clan PA have probably evolved from serine peptidases, which still form the majority of enzymes in the clan. The cysteine peptidase activities in clans PB and CH are autolytic only. In conclusion, we suggest that although almost all the cysteine peptidases depend for activity on catalytic dyads of cysteine and histidine, it is worth noting some important differences that they have inherited from their distant ancestral peptidases.     

Microbial starch-binding domain

Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.     

Classification of Protein Kinases on the Basis of Both Kinase and Non-Kinase Regions

Background

Protein phosphorylation is a generic way to regulate signal transduction pathways in all kingdoms of life. In many organisms, it is achieved by the large family of Ser/Thr/Tyr protein kinases which are traditionally classified into groups and subfamilies on the basis of the amino acid sequence of their catalytic domains. Many protein kinases are multi-domain in nature but the diversity of the accessory domains and their organization are usually not taken into account while classifying kinases into groups or subfamilies.

Methodology

Here, we present an approach which considers amino acid sequences of complete gene products, in order to suggest refinements in sets of pre-classified sequences. The strategy is based on alignment-free similarity scores and iterative Area Under the Curve (AUC) computation. Similarity scores are computed by detecting common patterns between two sequences and scoring them using a substitution matrix, with a consistent normalization scheme. This allows us to handle full-length sequences, and implicitly takes into account domain diversity and domain shuffling. We quantitatively validate our approach on a subset of 212 human protein kinases. We then employ it on the complete repertoire of human protein kinases and suggest few qualitative refinements in the subfamily assignment stored in the KinG database, which is based on catalytic domains only. Based on our new measure, we delineate 37 cases of potential hybrid kinases: sequences for which classical classification based entirely on catalytic domains is inconsistent with the full-length similarity scores computed here, which implicitly consider multi-domain nature and regions outside the catalytic kinase domain. We also provide some examples of hybrid kinases of the protozoan parasite Entamoeba histolytica.

Conclusions

The implicit consideration of multi-domain architectures is a valuable inclusion to complement other classification schemes. The proposed algorithm may also be employed to classify other families of enzymes with multi-domain architecture.     

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.     

Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein

Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase," different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.     

Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog

Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 A structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.     

Identification and analysis of catalytic TIM barrel domains in seven further glycoside hydrolase families

Fold recognition results allocate catalytic triose phosphate isomerase (TIM) barrels to seven previously unassigned glycoside hydrolase (GH) families, numbers 29, 44, 50, 71, 84, 85 and 89, enabling prediction of catalytic residues. Modelling of GH family 50 suggests that it may be the common evolutionary ancestor of families 42 and 14. TIM barrels now comprise the catalytic domains of more than half of the assigned GH families, and catalyse a much larger variety of GH reactions than any other catalytic domain architecture. Only 327 GH sequences still have no structurally identified catalytic domain.