Eqolisins are rare acid proteases found in archaea, bacteria and fungi Certain fungi secrete acids as part of their lifestyle and interestingly these also have many eqolisin paralogs, up to nine paralogs have been recorded This suggests a process of functional redundancy and diversification has occurred, which was the subject of the research we performed and describe here We identified eqolisin homologs by means of iterative HMMER analysis of the NR database The identified sequences were scrutinized for which new hallmarks were identified by molecular dynamics simulations of mutants in highly conserved positions, using the structure of an eqolisin that was crystallized in the presence of a transition state inhibitor Four conserved glycines were shown to be important for functionality A substitution of W67F is shown to be accompanied by the L105W substitution Molecular dynamics shows that the W67 binds to the substrate via a π-π stacking and a salt bridge, the latter being stronger in a virtual W67F/L105W double mutant of the resolved structure of Scytalido-carboxyl peptidase-B (PDB ID: 2IFW) Additional problematic mutations are discussed Upon sequence scrutiny we obtained a set of 233 sequences that was used to reconstruct a Bayesian phylogenetic tree We identified 14 putative specificity determining positions (SDPs) of which four are explained by mere structural explanations and nine seem to correspond to functional diversification related with substrate binding and specificity A first sub-network of SDPs is related to substrate specificity whereas the second sub-network seems to affect the dynamics of three loops that are involved in substrate binding The eqolisins form a small superfamily of acid proteases with nevertheless many paralogs in acidic fungi Functional redundancy has resulted in diversification related to substrate specificity and substrate binding

/pdf/molecular-dynamics-and-structure-function-analysis-show-that-1004d5vxug.pdf

Molecular dynamics and structure function analysis show that substrate binding and specificity are major forces in the functional diversification of Eqolisins.

Sedolisin is a proteolytic enzyme, listed in the peptidase database MEROPS as a founding member of clan SB, family S53. This enzyme, although active at low pH, was originally shown not to be inhibited by an aspartic peptidase specific inhibitor, S-PI (pepstatin Ac). In this Perspective, the S53 family is described from the moment of original identification to evolution. The representative enzymes of the family are sedolisin, kumamolisin, and TPP-1. They exhibit the following unique features. (1) The fold of the molecule is similar to that of subtilisin, but the catalytic residues consist of a triad, Ser/Glu/Asp, that is unlike the Ser/His/Asp triad of subtilisin. (2) The molecule is expressed as a pro-form composed of the amino-terminal prosegment and the active domain. Additionally, some members of this family have an additional, carboxy-terminal prosegment. (3) Their optimum pH for activity is in the acidic region, not in the neutral to alkaline region where subtilisin is active. (4) Their distribution in nature is very broad across the three kingdoms of life. (5) Some of these enzymes from fungi and bacteria are pathogens to plants. (6) Some of them have significant potential applications for industry. (7) The lack of a TPP-1 gene in human brain is the cause of incurable juvenile neuronal ceroid lipofuscinosis (Batten's disease).

Serine-Carboxyl Peptidases, Sedolisins: From Discovery to Evolution.

Identification of novel effectors with conserved features has always remained a challenge in plant-pathogen interaction studies. The introduction of the genomics era in plant-pathogen studies has led to the identification of significant candidate effectors with novel motifs such as RxLR and dEER motifs. However, in the case of fungal pathogens, limited conserved motifs associated with effectors have been discovered yet. In the present study, we have performed comparative secretome analysis for major plant pathogens of diverse nutrition mechanisms with the aim of dissecting the features underlying their corresponding secretome and conserved motifs. We showed that rust fungi possess the lowest Cell wall degrading enzymes (CWDEs) consortium lower than other biotrophic pathogens. We also showed rust fungi possess the highest secretory superoxide dismutase (SOD) than other studied plant pathogens. Further, we prioritized the candidate secretory effectors proteins (CSEPs) of all the studied pathogens by combining various effector mining parameters to highlight the candidates with potential effector features. A novel WAxR motif in conjugation with the Y/F/WxC (FGC) motif was identified in the effectors of various P. striiformis races present globally. The WAxR/WAxR like motifs ( WxxR, WAxx, xAxR) containing effectors were also found in the secretome of other rust fungi. Further, the functional validation of two candidate effectors with WAxR motif from P. striiformis Yr9 showed that these effectors localize to the nucleus as well as cytoplasm, and are able to suppress BAX induced cell death in Nicotiana benthamiana. The mutation analysis of individual residues of the WAxR motif (W, A, R) however did not affect the cell death suppression nor subcellular localization of these effectors. Overall, the current study reports the presence of novel motifs in large numbers of effectors of rust fungi with cell death suppression features. HighlightsO_LISecretome analysis of various plant pathogens performed C_LIO_LIA prioritization list for candidate effectors designed C_LIO_LIA novel WAxR motif was discovered in rust fungal effector proteins C_LIO_LITwo WAxR candidate effectors were functionally validated C_LI

/pdf/comparative-secretomics-identifies-conserved-waxr-motif-aldy2apkwi.pdf

Comparative secretomics identifies conserved WAxR motif-containing effectors in rust fungi that suppress cell death in plants

Background: Eqolisins are rare acid proteases found in archaea, bacteria and fungi. Certain fungi secrete acids as part of their lifestyle and interestingly these also have many eqolisin paralogs, up to seven paralogs have been recorded. This suggest functional redundancy and diversification, which was the subject of the research we performed and describe here. Results: We identified eqolisin homologs by means of iterative HMMER analysis of the NR database. The identified sequences were scrutinized for which we defined novel hallmarks, identified by molecular dynamics simulations of mutants of highly conserved positions, using the structure of an eqolisin that was crystallized in the presence of a transition state inhibitor. Four conserved glycines were shown to be required for functionality. A mutation of W67F is shown to be accompanied by the L105W mutation. Molecular dynamics shows that the W67 binds to the substrate via a π-π stacking and a salt bridge. The latter is stronger in the W67F/L105W double mutant. Additional likely fatal mutants are discussed. Upon sequence scrutiny we obtained a set of 233 sequences that in all likelihood lacks false positives. This was used to reconstruct a Bayesian phylogenetic tree. We identified 14 specificity determining positions. These occur in two pairs that are explained by structural constraints as well as two sub-networks of real specificity determining positions. The first sub-network is related to substrate specificity whereas the second sub-network seems to affect the dynamics of three loops that are involved in substrate binding.

