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Istvan Venekei - One of the best experts on this subject based on the ideXlab platform.
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enzymatic characterization of a Serralysin like metalloprotease from the entomopathogen bacterium xenorhabdus
Biochimica et Biophysica Acta, 2011Co-Authors: Mustafa K Massaoud, Judit Marokhazi, Istvan VenekeiAbstract:Abstract We investigated the enzymatic properties of a Serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest k cat / K M value. The pH profile of activity, similarly to other Serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu 2+ and Co 2+ ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn 2+ was inhibitory (both are unusual among Serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn 2+ , Cu 2+ and Co 2+ up to 90–200% of its original value, while Zn 2+ was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the Serralysins contain Asn.
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Enzymatic characterization of a Serralysin-like metalloprotease from the entomopathogen bacterium, Xenorhabdus Proteins and proteomics
Biochimica et Biophysica Acta, 2011Co-Authors: Mustafa K Massaoud, Judit Marokhazi, Istvan VenekeiAbstract:We investigated the enzymatic properties of a Serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest kcₐₜ/KM value. The pH profile of activity, similarly to other Serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu²⁺ and Co²⁺ ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn²⁺ was inhibitory (both are unusual among Serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn²⁺, Cu²⁺ and Co²⁺ up to 90–200% of its original value, while Zn²⁺ was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the Serralysins contain Asn.
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proteolytic enzyme production by strains of the insect pathogen xenorhabdus and characterization of an early log phase secreted protease as a potential virulence factor
Applied and Environmental Microbiology, 2010Co-Authors: Mustafa K Massaoud, Judit Marokhazi, Istvan VenekeiAbstract:As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa Serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.
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identification of natural target proteins indicates functions of a Serralysin type metalloprotease prta in anti immune mechanisms
Applied and Environmental Microbiology, 2009Co-Authors: G Felfoldi, Judit Marokhazi, Miklos Kepiro, Istvan VenekeiAbstract:Serralysins are generally thought to function as pathogenicity factors of bacteria, but so far no hard evidence of this (e.g., specific substrate proteins that are sensitive to the cleavage by these proteases) has been found. We have looked for substrate proteins to a Serralysin-type proteinase, PrtA, in a natural host-pathogen molecular interaction system involving Manduca sexta and Photorhabdus luminescens. The exposure in vitro of hemolymph to PrtA digestion resulted in selective cleavage of 16 proteins, provisionally termed PAT (PrtA target) proteins. We could obtain sequence information for nine of these PrtA sensitive proteins, and by searching databases, we could identify six of them. Each has immune-related function involving every aspect of the immune defense: beta-1,3 glucan recognition protein 2 (immune recognition), hemocyte aggregation inhibitor protein (HAIP), serine proteinase homolog 3, six serpin-1 variants, including serpin-1I (immune signaling and regulation), and scolexins A and B (coagulation cascade effector function). The functions of the identified PrtA substrate proteins shed new light on a possible participation of a Serralysin in the virulence mechanism of a pathogen. Provided these proteins are targets of PrtA in vivo, this might represent, among others, a complex suppressive role on the innate immune response via interference with both the recognition and the elimination of the pathogen during the first, infective stage of the host-pathogen interaction. Our results also raise the possibility that the natural substrate proteins of Serralysins of vertebrate pathogens might be found among the components of the innate immune system.
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cleavage site analysis of a Serralysin like protease prta from an insect pathogen photorhabdus luminescens and development of a highly sensitive and specific substrate
FEBS Journal, 2007Co-Authors: Judit Marokhazi, Nikolett Mihala, Ferenc Hudecz, Andras Fodor, Laszlo Graf, Istvan VenekeiAbstract:The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a Serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and β-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1′ and P2′. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1′ > P2 > P2′. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4′(dimethylamino)phenylazo]benzoyl–EVYAVES−5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The ∼ 4 × 106 m−1·s−1 specificity constant of PrtA (at Km ∼ 5 × 10−5 m and kcat ∼ 2 × 102 s−1) on this substrate was the highest activity for a Serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other Serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.
Judit Marokhazi - One of the best experts on this subject based on the ideXlab platform.
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enzymatic characterization of a Serralysin like metalloprotease from the entomopathogen bacterium xenorhabdus
Biochimica et Biophysica Acta, 2011Co-Authors: Mustafa K Massaoud, Judit Marokhazi, Istvan VenekeiAbstract:Abstract We investigated the enzymatic properties of a Serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest k cat / K M value. The pH profile of activity, similarly to other Serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu 2+ and Co 2+ ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn 2+ was inhibitory (both are unusual among Serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn 2+ , Cu 2+ and Co 2+ up to 90–200% of its original value, while Zn 2+ was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the Serralysins contain Asn.
