Characterization of the catalytic disulfide bond in E. coli 4-thiouridine synthetase to elucidate its functional quaternary structure.
4-Thiouridine at position 8 in prokaryotic tRNA serves as a photosensor for near-UV light, and the posttranscriptional conversion of uridine to 4-thiouridine is catalyzed by the 4-thiouridine synthetases (s(4) US, also named ThiI), which fall into two classes that differ in the presence of a C-terminal rhodanese homology domain. A cysteine residue in this domain first bears a persulfide group and then forms a disulfide bond with a cysteine residue that is conserved in both classes of s(4) US. Recent crystal structures suggest that s(4) US dimerizes in the presence of RNA substrate with domains from each subunit contributing to the binding and reaction of one RNA molecule, which raises the question of whether the catalytic disulfide bond in the longer class of s(4) US is formed within or between subunits. The E. coli enzyme is the best-characterized member of the longer class of s(4) US, and it was examined after quantitative installation of the disulfide bond during a single catalytic turnover. Gel electrophoresis and proteolysis/MALDI-MS results strongly imply that the disulfide bond forms within a single subunit, which provides a vital constraint for the structural modeling of the class of s(4) US with an appended rhodanese homology domain and the design and interpretation of experiments to probe the dynamics of the domains during catalysis.
Department of Chemistry, University of Louisville, Louisville, Kentucky, 40205.
Govardhan Reddy
GR
Veerareddygari
Department of Chemistry, University of Louisville, Louisville, Kentucky, 40205.
Thomas C
TC
Klusman
Department of Chemistry, University of Louisville, Louisville, Kentucky, 40205.
Eugene G
EG
Mueller
D002384:Catalysis; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D000072417:Protein Domains; D020836:Protein Structure, Quaternary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013891:Thiouridine / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D029968:Escherichia coli Proteins; D013891:Thiouridine; D008025:Ligases
4-thiouridine; RNA modification; ThiI; catalytic disulfide; enzyme mechanism; persulfide; rhodanese