Effects of insertions and deletions in a beta-bulge region of Escherichia coli dihydrofolate reductase

The role of a beta-bulge in Escherichia coli dihydrofolate reductase (DHFR)has been explored by a series of insertion and deletion mutations.Insertion of a seven amino acid sequence from a structurally equivalent'beta-blowout' sequence from human DHFR destabilizes E. coli DHFR by 3.6kcal/mol and decreases catalytic efficiency (kcat/K(m)) 34- fold. Deletionof F137, delta 137, the looped out residue in the bulge, also destabilizesE. coli DHFR by 2.8 kcal/mol but only decreases catalytic efficiencythreefold. Concurrent deletion of F137 and mutation of, V136 to proline totry and maintain the strand twist associated with the beta-bulge decreasesprotein stability by 3.4 kcal/mol and decreases catalytic efficiency84-fold. These insertion/deletion mutations were also constructed in a D27SDHFR background. The D27S mutation has been described previously andproposed to remove the catalytic acid from the active site. The delta 137mutation partially suppresses the effect of the D27S mutation as itdecreases the K(m) for substrate, dihydrofolate, twofold. Non-additiveeffects are observed for the insertion/deletion mutations in wild-typeversus D27S DHFR backgrounds, consistent with structural changes.