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Enzymatic characterization of a Serralysin-like metalloprotease from the entomopathogen bacterium, Xenorhabdus Proteins and proteomics
Biochimica et Biophysica Acta, 2011Co-Authors: Mustafa K Massaoud, Judit Marokhazi, Istvan VenekeiAbstract:We investigated the enzymatic properties of a Serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest kcₐₜ/KM value. The pH profile of activity, similarly to other Serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu²⁺ and Co²⁺ ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn²⁺ was inhibitory (both are unusual among Serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn²⁺, Cu²⁺ and Co²⁺ up to 90–200% of its original value, while Zn²⁺ was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the Serralysins contain Asn.
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proteolytic enzyme production by strains of the insect pathogen xenorhabdus and characterization of an early log phase secreted protease as a potential virulence factor
Applied and Environmental Microbiology, 2010Co-Authors: Mustafa K Massaoud, Judit Marokhazi, Istvan VenekeiAbstract:As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa Serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.
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identification of natural target proteins indicates functions of a Serralysin type metalloprotease prta in anti immune mechanisms
Applied and Environmental Microbiology, 2009Co-Authors: G Felfoldi, Judit Marokhazi, Miklos Kepiro, Istvan VenekeiAbstract:Serralysins are generally thought to function as pathogenicity factors of bacteria, but so far no hard evidence of this (e.g., specific substrate proteins that are sensitive to the cleavage by these proteases) has been found. We have looked for substrate proteins to a Serralysin-type proteinase, PrtA, in a natural host-pathogen molecular interaction system involving Manduca sexta and Photorhabdus luminescens. The exposure in vitro of hemolymph to PrtA digestion resulted in selective cleavage of 16 proteins, provisionally termed PAT (PrtA target) proteins. We could obtain sequence information for nine of these PrtA sensitive proteins, and by searching databases, we could identify six of them. Each has immune-related function involving every aspect of the immune defense: beta-1,3 glucan recognition protein 2 (immune recognition), hemocyte aggregation inhibitor protein (HAIP), serine proteinase homolog 3, six serpin-1 variants, including serpin-1I (immune signaling and regulation), and scolexins A and B (coagulation cascade effector function). The functions of the identified PrtA substrate proteins shed new light on a possible participation of a Serralysin in the virulence mechanism of a pathogen. Provided these proteins are targets of PrtA in vivo, this might represent, among others, a complex suppressive role on the innate immune response via interference with both the recognition and the elimination of the pathogen during the first, infective stage of the host-pathogen interaction. Our results also raise the possibility that the natural substrate proteins of Serralysins of vertebrate pathogens might be found among the components of the innate immune system.
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cleavage site analysis of a Serralysin like protease prta from an insect pathogen photorhabdus luminescens and development of a highly sensitive and specific substrate
FEBS Journal, 2007Co-Authors: Judit Marokhazi, Nikolett Mihala, Ferenc Hudecz, Andras Fodor, Laszlo Graf, Istvan VenekeiAbstract:The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a Serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and β-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1′ and P2′. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1′ > P2 > P2′. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4′(dimethylamino)phenylazo]benzoyl–EVYAVES−5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The ∼ 4 × 106 m−1·s−1 specificity constant of PrtA (at Km ∼ 5 × 10−5 m and kcat ∼ 2 × 102 s−1) on this substrate was the highest activity for a Serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other Serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.
Ferenc Hudecz - One of the best experts on this subject based on the ideXlab platform.
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cleavage site analysis of a Serralysin like protease prta from an insect pathogen photorhabdus luminescens and development of a highly sensitive and specific substrate
FEBS Journal, 2007Co-Authors: Judit Marokhazi, Nikolett Mihala, Ferenc Hudecz, Andras Fodor, Laszlo Graf, Istvan VenekeiAbstract:The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a Serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and β-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1′ and P2′. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1′ > P2 > P2′. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4′(dimethylamino)phenylazo]benzoyl–EVYAVES−5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The ∼ 4 × 106 m−1·s−1 specificity constant of PrtA (at Km ∼ 5 × 10−5 m and kcat ∼ 2 × 102 s−1) on this substrate was the highest activity for a Serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other Serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.
