Substrate Specificity of the Integral Membrane Protease OmpT Determined by Spatially Addressed Peptide Libraries

Abstract

Escherichia coli outer membrane protease T (OmpT) is an endopeptidase that specifically cleaves between two consecutive basic residues. In this study we have investigated the substrate specificity of OmpT using spatially addressed SPOT peptide libraries. The peptide acetyl-Dap(dnp)-Ala-Arg/Arg-Ala-Lys(Abz)-Gly was synthesized directly onto cellulose membrane. The peptide contained the aminobenzoyl (Abz) fluorophore, which was internally quenched by the dinitrophenyl (dnp) moiety. Treatment of the SPOT membrane with the small, water-soluble protease trypsin resulted in highly fluorescent peptide SPOTs. However, no peptide cleavage was observed after incubation with detergent-solubilized OmpT, a macromolecular complex with an estimated molecular mass of 180 kDa. This problem could be solved by the introduction of a long, polar polyoxyethylene glycol linker between the membrane support and the peptide. Peptide libraries for the P(2), P(1), P(1)', and P(2)' positions in the substrate were screened with OmpT, and peptides of positive SPOTs were resynthesized and subjected to kinetic measurements in solution. The best substrate Abz-Ala-Lys-Lys-Ala-Dap(dnp)-Gly had a turnover number k(cat) of 40 s(-)(1), which is 12-fold higher than the starting substrate. Peptides containing an acidic residue at P(2) or P(2)' were not substrates for OmpT, suggesting that long-range electrostatic interactions are important for the formation of the enzyme-substrate complex. OmpT was highly selective toward L-amino acids at P(1) but was less so at P(1)' where a peptide with D-Arg at P(1)' was a competitive inhibitor (K(i) of 19 microM). An affinity chromatography resin based on these findings was developed, which allowed for the one-step purification of OmpT from a bacterial lysate. The implications of the determined consensus substrate sequence (Arg/Lys)/(Arg/Lys)-Ala for the proposed biological function of OmpT in defense against antimicrobial peptides are discussed.

Role of theompTMutation in Stimulated Decrease in Colony-Forming Ability Due to Intracellular Protein Aggregate Formation inEscherichia coliStrain BL21

“…This result was in accord with the previous belief that OmpT protease is an outer-membrane protease. [19][20][21] At the same time, it was clearly seen that the supernatant contained a substantial amount OmpT protease. To further confirm the intactness of OmpT protease induced by plasmid pOmpT-Tet, we examined OmpT protease activity.…”

“…It is well-established that OmpT protease specifically cleaves between two consecutive basic residues. 13,21,22) Taking this into account, we examined the amino acid sequence of Sup35NM and found that it had no arginine (R), and that it had four pairs of lysine (K) in the M domain (Fig. 6B).…”

Capsular Antigen Fraction 1 and Pla Modulate the Susceptibility of Yersinia pestis to Pulmonary Antimicrobial Peptides Such as Cathelicidin

“…This specificity could not be due to different active sites on Pla, since a substitution mutation inactivating the enzymatic site of Pla for plasminogen cleavage also affected the inactivation of LL-37 or CRAMP. OmpT and PgtE cleave preferentially between two basic amino acid residues (14,17), explaining why CAMPs such as cathelicidins, with their repeated pairs of Arg and/or Lys residues, are optimal targets for these omptins (24,59). The efficiency (k cat /K m ) of OmpT for a peptide with an Arg2Arg cleavage site was fourfold better than that for a similar peptide with an Arg2Val cleavage site (42,65).…”

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

“…3) [21], resulted in at least sevenfold lower activity. OmpT preferentially cleaves peptides between two basic amino acids [5], therefore the anionic nature of the cleft is probably a major determinant of its substrate speci¢city. More speci¢cally, we proposed earlier, based on the crystal structure of OmpT [21], that Glu 27 and Asp 208 may de¢ne the high speci¢city of OmpT for Arg or Lys at position P1 in the substrate (nomenclature as in reference [27]).…”