title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation.
abstract: Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB's catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.
authors.affiliation: Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge AB T1K 3M4, Canada.
authors.forename: Jenna
authors.initials: J
authors.lastname: Friedt
authors.forename: Fern M V
authors.initials: FM
authors.lastname: Leavens
authors.forename: Evan
authors.initials: E
authors.lastname: Mercier
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Wieden
authors.forename: Ute
authors.initials: U
authors.lastname: Kothe
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D055162:Biocatalysis; D020134:Catalytic Domain; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011560:Pseudouridine / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D055672:Static Electricity
chemical_list: D011560:Pseudouridine; D001224:Aspartic Acid; D012343:RNA, Transfer; D001120:Arginine; D019751:Intramolecular Transferases; C096995:pseudouridine synthases