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cleavage site analysis of a Serralysin like protease prta from an insect pathogen photorhabdus luminescens and development of a highly sensitive and specific substrate
FEBS Journal, 2007Co-Authors: Judit Marokhazi, Nikolett Mihala, Ferenc Hudecz, Andras Fodor, Laszlo Graf, Istvan VenekeiAbstract:The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a Serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and beta-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1' and P2'. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1' > P2 > P2'. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4'(dimethylamino)phenylazo]benzoyl-EVYAVES-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The approximately 4 x 10(6) M(-1) x s(-1) specificity constant of PrtA (at K(m) approximately 5 x 10(-5) M and k(cat) approximately 2 x 10(2) s(-1)) on this substrate was the highest activity for a Serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other Serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.
Laszlo Graf - One of the best experts on this subject based on the ideXlab platform.
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cleavage site analysis of a Serralysin like protease prta from an insect pathogen photorhabdus luminescens and development of a highly sensitive and specific substrate
FEBS Journal, 2007Co-Authors: Judit Marokhazi, Nikolett Mihala, Ferenc Hudecz, Andras Fodor, Laszlo Graf, Istvan VenekeiAbstract:The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a Serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and β-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1′ and P2′. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1′ > P2 > P2′. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4′(dimethylamino)phenylazo]benzoyl–EVYAVES−5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The ∼ 4 × 106 m−1·s−1 specificity constant of PrtA (at Km ∼ 5 × 10−5 m and kcat ∼ 2 × 102 s−1) on this substrate was the highest activity for a Serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other Serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.
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cleavage site analysis of a Serralysin like protease prta from an insect pathogen photorhabdus luminescens and development of a highly sensitive and specific substrate
FEBS Journal, 2007Co-Authors: Judit Marokhazi, Nikolett Mihala, Ferenc Hudecz, Andras Fodor, Laszlo Graf, Istvan VenekeiAbstract:The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a Serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and beta-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1' and P2'. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1' > P2 > P2'. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4'(dimethylamino)phenylazo]benzoyl-EVYAVES-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The approximately 4 x 10(6) M(-1) x s(-1) specificity constant of PrtA (at K(m) approximately 5 x 10(-5) M and k(cat) approximately 2 x 10(2) s(-1)) on this substrate was the highest activity for a Serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other Serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.
Trillo Muyo Sergio - One of the best experts on this subject based on the ideXlab platform.
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Estudio estructural y funcional de un inhibidor proteínico monodominio de doble faz, sermetstatina, en complejo con dos peptidasas de diferente clase, subtilisina y esnapalisina
[Barcelona] : Universitat Autònoma de Barcelona, 2013Co-Authors: Trillo Muyo Sergio, Universitat Autònoma De Barcelona. Institut De Biotecnologia I De BiomeAbstract:A portada: Universitat Autònoma de Barcelona, Institut de Biotecnologia i Biomedicina: Consejo Superior de Investigaciones Científicas: Institut de Biologia Molecular de Barcelona, Departament de Biologia Estructural, Proteolysis LaboratoryLos Inhibidores de peptidasas representan un mecanismo fisiológico para la regulación de las enzimas proteolíticas. Mientras que mayoría de los inhibidores monodominio tienen un único sitio reactivo mediante el cual interaccionan con sus peptidasas dianas de un tipo catalítico específico, algunos de ellos inhiben dos moléculas de peptidasa simultáneamente, dando lugar a la formación de complejos ternarios. Para estudiar este tipo de inhibidores se analizó la función de uno de ellos, sermetstatina. Este inhibidor forma un dímero que une fuertemente serín peptidasas y metalopeptidasas. La estructura del dímero de inhibidor fue determinada revelando que sermetstatina presenta una conformación en α/β-sándwich alargado constituido por cinco hebras β antiparalelas conectadas entre sí (β3-β2-β1-β4-β5; conectividad -1, -1, +3, +1) que dan lugar a una hoja β girada ∼30o, en cuya cara cóncava se acomodan dos hélices α (α1 y α2) y una hélice 310. Además, las estructuras de los complejos heterotetraméricos de sermetstatina con la serín peptidasa