ACTIVATION OF METHYLESTERASE CHEB - EVIDENCE OF A DUAL ROLE FOR THE REGULATORY DOMAIN

The response regulator CheB functions within the bacterial chemotaxis system together with the methyltransferase CheR to control the level o f chemoreceptor methylation, influencing the signaling activities of t he receptors. CheB catalyzes demethylation of specific methylglutamate residues introduced into the chemoreceptors by CheR. CheB has a two-d omain architecture consisting of an N-terminal regulatory domain joine d by a linker to a C-terminal effector domain. In the unphosphorylated state of the response regulator, the regulatory domain inhibits the m ethylesterase activity of the effector domain. Upon phosphorylation of a specific aspartate residue within the regulatory domain, the C-term inal methylesterase activity is stimulated, resulting in the subsequen t demethylation of the chemoreceptors. We have investigated the mechan ism of regulation of CheB activity by the N-terminal regulatory domain . First, we have found that phosphorylation of the N-terminal domain n ot only relieves inhibition of the C-terminal methylesterase activity but also provides an enhancement of this activity above that seen for the C-terminal effector domain alone. Second, we have identified mutat ions in CheB that show an enhancement of methylesterase activity in th e absence of phosphorylation. Most of these single-site mutations are localized in the linker region joining the regulatory and effector dom ains. On the basis of these observations, we propose a model for activ ation of CheB in which phosphorylation of the regulatory domain result s in a reorganization of the domain interface, allowing exposure of th e active site to the receptor substrate and simultaneously stimulating methylesterase activity.