Structural and Kinetic Characterization of Escherichia coli TadA, the Wobble-Specific tRNA Deaminase

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 (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.

The C-terminal end of the Trypanosoma brucei editing deaminase plays a critical role in tRNA binding

“…A similar K M value of 0.72 +/À 0.11 mM was observed for the partially purified (600-fold pure) native enzyme, suggesting that our recombinant enzyme is a fair representation of that natively expressed in T. brucei (data not shown). The observed K M is also within the range of the E. coli ADATa enzyme (K M = 0.83 mM) (Kim et al 2006). However, the E. coli enzyme shows a 10-fold better k cat (Kim et al 2006).…”

“…The observed K M is also within the range of the E. coli ADATa enzyme (K M = 0.83 mM) (Kim et al 2006). However, the E. coli enzyme shows a 10-fold better k cat (Kim et al 2006). The ADAT2 C-terD5 had a K M of 0.76 +/À 0.21 mM and a k cat of 0.14 +/À 0.05 min À1 (Table 2 ; Fig.…”

A Single Zinc Ion Is Sufficient for an Active Trypanosoma brucei tRNA Editing Deaminase

“…Again there are two viable possibilities. One possibility would be that Zn 2ϩ coordination is still standard, as in the case of bacterial ADATa (13)(14)(15)18), where each subunit binds one Zn 2ϩ ( Fig. 5A and supplemental Fig.…”

“…34 is generated by bacterial homodimeric ADATa with each monomer coordinating one Zn 2ϩ and containing all the additional amino acids needed for catalysis (13)(14)(15). Inosine has not been described in mitochondria, but a recent report showed that a bacteria-type ADATa enzyme plays key functions in inosine formation and thus translation in chloroplasts (16,17).…”

Mini Review From the Molecular Base to the Diagnostic Value of Adenosine Deaminase

“…This enzyme destabilizes double-stranded RNA through conversion of adenosine to inosine (10). Likewise, ADAT is a tRNA-specific ADA, changing tRNA to allow for a wobble base pairing (11). The total plasma ADA can be determined by measurement of the ammonia liberated from adenosine.…”