Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

Abstract

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-AlaGln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a K m of 4.6 µM, a k cat of 1.73 s -1 , and a catalytic efficiency of 376 (mM s) -1 . Another good substrate for ZapA was peptide 6 (AbzArg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a K m of 13.6 µM, a k cat of 3.96 s -1 and a catalytic efficiency of 291 (mM s) -1 . The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P 1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P 2 .

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

“…These peptides contain an Abzgroup (ortho-aminobenzoyl) at the N-terminus, and EDDnp (2,4, dinitrophenylethylenediamine) at the C-terminus, as a fluorescent and quencher pair, respectively. The peptides used in this study were the bradykinin-derived peptide (Abz-R-P-P-G-F-S-P-F-R-EDDnp) and the dynorphin-derived peptide (Abz-G-G-F-L-R-R-Q-EDDnp) (14).…”

“…The kinetic parameters were calculated by the method of Wilkinson (19). The activities of the natural and recombinant enzymes were determined using the operational substrate described by Fernandes et al (14). One unit of ZapA activity is the amount of enzyme which hydrolyzes 1 µmol of the peptide substrate in 1 min.…”

“…In order to investigate whether ZapA should be included among specific bacterial metalloproteases, or whether it displays broad substrate specificy, we employed the newly developed fluorogenic operational substrate (14) to characterize putative biological substrates of ZapA.…”

Cloning, characterization and molecular analysis of a metalloprotease from Proteus mirabilis

“…The substrate specificity of ZapA had been characterized using fluorescent peptides derived from bioactive peptides and the oxidized β-chain of insulin. As one of the most important virulence factors of P. mirabilis, the role of ZapA in pathogenesis was probably due to the destruction of IgA, antimicrobial peptides, bioactive molecules, and structural components of the host cells (Fernandes et al 2000;Anéas et al 2001;Belas et al 2004).…”

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

“…There are a number of enzymes in M10B, also called serralysins, which are secreted by a wide range of microorganisms, including plant and human pathogens. Serralysin-type proteases are generally supposed to be nonspecific enzymes, involved in, e.g., bioconversion of the host tissues, because they cleave with a relaxed residue preference in a variety of synthetic peptide substrates and denatured oligopeptides of biological origin (1,2,9,22,23,25,30). However, serralysins, like most of the proteases, encounter substrate proteins under physiological conditions, where they are in native conformation.…”