Improving Escherichia 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) a lkaline phosphatase (AP) the high catalytic activity of the mammalian enzym e while maintaining its high thermostability First, we identified mutations , at positions other than those Occupied by essential catalytic residues, w hich inactivate the bacterial enzyme without destroying its overall conform ation. We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being o utside. Second, the gene encoding the inactive mutant was submitted to rand om mutagenesis. Enzyme activity was restored upon the single mutation D330N , at a position that is 12 Angstrom away from the center of the catalytic p ocket. Third, this mutation was combined with other mutations previously re ported 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 enzy me, the, double mutant D330N/D153G remained highly stable (T-m = 87 degrees C). Moreover, when combining the phosphatase and transferase activities, th e 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)). D ue, to the simultaneous increase in K-m, the resulting k(cat)/k(m) value wa s 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 d ata of a covalent D330N/D153G, enzyme-phosphate complex show that the phosp hate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant, previously reported.