The Structural Permissiveness of Triosephosphate Isomerase (TpiA) of Escherichia coli.

Triosephosphate isomerase (TpiA) is widely regarded as an example of an optimally evolved enzyme due to its essential role in biological systems, its structural conservation, and its near-perfect kinetic parameters. In this study, we investigated the structural robustness of the archetypal TpiA variant from Escherichia coli using an in vitro 5-amino acid linker scanning method. The resulting library was introduced into a tpiA mutant strain for functional complementation. From this library, 16 TpiA variants that were phenotypically indistinguishable from the wild-type enzyme were selected for further analysis. Although all variants retained enzymatic activities within the wild-type range, several insertions were found in highly structured protein domains where the linker was expected to cause significant structural perturbations. Despite these potentially disruptive additions, the enzymes maintained their activity even when expressed in a dnaK mutant, suggesting that chaperones did not compensate for structural abnormalities in vivo. Additionally, when these mutant TpiA variants were produced using an in vitro transcription/translation system, they exhibited enzymatic activity comparable to, and in some cases exceeding, that of the non-mutated enzyme. AlphaFold2 exposed that insertions reconstructed the local architecture of the nearby amino acid sequences. The evolutionary implications of this remarkable structural resilience are discussed.