Partial Consensus Design and Enhancement of Protein Function by Secondary-Structure-Guided Consensus Mutations

Consensus design (CD) is a representative sequence-based protein design method that enables the design of highly functional proteins by analyzing vast amounts of protein sequence data. This study proposes a partial consensus design (PCD) of a protein as a derivative approach of CD. The method replaces the target protein sequence with a consensus sequence in a secondary-structure-dependent manner (i.e., regionally dependent and divided into α-helix, β-sheet, and loop regions). In this study, we generated several artificial partial consensus l-threonine 3-dehydrogenases (PcTDHs) by PCD using the TDH from Cupriavidus necator (CnTDH) as a target protein. Structural and functional analysis of PcTDHs suggested that thermostability would be independently improved when consensus mutations are introduced into the loop region of TDHs. On the other hand, enzyme kinetic parameters (k cat/K m) and average productivity would be synergistically enhanced by changing the combination of the mutationsreplacement of one region of CnTDH with a consensus sequence provided only negative effects, but the negative effects were nullified when the two regions were replaced simultaneously. Taken together, we propose the hypothesis that there are protein regions that encode individual protein properties, such as thermostability and activity, and that the introduction of consensus mutations into these regions could additively or synergistically modify their functions.