Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT.
OmpT is an integral outer membrane protease of Escherichia coli. Overexpression of OmpT in E. coli and subsequent in vitro folding of the produced inclusion bodies yielded protein with a native-like structure. However, enzymatically active protease was only obtained after addition of the outer membrane lipid lipopolysaccharide (LPS). OmpT is the first example of an enzyme that requires LPS for activity. In this study, we investigated the nature of this activation. Circular dichroism analysis showed that binding of LPS did not lead to large structural changes. Titration of OmpT with LPS and determining the resulting OmpT activity with a fluorimetric assay yielded a dissociation constant of 10-4 m for E. coli K-12 LPS. Determining the dissociation constants for different LPS chemotypes revealed that a fully acylated lipid A part is minimally required for activation of OmpT. The heptose-bound phosphates in the inner core region were also important for activation. The affinity for LPS was not dependent on the concentration of substrate, neither was affinity for the substrate influenced by the concentration of LPS. This indicated that LPS most likely does not act at the level of substrate binding. We hypothesize that LPS induces a subtle conformational change in the protein that is required for obtaining a native active site geometry.
Kramer
R Arjen
RA
Department of Enzymology and Protein Engineering, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, the Netherlands.
Brandenburg
Klaus
K
Vandeputte-Rutten
Lucy
L
Werkhoven
Marjolein
M
Gros
Piet
P
Dekker
Niek
N
Egmond
Maarten R
MR
D002942:Circular Dichroism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D008070:Lipopolysaccharides; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease