Purification and characterization of the Myxococcus xanthus FrzE protein shows that it has autophosphorylation activity

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Abstract
Myxococcus xanthus exhibits multicellular interactions during vegetative growth and fruiting body formation. Gliding motility is needed for these interactions. The frizzy (frz) genes are required to control directed motility. FrzE is homologous to both CheA and CheY from Salmonella typhimurium. We used polyclonal antiserum raised against a fusion protein to detect FrzE in M. xanthus extracts by Western immunoblot analysis. FrzE was clearly present during vegetative growth and at much lower levels during development. A recombinant FrzE protein was overproduced in Escherichia coli, purified from inclusion bodies, and renatured. FrzE was autophosphorylated when it was incubated in the presence of [gamma-32P]ATP and MnCl2. Chemical analyses of the phosphorylated FrzE protein indicated that it contained an acylphosphate; probably phosphoaspartate. FrzE was phosphorylated in an intramolecular reaction. Based on these observations, we propose a model of the mechanism of FrzE phosphorylation in which autophosphorylation initially occurs at a conserved histidine residue within the "CheA" domain and then, via an intramolecular transphosphorylation, is transferred to a conserved aspartate residue within the "CheY" domain.