Construção de modelos de interação in silico e in vitro do inibidor do tipo Kunitz de Adenanthera pavonina L. para as enzimas cisteínicas e serínicas

Serines proteinases inhibitors (PIs) are widely distributed in nature and are able to inhibit both in vitro and in vivo enzymatic activites. Seed PIs in than leguminous are classified in seven families, Bowman-Birk and Kunitz type families that most studied representing an important role in the first line of defense toward insects pests. Some Kunitz type inhibitors possess activities serine and cysteine for proteinases named bifunctional inhibitor, as ApTKI the inhibitor isolate from seed of Adenanthera pavonina. The A. pavonina inhibitor presenting the uncommon property and was used for interaction studies between proteinases serine (trypsin) and cysteine (papain). In order to determinate the in vitro interaction of ApTKI against enzymes inhibitor purification was carried cut by using chromatographic techniques and inhibition assays. The 3D model of the bifunctional inhibitor ApTKI was constructed SWISS-MODEL program by homology modeling using soybean trypsin inhibitor (STI, pdb:1ba7), as template which presented 40% of identity to A. pavonina inhibitor. Model quality was evaluated by PROCHECK program. Moreover in silico analyzes of formed complex between the enzymes and ApTKI was evaluated by HEX 4.5 program. In vitro results confirmed the inhibitory assays, where the inhibitor presented the ability to simultaneously inhibit trypsin and papain. The residues encountered in the inhibitor model of folder structural three-dimensional that make contact to enzymes target coud explain the specificity pattern against serine and cysteine proteinases

Glutathione Transferases as Detoxification Agents: Structural and Functional Studies

Glutathione transferases (GSTs) are a diverse family of enzymes that catalyze the glutathione-dependent detoxification of toxic compounds. GSTs are responsible for the conjugation of the tripeptide glutathione (GSH) to a wide range of electrophilic substrates. These include industrial pollutants, drugs, genotoxic carcinogen metabolites, antibiotics, insecticides and herbicides. In light of applications in biomedicine and biotechnology as cellular detoxification agents, detailed structural and functional studies of GSTs are required. Plant tau class GSTs play crucial catalytic and non-catalytic roles in cellular xenobiotic detoxification process in agronomically important crops. The abundant existence of GSTs in Glycine max and their ability to provide resistance to abiotic and biotic stresses such as herbicide tolerance is of great interest in agriculture because they provide effective and suitable tools for selective weed control. Structural and catalytic studies on tau class GST isoenzymes from Glycine max (GmGSTU10-10, GmGSTU chimeric clone 14 (Sh14), and GmGSTU2-2) were performed. Crystal structures of GmGSTU10-10 in complex with glutathione sulfenic acid (GSOH) and Sh14 in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH) were determined by molecular replacement at 1.6 Å and 1.75 Å, respectively. Major structural variations that affect substrate recognition and catalytic mechanism were revealed in the upper part of helix H4 and helix H9 of GmGSTU10-10. Structural analysis of Sh14 showed that the Trp114Cys point mutation is responsible for the enhanced catalytic activity of the enzyme. Furthermore, two salt bridges that trigger an allosteric effect between the H-sites were identified at the dimer interface between Glu66 and Lys104. The 3D structure of GmGSTU2-2 was predicted using homology modeling. Structural and phylogenetic analysis suggested GmGSTU2-2 shares residues that are crucial for the catalytic activity of other tau class GSTs–Phe10, Trp11, Ser13, Arg20, Tyr30, Leu37, Lys40, Lys53, Ile54, Glu66 and Ser67. This indicates that the catalytic and ligand binding site in GmGSTU2-2 are well-conserved. Nevertheless, at the ligandin binding site a significant variation was observed. Tyr32 is replaced by Ser32 in GmGSTU2-2 and thismay affect the ligand recognition and binding properties of GmGSTU2-2. Moreover, docking studies revealed important amino acid residues in the hydrophobic binding site that can affect the substrate specificity of the enzyme. Phe10, Pro12, Phe15, Leu37, Phe107, Trp114, Trp163, Phe208, Ile212, and Phe216 could form the hydrophobic ligand binding site and bind fluorodifen. Additionally, side chains of Arg111 and Lys215 could stabilize the binding through hydrogen bonds with the –NO2 groups of fluorodifen. GST gene family from the pathogenic soil bacterium Agrobacterium tumefaciens C58 was characterized and eight GST-like proteins in A. tumefaciens (AtuGSTs) were identified. Phylogenetic analysis revealed that four members of AtuGSTs belong to a previously recognized bacterial beta GST class and one member to theta class. Nevertheless, three AtuGSTs do not belong to any previously known GST classes. The 3D structures of AtuGSTs were predicted using homology modeling. Comparative structural and sequence analysis of the AtuGSTs showed local sequence and structural characteristics between different GST isoenzymes and classes. Interactions at the G-site are conserved, however, significant variations were seen at the active site and the H5b helix at the C-terminal domain. H5b contributes to the formation of the hydrophobic ligand binding site and is responsible for recognition of the electrophilic moiety of the xenobiotic. It is noted that the position of H5b varies among models, thus providing different specificities. Moreover, AtuGSTs appear to form functional dimers through diverse modes. AtuGST1, AtuGST3, AtuGST4 and AtuGST8 use hydrophobic ‘lock–and–key’-like motifs whereas the dimer interface of AtuGST2, AtuGST5, AtuGST6 and AtuGST7 is dominated by polar interactions. These results suggested that AtuGSTs could be involved in a broad range of biological functions including stress tolerance and detoxification of toxic compounds.

Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase: a potential target to reduce Fusarium head blight disease.

2016

Diaryl dihydropyrazole-3-carboxamides with significant in vivo antiobesity activity related to CB1 receptor antagonism: Synthesis, biological evaluation, and molecular modeling in the homology model

A number of analogues of diaryl dihydropyrazole-3-carboxamides have been synthesized. Their activities were evaluated for appetite suppression and body weight reduction in animal models. Depending on the chemical modification of the selected dihydropyrazole scaffold, the lead compoundsthe bisulfate salt of (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 26 and the bisulfate salt of (−)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 30showed significant body weight reduction in vivo, which is attributed to their CB1 antagonistic activity and exhibited a favorable pharmacokinetic profile. The molecular modeling studies also showed interactions of two isomers of (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 9 with CB1 receptor in the homology model similar to those of N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (rimonabant) 1 and 4S-(−)-3-(4-chlorophenyl)-N-methyl-N‘-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (SLV-319) 2.

Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing tile PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive width MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

P2Y(1) antagonists: Combining receptor-based modeling and QSAR for a quantitative prediction of the biological activity based on consensus scoring

P2Y(1) is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y(1) homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.

Advances in GPCR modeling evaluated by the GPCR Dock 2013 assessment:Meeting new challenges

Despite tremendous successes of GPCR crystallography, the receptors with available structures represent only a small fraction of human GPCRs. An important role of the modeling community is to maximize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock assessment was established to stimulate and monitor the progress in molecular modeling and ligand docking for GPCRs. The four targets in the present third assessment round presented new and diverse challenges for modelers, including prediction of allosteric ligand interaction and activation states in 5-hydroxytryptamine receptors 1B and 2B, and modeling by extremely distant homology for smoothened receptor. Forty-four modeling groups participated in the assessment. State-of-the-art modeling approaches achieved close-to-experimental accuracy for small rigid orthosteric ligands and models built by close homology, and they correctly predicted protein fold for distant homology targets. Predictions of long loops and GPCR activation states remain unsolved problems.

Ab initio modeling and molecular dynamics simulation of the alpha 1b-adrenergic receptor activation.

This work describes the ab initio procedure employed to build an activation model for the alpha 1b-adrenergic receptor (alpha 1b-AR). The first version of the model was progressively modified and complicated by means of a many-step iterative procedure characterized by the employment of experimental validations of the model in each upgrading step. A combined simulated (molecular dynamics) and experimental mutagenesis approach was used to determine the structural and dynamic features characterizing the inactive and active states of alpha 1b-AR. The latest version of the model has been successfully challenged with respect to its ability to interpret and predict the functional properties of a large number of mutants. The iterative approach employed to describe alpha 1b-AR activation in terms of molecular structure and dynamics allows further complications of the model to allow prediction and interpretation of an ever-increasing number of experimental data.

Molecular modeling databases: A new way in the search of protein targets for drug development

DBMODELING is a relational database of annotated comparative protein structure models and their metabolic, pathway characterization. It is focused on enzymes identified in the genomes of Mycobacterium tuberculosis and Xylella fastidiosa. The main goal of the present database is to provide structural models to be used in docking simulations and drug design. However, since the accuracy of structural models is highly dependent on sequence identity between template and target, it is necessary to make clear to the user that only models which show high structural quality should be used in such efforts. Molecular modeling of these genomes generated a database, in which all structural models were built using alignments presenting more than 30% of sequence identity, generating models with medium and high accuracy. All models in the database are publicly accessible at http://www.biocristalografia.df.ibilce.unesp.br/tools. DBMODELING user interface provides users friendly menus, so that all information can be printed in one stop from any web browser. Furthermore, DBMODELING also provides a docking interface, which allows the user to carry out geometric docking simulation, against the molecular models available in the database. There are three other important homology model databases: MODBASE, SWISSMODEL, and GTOP. The main applications of these databases are described in the present article. © 2007 Bentham Science Publishers Ltd.

Modeling of human P450 oxidoreductase structure by in silico mutagenesis and MD simulation

P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450s and mutations in POR cause several metabolic disorders. We have modeled the structure of human P450 oxidoreductase by in silico amino acid replacements in the rat POR crystal structure. The rat POR has 94% homology with human POR and 38 amino acids were replaced to make its sequence identical to human POR. Several rounds of molecular dynamic simulations refined the model and removed structural clashes from side chain alterations of replaced amino acids. This approach has the advantage of keeping the cofactor contacts and structural features of the core enzyme intact which could not be achieved by homology based approaches. The final model from our approach was of high quality and compared well with experimentally determined structures of other PORs. This model will be used for analyzing the structural implications of mutations and polymorphisms in human POR.

Modeling Techniques for the High-Resolution Interpretation of Cryo-Electron Microscopy Reconstructions

Essential biological processes are governed by organized, dynamic interactions between multiple biomolecular systems. Complexes are thus formed to enable the biological function and get dissembled as the process is completed. Examples of such processes include the translation of the messenger RNA into protein by the ribosome, the folding of proteins by chaperonins or the entry of viruses in host cells. Understanding these fundamental processes by characterizing the molecular mechanisms that enable then, would allow the (better) design of therapies and drugs. Such molecular mechanisms may be revealed trough the structural elucidation of the biomolecular assemblies at the core of these processes. Various experimental techniques may be applied to investigate the molecular architecture of biomolecular assemblies. High-resolution techniques, such as X-ray crystallography, may solve the atomic structure of the system, but are typically constrained to biomolecules of reduced flexibility and dimensions. In particular, X-ray crystallography requires the sample to form a three dimensional (3D) crystal lattice which is technically di‑cult, if not impossible, to obtain, especially for large, dynamic systems. Often these techniques solve the structure of the different constituent components within the assembly, but encounter difficulties when investigating the entire system. On the other hand, imaging techniques, such as cryo-electron microscopy (cryo-EM), are able to depict large systems in near-native environment, without requiring the formation of crystals. The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail. In this dissertation, several modeling methods are introduced to either integrate cryo-EM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one cannot reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the different biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative. Three manuscripts are presented as part of the PhD dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The first manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions. The first manuscript, titled An assembly model for Rift Valley fever virus, was submitted for publication in the Journal of Molecular Biology. The cryo-EM structure of the Rift Valley fever virus was previously solved at 27Å-resolution by Dr. Freiberg and collaborators. Such reconstruction shows the overall shape of the virus envelope, yet the reduced level of detail prevents the direct atomic interpretation. High-resolution structures are not yet available for the entire virus nor for the two different component glycoproteins that form its envelope. However, homology models may be generated for these glycoproteins based on similar structures that are available at atomic resolutions. The manuscript presents the steps required to identify an atomic model of the entire virus envelope, based on the low-resolution cryo-EM map of the envelope and the homology models of the two glycoproteins. Starting with the results of the exhaustive search to place the two glycoproteins, the model is built iterative by running multiple multi-body refinements to hierarchically generate models for the different regions of the envelope. The generated atomic model is supported by prior knowledge regarding virus biology and contains valuable information about the molecular architecture of the system. It provides the basis for further investigations seeking to reveal different processes in which the virus is involved such as assembly or fusion. The second manuscript was recently published in the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the different components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the different components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the benefit of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å. The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the different alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data. The three manuscripts presented in this PhD dissertation cover different computation methods for the integration and interpretation of cryo-EM reconstructions. The methods were developed in the molecular modeling software Sculptor (http://sculptor.biomachina.org) and are available for the scientific community interested in the multi-resolution modeling of cryo-EM data. The work spans a wide range of resolution covering multi-body refinement and registration at low-resolution along with annotation of consistent patterns at high-resolution. Such methods are essential for the modeling of cryo-EM data, and may be applied in other fields where similar spatial problems are encountered, such as medical imaging.

