A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst.
tRNA pseudouridine synthase I catalyzes the conversion of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon loop of many tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins. Fluorouracil-substituted tRNA caused a time-dependent inactivation of pseudouridine synthase I and formed a covalent complex with the enzyme that involved the FUMP at position 39. Asp60, conserved in all known and putative pseudouridine synthases, was mutated to amino acids with diverse side chains. All Asp60 mutants bound tRNA but were catalytically inactive and failed to form covalent complexes with fluorouracil-substituted tRNA. We conclude that the conserved Asp60 is essential for pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles.
Huang
L
L
Department of Biochemistry, University of California, San Francisco 94143-0448, USA.
Pookanjanatavip
M
M
Gu
X
X
Santi
D V
DV
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli; D005472:Fluorouracil / Q000494:pharmacology; D005822:Genetic Vectors / Q000378:metabolism; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010761:Phosphorus Radioisotopes; D012349:RNA, Transfer, Asp / Q000235:genetics / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000494:pharmacology; D014498:Uracil / Q000494:pharmacology
D010761:Phosphorus Radioisotopes; D012349:RNA, Transfer, Asp; D012360:RNA, Transfer, Phe; D001224:Aspartic Acid; D014498:Uracil; D019751:Intramolecular Transferases; C023199:tRNA-pseudouridine synthase I; D005472:Fluorouracil