subtilisina y la metalopeptidasa esnapalisina fueron también determinadas, mostrando que la inhibición ocurre a través de lazos reactivos distales independientes. La interacción entre subtilisina y sermetstatina se produce mediante el lazo reactivo 2, posicionado adecuadamente por su hélice de anclaje, y la hendidura del centro activo de la enzima. El lazo se inserta a modo de cuña mimetizando un substrato en conformación extendida y canónica en la hendidura del centro activo de la enzima, siguiendo el mecanismo estándar de inhibición. Por otro lado, la interacción entre esnapalisina y sermetstatina se produce mediante el extremo N-terminal, el lazo reactivo 1, la hélice α1 y la región Lβ4β5 del inhibidor; y la hendidura del centro activo de la enzima y exositios presentes en la superficie de la proteasa. Este modo de inhibición sigue un mecanismo novedoso en inhibidores de metalopeptidasas, siendo este una reminiscencia distante del modo inhibitorio de los TIMPs en su unión con 07Ps así como del modo inhibitorio del inhibidor de serralisina sobre la metalopeptidasa serralisina. Estas estructuras y el modelo del complejo heterohexamérico proporcionan por primera vez una visión detallada del mecanismo molecular de la inhibición simultánea de peptidasas pertenecientes a dos clases mecanísticamente diferentes por un inhibidor monodominio. En resumen, en el presente trabajo se ha determinado que sermetstatina es un inhibidor de doble faz genuino monodominio que ha evolucionado a partir de inhibidores de serín peptidasas de la familia MEROPS I16 con un único sitio reactivo que siguen el mecanismo estándar de inhibición. Dicha evolución ha dado lugar a una proteína bifuncional capaz de inhibir simultáneamente diferentes serín peptidasas y una metalopeptidasa específica a través de dos sitios reactivos distales compatibles.Protein inhibitors provide a physiological mechanism for the regulation of proteolytic enzymes. While most single-domain inhibitors have one reactive site with which they target peptidases of a specific catalytic class, selected specimens inhibit two peptidase molecules simultaneously, thus giving rise to ternary complexes. To study such inhibition, the function of one of these proteins, sermetstatin, was analyzed. This inhibitor strongly binds as a dimer to serine peptidases and a metallopeptidase. The structure of the isolated inhibitor dimer was determined revealing that sermetstatin is an elongated α/β-sandwich. It consists of a five-stranded antiparallel β-sheet (β3-β2-β1-β4-β5; connectivity -1,-1,+3,+1) twisted by ∼30o, whose concave face accommodates two α-helices (α1 and α2) and a 310-helix. In addition, the structures of the heterotetrameric complexes with the serine peptidase subtilisin and the metallopeptidase snapalysin were equally determined, showing that inhibition occurs through two independent distal reactive sites. The subtilisin-sermetstatin interaction is made by reactive-site loop 2, adequately positioned by its scaffold helix, and the active-site cleft of the enzyme. The loop is inserted wedge-like mimicking a substrate in extended, "canonical" conformation in the active-site cleft of the enzyme following the "standard mechanism". On the other hand, the snapalysin-sermetstatin interaction involves the N-terminal tail, reactive site loop 1, helix α1 and Lβ4β5 of the inhibitor; and the active-site cleft of the enzyme and some exosites on the protease surface. This inhibition modus follows a novel mechanism for metallopeptidase inhibitors only distantly reminiscent of the inhibitory mode of tissue inhibitors of metalloproteinases on their target matrix metalloproteinases and of Serralysin inhibitors on their cognate Serralysin MPs. These structures and the derived model for the heterohexameric complex provide for the first time a detailed view of the molecular mechanism of simultaneous inhibition of proteinases belonging to two distinct mechanistic classes by a single-domain protein. In summary, it was determined that sermetstatin is a genuine Janus-faced single-domain inhibitor which has evolved from single-site standard-mechanism serine peptidase inhibitors of family I16 to give a protein capable of simultaneous inhibition of serine peptidase in general and a specific metallopeptidase through distinct but compatible sites
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Estudio estructural y funcional de un inhibidor proteínico monodominio de doble faz, sermetstatina, en complejo con dos peptidasas de diferente clase, subtilisina y esnapalisina
