Catalytic activity of the N-terminal domain of Escherichia coli asparagine synthetase B can be reengineered by single-point mutation

The development of mechanistic strategies for the modification of enzyme function is of considerable biotechnological interest. We now report that replacement of the catalytically important residue Asn-74 by aspartic acid (N74D) in the N-terminal domain of Escherichia coli asparagine synthetase B (AS-B) confers nitrile hydratase activity upon the mutant enzyme. Furthermore, while wild type AS-B can efficiently catalyze the hydrolysis of glutamine to glutamate, the N74D AS-B mutant exhibits very low glutaminase activity. These results are consistent with similar experiments on papain, supporting the hypothesis that mutation of a critical active site residue to affect the partitioning of an intermediate common to multiple reaction mechanisms may represent an approach by which enzymes can be obtained with different catalytic function. Our experiments also provide the first direct chemical evidence for a close mechanistic relationship between papain, a thiol protease, and AS-B, a class II Ntn amidotransferase. These enzymes are not likely to have arisen by evolution from a common ancestral protein.