title: Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea.
abstract: 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 that 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 autoregulation. Cysteine residues, distinct from the canonical E1 'active site' cysteine, were found important in UbaA function supporting a model that this noncanonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
authors.forename: Nathaniel L
authors.initials: NL
authors.lastname: Hepowit
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA.
authors.forename: Ian Mitchelle S
authors.initials: IM
authors.lastname: de Vera
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Cao
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
authors.forename: Xian
authors.initials: X
authors.lastname: Fu
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
authors.forename: Yifei
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
authors.forename: Sivakumar
authors.initials: S
authors.lastname: Uthandi
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
authors.forename: Nikita E
authors.initials: NE
authors.lastname: Chavarria
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Englert
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Su
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.|Department of Chemistry, Yale University, New Haven, CT, USA.
authors.forename: Dieter
authors.initials: D
authors.lastname: Sӧll
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Kojetin
authors.affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA. jmaupin@ufl.edu.|Genetics Institute, University of Florida, Gainesville, FL, USA. jmaupin@ufl.edu.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Maupin-Furlow
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000502:physiology; D019622:Haloferax volcanii / Q000201:enzymology; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D000072417:Protein Domains; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism*; D013816:Thermodynamics; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D019843:Archaeal Proteins; D008024:Ligands; D025841:Small Ubiquitin-Related Modifier Proteins; D013438:Sulfhydryl Compounds; D013455:Sulfur; D000255:Adenosine Triphosphate; D044764:Ubiquitin-Activating Enzymes; D003545:Cysteine
keywords: molybdopterin biosynthesis; posttranslational modification; proteasomes; sulfur relay; tRNA thiolation