The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu

• Published in: Nature chemical biology Vol. 9; no. 12; pp. 811 - 817
• Main Authors: Castro-Roa, Daniel, Garcia-Pino, Abel, De Gieter, Steven, van Nuland, Nico A J, Loris, Remy, Zenkin, Nikolay
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.12.2013; Nature Publishing Group
• Subjects: 101/58; 101/6; 38/70; 631/326/41; 631/337/574; 631/535/878; 631/92/275; 82/16; 82/29; 82/80; Antitoxins; Bacteria; Bacterial physiology; Biochemical Engineering; Biochemistry; Bioorganic Chemistry; Cell Biology; Chemistry; Chemistry/Food Science; Enzymatic activity; Enzymes; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression Regulation, Bacterial; Guanosine Triphosphate - genetics; Guanosine Triphosphate - metabolism; Models, Molecular; Mutation; Nucleotidyltransferases - genetics; Nucleotidyltransferases - metabolism; Peptide Elongation Factor Tu - genetics; Peptide Elongation Factor Tu - metabolism; Phosphorylation; Physiology; Plasticity; Protein Folding; Proteins; RNA, Transfer - genetics; RNA, Transfer - metabolism; Substrates; Toxins
• ISSN: 1552-4450; 1552-4469
• DOI: 10.1038/nchembio.1364

The Doc-Phd toxin-antitoxin system inhibits bacterial translation via an unknown mechanism. Functional and structural analyses now show that Doc, which has an active site like AMPylating Fic proteins, actually works as a kinase, phosphorylating EF-Tu to block translation. Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc - phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.