/pdf/substrate-binding-and-specificity-appear-as-major-forces-in-2uixemcc2p.pdf

Substrate binding and specificity appear as major forces in the functional diversification of eqolisins.

Seqrutinator is an objective, flexible pipeline that removes sequences with sequencing and/or gene model errors and sequences from pseudogenes from complex, eukaryotic protein superfamilies. Testing Seqrutinator on major superfamilies BAHD, CYP, and UGT removes only 1.94% of SwissProt entries, 14% of entries from the model plant Arabidopsis thaliana, but 80% of entries from Pinus taeda's recent complete proteome. Application of Seqrutinator on crude BAHDomes, CYPomes, and UGTomes obtained from 16 plant proteomes shows convergence of the numbers of paralogues. MSAs, phylogenies, and particularly functional clustering improve drastically upon Seqrutinator application, indicating good performance. 

pdf/seqrutinator-scrutiny-of-large-protein-superfamily-sequence-37y4t8iybaqq.pdf

Seqrutinator: scrutiny of large protein superfamily sequence datasets for the identification and elimination of non-functional homologues

Sedolisins are acid proteases that are related to the basic subtilisins. They have been identified in all three superkingdoms but are not ubiquitous, although fungi that secrete acids as part of their lifestyle can have up to six paralogs. Both TriPeptidyl Peptidase (TPP) and endopeptidase activity have been identified and it has been suggested that these correspond to separate subfamilies. We studied eukaryotic sedolisins by computational analysis. A maximum likelihood tree shows one major clade containing non-fungal sequences only and two major as well as two minor clades containing only fungal sequences. One of the major fungal clades contains all known TPPs whereas the other contains characterized endosedolisins. We identified four Cluster Specific Inserts (CSIs) in endosedolisins, of which CSIs 1, 3 and 4 appear as solvent exposed according to structure modeling. Part of CSI2 is exposed but a short stretch forms a novel and partially buried α-helix that induces a conformational change near the binding pocket. We also identified a total of 15 specificity determining positions (SDPs) of which five, identified in two independent analyses, form highly connected SDP sub-networks. Modeling of virtual mutants suggests a key role for the W307A or F307A substitution. The remaining four key SDPs physically interact at the interface of the catalytic domain and the enzyme’s prosegment. Modeling of virtual mutants suggests these SDPs are indeed required to compensate the conformational change induced by CSI2 and the A307. One of the two small fungal clades concerns a subfamily that contains 213 sequences, is mostly similar to the major TPP subfamily but differs, interestingly, in position 307, showing mostly isoleucine and threonine. Analysis confirms there are at least two sedolisin subfamilies in fungi: TPPs and endopeptidases, and suggests a third subfamily with unknown characteristics. Sequence and functional diversification was centered around buried SDP307 and resulted in a conformational change of the pocket. Mutual Information network analysis forms a useful instrument in the corroboration of predicted SDPs.

https://link.springer.com/content/pdf/10.1186/s12859-018-2404-y.pdf

Structure-function analysis of Sedolisins: evolution of tripeptidyl peptidase and endopeptidase subfamilies in fungi.

Sedolisins are acid proteases that are related to the basic subtilisins. They have been identified in all three superkingdoms but are not ubiquitous, although fungi that secrete acids as part of their lifestyle can have up to six paralogs. Both tripeptidyl peptidase (TPP) and endopeptidase activity have been identified and it has been suggested that these correspond to separate subfamilies.
	We studied eukaryotic sedolisins by computational analysis. A maximum likelihood tree shows three major clades of which two contain only fungal sequences. One fungal clade contains all known TPPs whereas the other contains the endosedolisins. We identified four cluster specific inserts (CSIs) in endosedolisins, of which CSIs 1, 3 and 4 appear as solvent exposed according to structure modeling. Part of CSI2 is exposed but a short stretch forms a novel and partially buried α-helix that induces a conformational change near the binding pocket. We also identified a total of 12 specificity determining positions (SDPs) divided over three SDP sub-networks. The major SDP network contains eight directly connected SDPs and modeling of virtual mutants suggests a key role for the W307A or F307A substitution. This substitution is accompanied by a group of four SDPs that physically interact at the interface of the catalytic domain and the enzyme9s prosegment. Modeling of virtual mutants suggests these SDPs are indeed required to compensate the conformational change induced by CSI2 and the A307. The additional major network SDPs as well as the two small SDP networks appear to be linked to this major substitution, all together explaining the hypothesized functional diversification of fungal sedolisins.

/pdf/functional-diversification-of-tripeptidyl-peptidase-and-2559tb7xqt.pdf

Functional Diversification of Tripeptidyl Peptidase and Endopeptidase Sedolisins in Fungi

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Structure-function analysis of Sedolisins: evolution of tripeptidyl peptidase and endopeptidase subfamilies in fungi.

Functional Diversification of Tripeptidyl Peptidase and Endopeptidase Sedolisins in Fungi