The GTPase activity of the Escherichia coli Ffh protein is important for normal growth.
The Escherichia coli (E. coli) Ffh protein is homologous to the 54kDa subunit of the eukaryotic signal recognition particle. We have examined an intrinsic GTPase activity of this protein and have created mutations in one sequence motif (GXXXXGK) of the putative GTP binding site. When glycine-112 was changed to valine (Ffh-G112V), Vmax was reduced to only 4% of the wildtype level. On the other hand, when glutamine-109 was altered to glycine (Ffh-Q109G), the major effect was a 50-fold increase in Km. These results show that the residues Q-109 and G-112 are essential for the binding and hydrolysis of GTP and that they are part of a catalytic site structurally related to that of many other GTPase proteins. Expression of the mutant protein Ffh-G112V in E. coli was highly toxic in the presence of the wildtype protein. In contrast, genetic complementation experiments showed that a viable strain could be constructed where the Ffh-Q109G mutant protein replaced wildtype Ffh. However, expression of the mutant protein had a negative effect on growth rate at 30 degrees C and resulted in elongated cells. These results demonstrate that the GTPase activity of the Ffh protein is required for proper function of the protein in vivo.
Samuelsson
T
T
Department of Medical Biochemistry, Göteborg University, Sweden.
Olsson
M
M
Wikström
P M
PM
Johansson
B R
BR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002455:Cell Division; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D017353:Gene Deletion; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D018271:Signal Recognition Particle / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D018271:Signal Recognition Particle; D020558:GTP Phosphohydrolases