Identification of active-site residues in Bradyrhizobium japonicum malonamidase E2

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Abstract
Malonamidase (MA) E2 was previously purified and characterized from Bradyrhizobium japonicum USDA 110. The gene encoding this enzyme has been cloned, sequenced and expressed in Escherichia coli. The recombinant MAE2 was purified to homogeneity from the transformed E. coli. The biochemical properties of the recombinant enzyme are essentially identical to those from wild-type B. japonicum. A database search showed that the MAE2 protein has a high sequence similarity with the common signature sequences of the amidase family. The only exception is that the aspartic residue in these signature sequences is replaced by a glutamine residue. In order to identify amino acid residues essential for enzyme activity, a series of site-directed mutagenesis studies and steady-state kinetic experiments were performed. Gln195, Ser199, Cys207 and Lys213 of the common signature sequences were selected for site-directed mutagenesis. Among the mutants, Q195D, Q195E and S199C showed less than 0.02% of the kcat value of the wild-type enzyme, and S199A, Q195L and Q195N exhibited no detectable catalytic activities. Mutants (K213L, K213R and K213H) obtained by replacement of the only conserved basic residue, Lys213, in the signature sequences, also displayed significant reductions (approx. 380-fold) in kcat value, whereas C207A kept full activity. These results suggest that MAE2 may catalyse hydrolysis of malonamate by a novel catalytic mechanism, in which Gln195, Ser199 and Lys213 are involved.