title: Characterization of the autophosphorylation property of HflX, a ribosome-binding GTPase from Escherichia coli.
abstract: Escherichia coli HflX belongs to the widely distributed but poorly characterized HflX family of translation factor-related GTPases that is conserved from bacteria to humans. A 426-residue polypeptide that binds 50S ribosomes and has both GTPase and ATPase activities, HflX also exhibits autophosphorylation activity. We show that HflX(C), a C-terminal fragment of HflX, has an enhanced autophosphorylation activity compared to the full-length protein. Using a chemical stability assay and thin layer chromatography, we have determined that phosphorylation occurs at a serine residue. Each of the nine serine residues of HflX(C) was mutated to alanine. It was found that all but S211A retained autophosphorylation activity, suggesting that S211, located in the P-loop, was the likely site for autophosphorylation. While the S211A mutant lacked the autophosphorylation site, it possessed strong GTP binding and GTPase activities.
authors.affiliation: Department of Biochemistry Bose Institute Kolkata India.
authors.forename: Aditi
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Department of Biochemistry Bose Institute KolkataIndia; Present address: CSIR-Institute of Microbial Technology Sector 39A Chandigarh 160036 India.
authors.forename: Dipak
authors.initials: D
authors.lastname: Dutta
authors.affiliation: Department of Biochemistry Bose Institute KolkataIndia; Present address: Department of Plant Biology The Samuel Roberts Noble Foundation Ardmore OK USA.
authors.forename: Kaustav
authors.initials: K
authors.lastname: Bandyopadhyay
authors.affiliation: Department of Biochemistry Bose Institute Kolkata India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Parrack
keywords: HflX(C); TRAFAC‐GTPase; autophosphorylation