Lipopeptide substrates for SpsB, the Staphylococcus aureus type I signal peptidase: design, conformation and conversion to alpha-ketoamide inhibitors.
Pre-protein sequence data was used to design substrates for SpsB, the bacterial signal peptidase I enzyme from Staphylococcus 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 P1 and P1' alanines with a k(cat)/K(m) of 2.3x10(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.
Bruton
Gordon
G
GlaxoSmithKline, New Frontiers Science Park, Essex CM19 5AW, Harlow, UK. gordon_bruton-1@gsk.com
Huxley
Anthony
A
O'Hanlon
Peter
P
Orlek
Barry
B
Eggleston
Drake
D
Humphries
John
J
Readshaw
Simon
S
West
Andrew
A
Ashman
Stephen
S
Brown
Murray
M
Moore
Keith
K
Pope
Andrew
A
O'Dwyer
Karen
K
Wang
Lei
L
D000577:Amides / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008074:Lipoproteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013997:Time Factors
D000577:Amides; D008074:Lipoproteins; D008565:Membrane Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; C026515:type I signal peptidase