Identification of active-site residues in Bradyrhizobium japonicum malonamidase E2.
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. Gln(195), Ser(199), Cys(207) and Lys(213) 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 k(cat) 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, Lys(213), in the signature sequences, also displayed significant reductions (approx. 380-fold) in k(cat) value, whereas C207A kept full activity. These results suggest that MAE2 may catalyse hydrolysis of malonamate by a novel catalytic mechanism, in which Gln(195), Ser(199) and Lys(213) are involved.
Koo
H M
HM
Department of Biochemistry, College of Science, Bioproducts Research Center, Yonsei University, Seoul 120-749, Korea.
Choi
S O
SO
Kim
H M
HM
Kim
Y S
YS
D000581:Amidohydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D020369:Bradyrhizobium / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008314:Malonates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism
D001426:Bacterial Proteins; D008314:Malonates; D005973:Glutamine; C013098:malonamic acid; D012694:Serine; D000581:Amidohydrolases; C064615:malonamidase E2 protein, Bradyrhizobium japonicum; D008239:Lysine