title: Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis.
abstract: Despite intensive research, the cyclodehydratase responsible for azoline biogenesis in thiazole/oxazole-modified microcin (TOMM) natural products remains enigmatic. The collaboration of two proteins, C and D, is required for cyclodehydration. The C protein is homologous to E1 ubiquitin-activating enzymes, whereas the D protein is within the YcaO superfamily. Recent studies have demonstrated that TOMM YcaOs phosphorylate amide carbonyl oxygens to facilitate azoline formation. Here we report the X-ray crystal structure of an uncharacterized YcaO from Escherichia coli (Ec-YcaO). Ec-YcaO harbors an unprecedented fold and ATP-binding motif. This motif is conserved among TOMM YcaOs and is required for cyclodehydration. Furthermore, we demonstrate that the C protein regulates substrate binding and catalysis and that the proline-rich C terminus of the D protein is involved in C protein recognition and catalysis. This study identifies the YcaO active site and paves the way for the characterization of the numerous YcaO domains not associated with TOMM biosynthesis.
authors.affiliation: 1] Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [2] Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [3].
authors.forename: Kyle L
authors.initials: KL
authors.lastname: Dunbar
authors.affiliation: 1] Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [2].
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Chekan
authors.affiliation: 1] Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [2] Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Courtney L
authors.initials: CL
authors.lastname: Cox
authors.affiliation: 1] Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [2] Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Burkhart
authors.affiliation: 1] Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [2] Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [3] Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Satish K
authors.initials: SK
authors.lastname: Nair
authors.affiliation: 1] Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [2] Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. [3] Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Mitchell
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D020816:Amino Acid Motifs; D001430:Bacteriocins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000737:chemistry / Q000378:metabolism; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001430:Bacteriocins; D029968:Escherichia coli Proteins; D010080:Oxazoles; D013844:Thiazoles; C020411:microcin; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D006836:Hydro-Lyases; D044764:Ubiquitin-Activating Enzymes