Selectivity of diacylhydrazine insecticides to the predatory bug Orius laevigatus: In vivo and modeling/docking experiments

BACKGROUND: Knowledge of pesticide selectivity to natural enemies is necessary for a successful implementation of biological and chemical control methods in integrated pest management (IPM) programs. Diacylhydrazine (DAH)-based ecdysone agonists also known as molting-accelerating compounds (MACs) are considered a selective group of insecticides, and their compatibility with predatory Heteroptera, which are used as biological control agents, is known. However, their molecular mode of action has not been explored in beneficial insects such as Orius laevigatus (Fieber) (Hemiptera: Anthocoridae). RESULTS: In this project in vivo toxicity assays demonstrated that the DAH-based RH-5849, tebufenozide and methoxyfenozide have no toxic effect against O. laevigatus. The ligand-binding domain (LBD) of the ecdysone receptor (EcR) of O. laevigatus was sequenced and a homology protein model was constructed which confirmed a cavity structure with 12 ?-helixes, harboring the natural insect molting hormone 20-hydroxyecdysone. However, docking studies showed that a steric clash occurred for the DAH-based insecticides due to a restricted extent of the ligand-binding cavity of the EcR of O. laevigatus. CONCLUSIONS: The insect toxicity assays demonstrated that MACs are selective for O. laevigatus. The modeling/docking experiments are indications that these pesticides do not bind with the LBD-EcR of O. laevigatus and support that they show no biological effects in the predatory bug. These data help in explaining the compatible use of MACs together with predatory bugs in IPM programs. Keywords: Orius laevigatus, selectivity, diacylhydrazine insecticides, ecdysone receptor, homology modelling, docking studies.

Catalytic mechanism of the adenylyl and guanylyl cyclases: Modeling and mutational analysis

The adenylyl and guanylyl cyclases catalyze the formation of 3′,5′-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5′-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase C2 catalytic domain was used to model by homology a mammalian adenylyl cyclase C1-C2 domain pair, a homodimeric adenylyl cyclase of Dictyostelium discoideum, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. Mg2+ATP or Mg2+GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a Mg2+ ion. The D310S and D310A mutants have 10-fold reduced Vmax and altered [Mg2+] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.

Similar antigenic surfaces, rather than sequence homology, dictate T-cell epitope molecular mimicry.

Molecular mimicry, normally defined by the level of primary-sequence similarities between self and foreign antigens, has been considered a key element in the pathogenesis of autoimmunity. Here we describe an example of molecular mimicry between two overlapping peptides within a single self-antigen, both of which are recognized by the same human self-reactive T-cell clone. Two intervening peptides did not stimulate the T-cell clone, even though they share nine amino acids with the stimulatory peptides. Molecular modeling of major histocompatibility complex class II-peptide complexes suggests that both of the recognized peptides generate similar antigenic surfaces, although these are composed of different sets of amino acids. The molecular modeling of a peptide shifted one residue from the stimulatory peptide, which was recognized in the context of the same HLA molecule by another T-cell clone, generated a completely different antigenic surface. Functional studies using truncated peptides confirmed that the anchor residues of the two "mimicking" epitopes in the HLA groove differ. Our results show, for two natural epitopes, how molecular mimicry can occur and suggest that studies of potential antigenic surfaces, rather than sequence similarity, are necessary for analyzing suspected peptide mimicry.

Gene cloning, molecular modeling, and phylogenetics of serine protease P32 and serine carboxypeptidase SCP1 from nematophagous fungi Pochonia rubescens and Pochonia chlamydosporia

The fungi Pochonia chlamydosporia and Pochonia rubescens are parasites of nematode eggs and thus are biocontrol agents of nematodes. Proteolytic enzymes such as the S8 proteases VCP1 and P32, secreted during the pathogenesis of nematode eggs, are major virulence factors in these fungi. Recently, expression of these enzymes and of SCP1, a new putative S10 carboxypeptidase, was detected during endophytic colonization of barley roots by these fungi. In our study, we cloned the genomic and mRNA sequences encoding P32 from P. rubescens and SCP1 from P. chlamydosporia. P32 showed a high homology with the serine proteases Pr1A from the entomopathogenic fungus Metarhizium anisopliae and VCP1 from P. chlamydosporia (86% and 76% identity, respectively). However, the catalytic pocket of P32 showed differences in the amino acids of the substrate-recognition sites compared with the catalytic pockets of Pr1A and VCP1 proteases. Phylogenetic analysis of P32 suggests a common ancestor with protease Pr1A. SCP1 displays the characteristic features of a member of the S10 family of serine proteases. Phylogenetic comparisons show that SCP1 and other carboxypeptidases from filamentous fungi have an origin different from that of yeast vacuolar serine carboxypeptidases. Understanding protease genes from nematophagous fungi is crucial for enhancing the biocontrol potential of these organisms.