ImprovingEscherichia coli Alkaline Phosphatase Efficacy by Additional Mutations inside and outside the Catalytic Pocket

We describe a strategy that allowed us to confer on a bacterial (E. coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability First, we identified mutations, at positions other than those Occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation. We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being outside. Second, the gene encoding the inactive mutant was submitted to random mutagenesis. Enzyme activity was restored upon the single mutation D330N, at a position that is 12 Angstrom away from the center of the catalytic pocket. Third, this mutation was combined with other mutations previously reported to increase AP activity slightly in the presence of magnesium. As a result, at pH 10.0 the phosphatase activity of both mutants D330N/D153H and D330N/D153G was 17-fold higher than that of the wild, type AP. Strikingly, although the two individual mutations D153H and D153G destabilize the enzyme, the, double mutant D330N/D153G remained highly stable (T-m = 87 degreesC). Moreover, when combining the phosphatase and transferase activities, the catalytic activity of the mutant D330N/D153G increased 40-fold (k(cat) = 3200 s(-1)) relative to that of the wild-type enzyme (k(cat) = 80 s(-1)). Due, to the simultaneous increase in K-m, the resulting k(cat)/k(m) value was only increased by a factor of two. Therefore, a single mutation occurring outside a catalytic pocket can dramatically control not only the activity of an enzyme, but also. its thermostability. Preliminary crystallographic data of a covalent D330N/D153G, enzyme-phosphate complex show that the phosphate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant, previously reported.