Id |
Subject |
Object |
Predicate |
Lexical cue |
T1 |
0-83 |
Sentence |
denotes |
Identification of active-site residues in Bradyrhizobium japonicum malonamidase E2. |
T2 |
84-186 |
Sentence |
denotes |
Malonamidase (MA) E2 was previously purified and characterized from Bradyrhizobium japonicum USDA 110. |
T3 |
187-278 |
Sentence |
denotes |
The gene encoding this enzyme has been cloned, sequenced and expressed in Escherichia coli. |
T4 |
279-357 |
Sentence |
denotes |
The recombinant MAE2 was purified to homogeneity from the transformed E. coli. |
T5 |
358-474 |
Sentence |
denotes |
The biochemical properties of the recombinant enzyme are essentially identical to those from wild-type B. japonicum. |
T6 |
475-611 |
Sentence |
denotes |
A database search showed that the MAE2 protein has a high sequence similarity with the common signature sequences of the amidase family. |
T7 |
612-724 |
Sentence |
denotes |
The only exception is that the aspartic residue in these signature sequences is replaced by a glutamine residue. |
T8 |
725-895 |
Sentence |
denotes |
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. |
T9 |
896-1016 |
Sentence |
denotes |
Gln(195), Ser(199), Cys(207) and Lys(213) of the common signature sequences were selected for site-directed mutagenesis. |
T10 |
1017-1199 |
Sentence |
denotes |
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. |
T11 |
1200-1378 |
Sentence |
denotes |
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. |
T12 |
1379-1439 |
Sentence |
denotes |
380-fold) in k(cat) value, whereas C207A kept full activity. |
T13 |
1440-1596 |
Sentence |
denotes |
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. |