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Title: Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea

Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of auto-regulation. Cysteine residues, distinct from the canonical E1 ‘active site’ cysteine, were found important in UbaA function supporting a model that thismore » non-canonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.« less
Authors:
 [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [3] ;  [3] ;  [3] ;  [2] ;  [1]
  1. Univ. of Florida, Gainesville, FL (United States)
  2. Scripps Research Inst., Jupiter, FL (United States)
  3. Yale Univ., New Haven, CT (United States)
Publication Date:
Grant/Contract Number:
FG02-05ER15650
Type:
Accepted Manuscript
Journal Name:
Federation of European Biochemical Societies (FEBS) Journal
Additional Journal Information:
Journal Name: Federation of European Biochemical Societies (FEBS) Journal; Journal Volume: 283; Journal Issue: 19; Journal ID: ISSN 1742-464X
Publisher:
Federation of European Biochemical Societies
Research Org:
Univ. of Florida, Gainesville, FL (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1463111
Alternate Identifier(s):
OSTI ID: 1401001

Hepowit, Nathaniel L., de Vera, Ian Mitchelle S., Cao, Shiyun, Fu, Xian, Wu, Yifei, Uthandi, Sivakumar, Chavarria, Nikita E., Englert, Markus, Su, Dan, Sӧll, Dieter, Kojetin, Douglas J., and Maupin-Furlow, Julie A.. Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea. United States: N. p., Web. doi:10.1111/febs.13819.
Hepowit, Nathaniel L., de Vera, Ian Mitchelle S., Cao, Shiyun, Fu, Xian, Wu, Yifei, Uthandi, Sivakumar, Chavarria, Nikita E., Englert, Markus, Su, Dan, Sӧll, Dieter, Kojetin, Douglas J., & Maupin-Furlow, Julie A.. Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea. United States. doi:10.1111/febs.13819.
Hepowit, Nathaniel L., de Vera, Ian Mitchelle S., Cao, Shiyun, Fu, Xian, Wu, Yifei, Uthandi, Sivakumar, Chavarria, Nikita E., Englert, Markus, Su, Dan, Sӧll, Dieter, Kojetin, Douglas J., and Maupin-Furlow, Julie A.. 2016. "Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea". United States. doi:10.1111/febs.13819. https://www.osti.gov/servlets/purl/1463111.
@article{osti_1463111,
title = {Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea},
author = {Hepowit, Nathaniel L. and de Vera, Ian Mitchelle S. and Cao, Shiyun and Fu, Xian and Wu, Yifei and Uthandi, Sivakumar and Chavarria, Nikita E. and Englert, Markus and Su, Dan and Sӧll, Dieter and Kojetin, Douglas J. and Maupin-Furlow, Julie A.},
abstractNote = {Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of auto-regulation. Cysteine residues, distinct from the canonical E1 ‘active site’ cysteine, were found important in UbaA function supporting a model that this non-canonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.},
doi = {10.1111/febs.13819},
journal = {Federation of European Biochemical Societies (FEBS) Journal},
number = 19,
volume = 283,
place = {United States},
year = {2016},
month = {10}
}