TY - JOUR
T1 - Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis
AU - Dunbar, Kyle L.
AU - Chekan, Jonathan R.
AU - Cox, Courtney L.
AU - Burkhart, Brandon J.
AU - Nair, Satish K.
AU - Mitchell, Douglas A.
N1 - Funding Information:
We are grateful to C. Deane and K. Taylor for the generation of select BalhD mutants and J. Melby for assistance with collecting MS/MS data. This work was supported by the US National Institutes of Health (NIH) (1R01 GM097142 to D.A.M., 1R01 GM102602 to S.K.N. and 2T32 GM070421 to K.L.D., B.J.B. and J.R.C.). Additional support was from the Harold R. Snyder Fellowship (University of Illinois at Urbana-Champaign (UIUC) Department of Chemistry to K.L.D.), the Robert C. and Carolyn J. Springborn Endowment (UIUC Department of Chemistry to B.J.B.), the National Science Foundation Graduate Research Fellowship (DGE-1144245 to B.J.B.) and the University of Illinois Distinguished Fellowship (UIUC Graduate College to J.R.C.) The Bruker UltrafleXtreme MALDI TOF/TOF mass spectrometer was purchased in part with a grant from the NIH–National Center for Research Resources (S10 RR027109 A).
Publisher Copyright:
© 2014 Nature America, Inc. All rights reserved.
PY - 2014/10/1
Y1 - 2014/10/1
N2 - 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.
AB - 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.
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U2 - 10.1038/nchembio.1608
DO - 10.1038/nchembio.1608
M3 - Article
C2 - 25129028
AN - SCOPUS:84922019867
SN - 1552-4450
VL - 10
SP - 823
EP - 829
JO - Nature Chemical Biology
JF - Nature Chemical Biology
IS - 10
ER -