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Phylogenetic spread of sequence data affects fitness of consensus enzymes: Insights from triosephosphate isomerase

The concept of consensus in multiple sequence alignments (MSAs) has been used to design and engineer proteins previously with some success. However, consensus design implicitly assumes that all amino acid positions function independently, whereas in reality, the amino acids in a protein interact with each other and work cooperatively to produce the optimum structure required for its function. Correlation analysis is a tool that can capture the effect of such interactions. In a previously published study, we made consensus variants of the triosephosphate isomerase (TIM) protein using MSAs that included sequences form both prokaryotic and eukaryotic organisms. These variants were not completely native‐like and were also surprisingly different from each other in terms of oligomeric state, structural dynamics, and activity. Extensive correlation analysis of the TIM database has revealed some clues about factors leading to the unusual behavior of the previously constructed consensus proteins. Among other things, we have found that the more ill‐behaved consensus mutant had more broken correlations than the better‐behaved consensus variant. Moreover, we report three correlation and phylogeny‐based consensus variants of TIM. These variants were more native‐like than the previous consensus mutants and considerably more stable than a wild‐type TIM from a mesophilic organism. This study highlights the importance of choosing the appropriate diversity of MSA for consensus analysis and provides information that can be used to engineer stable enzymes.