Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate

Judit Marokházi, Nikolett Mihala, F. Hudecz, A. Fodor, L. Gráf, I. Venekei

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16 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1946-1956
Number of pages11
JournalFEBS Journal
Volume274
Issue number8
DOIs
Publication statusPublished - Apr 2007

Fingerprint

serralysin
Photorhabdus
Pathogens
Insects
Peptide Hydrolases
Substrates
Peptides
Hydrolysis
beta-Lipotropin
Amino Acids
Bioconversion
Peptide Fragments
Enzymes
Infection
Microorganisms
Larva

Keywords

  • Cleavage site
  • Metalloprotease
  • PrtA of Photorhabdus
  • Serralysin
  • Specific substrate

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{90d5e487c95b40689d6071ee329d256c,
title = "Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate",
abstract = "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.",
keywords = "Cleavage site, Metalloprotease, PrtA of Photorhabdus, Serralysin, Specific substrate",
author = "Judit Marokh{\'a}zi and Nikolett Mihala and F. Hudecz and A. Fodor and L. Gr{\'a}f and I. Venekei",
year = "2007",
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T1 - Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate

AU - Marokházi, Judit

AU - Mihala, Nikolett

AU - Hudecz, F.

AU - Fodor, A.

AU - Gráf, L.

AU - Venekei, I.

PY - 2007/4

Y1 - 2007/4

N2 - 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.

AB - 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.

KW - Cleavage site

KW - Metalloprotease

KW - PrtA of Photorhabdus

KW - Serralysin

KW - Specific substrate

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DO - 10.1111/j.1742-4658.2007.05739.x

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SP - 1946

EP - 1956

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JF - FEBS Journal

SN - 1742-464X

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