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 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.

Identification of Natural Target Proteins Indicates Functions of a Serralysin-Type Metalloprotease, PrtA, in Anti-Immune Mechanisms

“…The presence of only a small number of hemolymph proteins we found to be cleaved by PrtA does not support the view that, as generally serralysins are thought to be, PrtA is a nonspecific protease having solely a role in, e.g., bioconversion. This is in accord with our former observation that PrtA did not hydrolyze native-state collagen, fibrin, and albumin (27). We found altogether 16 PAT proteins, most of which are minor components of the hemolymph.…”

“…The mechanism of this pathogenicity, the way the bacterium can evade the immune defense, is unknown, but PrtA might take part in it. Supporting this assumption are the facts that Photorhabdus starts producing PrtA early during infection (4,5,27,34) and that PrtA does not exhibit activity on native proteins (fibrinogen, albumin, and collagen types I and IV) (27), which might have been expected if it participates in the bioconversion of host tissues as a nonspecific protease (5,6) or if it is involved in the degradation of extracellular matrix (36). The contribution of PrtA to pathogenicity does not include a direct toxic effect either (5), at variance with several other metalloproteases which are lethal toxins.…”

“…PrtA was purified from Photorhabdus luminescens strain Brecon as described previously (27). Recombinant PrtA was purified from Escherichia coli Hb101 strains transformed with pUC19 plasmid containing Photorhabdus PrtA (a generous gift from Richard ffrench-Constant, Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom).…”

Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

“…Given its ability to hydrolyze environmental proteins, PrtA is presumably involved in nutrient utilization. Due to its low degree of selectivity, a polypeptide of as few as six amino acids can be a substrate (28). Therefore, it is believed that PrtA hydrolyzes most proteins in the extracellular media.…”

Estudo de atividades amidásicas na linhagem MN7 de Photorhabdus luminescens luminescens, isolada da linhagem LPP7 de Heterorhabditis baujardi.

“…De acordo com a literatura, PrtA tem a sua clivagem determinada principalmente pelos resíduos presentes nas posições P1' e P2 do substrato (MAROKHAZI et al, 2007). A similaridade das características químicas entre a valina (determinada como resíduo ótimo para a posição P2) e a metionina (observada em nossos ensaios como resíduo que favorece a clivagem do substrato) podem justificar os resultados observados neste trabalho com MN7.…”