Modular mutagenesis of a TIM-barrel enzyme: the crystal structure of a chimeric E. coli TIM having the eighth beta alpha-unit replaced by the equivalent unit of chicken TIM

The crystal structure of a hybrid Escherichia coli triosephosphate isomerase (TIM) has been determined at 2.8 A resolution. The hybrid TIM (ETIM8CHI) was constructed by replacing the eighth beta alpha-unit of E. coli TIM with the equivalent unit of chicken TIM. This replacement involves 10 sequence changes. One of the changes concerns the mutation of a buried alanine (Ala232 in strand 8) into a phenylalanine. The ETIM8CHI structure shows that the A232F sequence change can be incorporated by a side-chain rotation of Phe224 (in helix 7). No cavities or strained dihedrals are observed in ETIM8CHI in the region near position 232, which is in agreement with the observation that ETIM8CHI and E.coli TIM have similar stabilities. The largest CA (C-alpha atom) movements, approximately 3 A, are seen for the C-terminal end of helix 8 (associated with the outward rotation of Phe224) and for the residues in the loop after helix 1 (associated with sequence changes in helix 8). From the structure it is not clear why the kcat of ETIM8CHI is 10 times lower than in wild type E.coli TIM.