Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase.
The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k(cat) = 13 +/- 1 min(-)(1) and K(M) = 0.83 +/- 0.22 microM). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (>10 microM). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.
Kim
Jungwook
J
Department of Biochemistry, Center for Synchrotron Biosciences, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Malashkevich
Vladimir
V
Roday
Setu
S
Lisbin
Michael
M
Schramm
Vern L
VL
Almo
Steven C
SC
D000243:Adenosine Deaminase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D003564:Cytidine Deaminase / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012343:RNA, Transfer; D005969:Glutamate Dehydrogenase; D000243:Adenosine Deaminase; C511772:TadA protein, E coli; D003564:Cytidine Deaminase