Lipopeptide substrates for SpsB, the Staphylococcus aureus type I signal peptidase:: design, conformation and conversion to α-ketoamide inhibitors

Pre-protein sequence data was used to design substrates for SpsB, the bacterial signal peptidase I enzyme from Staphylococcus aureus. aureus. Key elements were an alkyl membrane anchor, proline at P5 and lysine at P2. The proline at P5 induced a helical turn in the lipopeptide. as deduced from NMR studies, from P6 to P2 in membrane mimetic solvents. The substrate Decanoyl-LTPTAKAASKIDD-OH was cleaved by SpsB, as expected, between the PI and Pl' alanines with a k(cat)/K-m of 2.3 x 10(6) M-1 s(-1) at pH 8.5. Insertion of proline at P1' converted substrates to competitive inhibitors, whilst the incorporation of an alpha-ketoamide at the cleavage site transformed substrates to time dependent inhibitors of SpsB. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.