Identification of active-site residues in Bradyrhizobium japonicum malonamidase E2

Malonamidase (MA) E2 was previously purified and characterized from Bradyrh izobium japonicum USDA 110. The gene encoding this enzyme has been cloned, sequenced and expressed in Escherichia coli. The recombinant MAE2 was purif ied to homogeneity from the transformed E. coli. The biochemical properties of the recombinant enzyme are essentially identical to those from wild-typ e B. japonicum. A database search showed that the MAE2 protein has a high s equence similarity with the common signature sequences of the amidase famil y. The only exception is that the aspartic residue in these signature seque nces is replaced by a glutamine residue. In order to identify amino acid re sidues essential for enzyme activity, a series of site-directed mutagenesis studies and steady-state kinetic experiments were performed. Gln(195), Ser (199), Cys(207) and Lys(213) of the common signature sequences were selecte d for site-directed mutagenesis. Among the mutants, Q195D, Q195E and S199C showed less than 0.02% of the k(cat) value of the wild-type enzyme, and S19 9A, Q195L and Q195N exhibited no detectable catalytic activities. Mutants ( K213L, K213R and K213H) obtained by replacement of the only conserved basic residue, Lys(213), in the signature sequences, also displayed significant reductions (approx. 380-fold) in K-cat value, whereas C207A kept Full activ ity. These results suggest that MAE2 may catalyse hydrolysis of malonamate by a novel catalytic mechanism, in which Gln(195), Ser(199) and Lys(213) ar e involved.