'Universitat Autonoma de Barcelona', 2013Co-Authors: Trillo Muyo SergioAbstract:Los inhibidores de peptidasas representan un mecanismo fisiológico para la regulación de las enzimas proteolíticas. Mientras que mayoría de los inhibidores monodominio tienen un único sitio reactivo mediante el cual interaccionan con sus peptidasas dianas de un tipo catalítico específico, algunos de ellos inhiben dos moléculas de peptidasa simultáneamente, dando lugar a la formación de complejos ternarios. Para estudiar este tipo de inhibidores se analizó la función de uno de ellos, sermetstatina. Este inhibidor forma un dímero que une fuertemente serín peptidasas y metalopeptidasas. La estructura del dímero de inhibidor fue determinada revelando que sermetstatina presenta una conformación en α/β-sándwich alargado constituido por cinco hebras β antiparalelas conectadas entre sí (β3-β2-β1-β4-β5; conectividad -1, -1, +3, +1) que dan lugar a una hoja β girada ∼30o, en cuya cara cóncava se acomodan dos hélices α (α1 y α2) y una hélice 310. Además, las estructuras de los complejos heterotetraméricos de sermetstatina con la serín peptidasa subtilisina y la metalopeptidasa esnapalisina fueron también determinadas, mostrando que la inhibición ocurre a través de lazos reactivos distales independientes. La interacción entre subtilisina y sermetstatina se produce mediante el lazo reactivo 2, posicionado adecuadamente por su hélice de anclaje, y la hendidura del centro activo de la enzima. El lazo se inserta a modo de cuña mimetizando un substrato en conformación extendida y canónica en la hendidura del centro activo de la enzima, siguiendo el mecanismo estándar de inhibición. Por otro lado, la interacción entre esnapalisina y sermetstatina se produce mediante el extremo N-terminal, el lazo reactivo 1, la hélice α1 y la región Lβ4β5 del inhibidor; y la hendidura del centro activo de la enzima y exositios presentes en la superficie de la proteasa. Este modo de inhibición sigue un mecanismo novedoso en inhibidores de metalopeptidasas, siendo este una reminiscencia distante del modo inhibitorio de los TIMPs en su unión con MMPs así como del modo inhibitorio del inhibidor de serralisina sobre la metalopeptidasa serralisina. Estas estructuras y el modelo del complejo heterohexamérico proporcionan por primera vez una visión detallada del mecanismo molecular de la inhibición simultánea de peptidasas pertenecientes a dos clases mecanísticamente diferentes por un inhibidor monodominio. En resumen, en el presente trabajo se ha determinado que sermetstatina es un inhibidor de doble faz genuino monodominio que ha evolucionado a partir de inhibidores de serín peptidasas de la familia MEROPS I16 con un único sitio reactivo que siguen el mecanismo estándar de inhibición. Dicha evolución ha dado lugar a una proteína bifuncional capaz de inhibir simultáneamente diferentes serín peptidasas y una metalopeptidasa específica a través de dos sitios reactivos distales compatibles.Protein inhibitors provide a physiological mechanism for the regulation of proteolytic enzymes. While most single-domain inhibitors have one reactive site with which they target peptidases of a specific catalytic class, selected specimens inhibit two peptidase molecules simultaneously, thus giving rise to ternary complexes. To study such inhibition, the function of one of these proteins, sermetstatin, was analyzed. This inhibitor strongly binds as a dimer to serine peptidases and a metallopeptidase. The structure of the isolated inhibitor dimer was determined revealing that sermetstatin is an elongated α/β-sandwich. It consists of a five-stranded antiparallel β-sheet (β3-β2-β1-β4-β5; connectivity -1,-1,+3,+1) twisted by ∼30o, whose concave face accommodates two α-helices (α1 and α2) and a 310-helix. In addition, the structures of the heterotetrameric complexes with the serine peptidase subtilisin and the metallopeptidase snapalysin were equally determined, showing that inhibition occurs through two independent distal reactive sites. The subtilisin-sermetstatin interaction is made by reactive-site loop 2, adequately positioned by its scaffold helix, and the active-site cleft of the enzyme. The loop is inserted wedge-like mimicking a substrate in extended, “canonical” conformation in the active-site cleft of the enzyme following the “standard mechanism”. On the other hand, the snapalysin-sermetstatin interaction involves the N-terminal tail, reactive site loop 1, helix α1 and Lβ4β5 of the inhibitor; and the active-site cleft of the enzyme and some exosites on the protease surface. This inhibition modus follows a novel mechanism for metallopeptidase inhibitors only distantly reminiscent of the inhibitory mode of tissue inhibitors of metalloproteinases on their target matrix metalloproteinases and of Serralysin inhibitors on their cognate Serralysin MPs. These structures and the derived model for the heterohexameric complex provide for the first time a detailed view of the molecular mechanism of simultaneous inhibition of proteinases belonging to two distinct mechanistic classes by a single-domain protein. In summary, it was determined that sermetstatin is a genuine Janus-faced single-domain inhibitor which has evolved from single-site standard-mechanism serine peptidase inhibitors of family I16 to give a protein capable of simultaneous inhibition of serine peptidase in general and a specific metallopeptidase through distinct but compatible sites
