Structural and kinetic characterization of Escherichia coli TadA, the wobble-Specific tRNA deaminase

Jungwook Kim, Vladimir Malashkevich, Setu Roday, Michael Lisbin, Vern L. Schramm, Steven C. Almo

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

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 (kcat = 13 ± 1 min-1 and KM = 0.83 ± 0.22 μM). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (> 10 μM). 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.

Original languageEnglish (US)
Pages (from-to)6407-6416
Number of pages10
JournalBiochemistry
Volume45
Issue number20
DOIs
StatePublished - May 23 2006

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Transfer RNA
Escherichia coli
Catalysis
Codon
Kinetics
Zinc
Substrates
Enzymes
RNA
Polynucleotides
Inosine
Deamination
Adenosine Deaminase
Adenosine
Nucleotides
Dimers
Ions
Assays
Water

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural and kinetic characterization of Escherichia coli TadA, the wobble-Specific tRNA deaminase. / Kim, Jungwook; Malashkevich, Vladimir; Roday, Setu; Lisbin, Michael; Schramm, Vern L.; Almo, Steven C.

In: Biochemistry, Vol. 45, No. 20, 23.05.2006, p. 6407-6416.

Research output: Contribution to journalArticle

Kim, Jungwook ; Malashkevich, Vladimir ; Roday, Setu ; Lisbin, Michael ; Schramm, Vern L. ; Almo, Steven C. / Structural and kinetic characterization of Escherichia coli TadA, the wobble-Specific tRNA deaminase. In: Biochemistry. 2006 ; Vol. 45, No. 20. pp. 6407-6416.
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