Endogenous microenvironmental engineering through targeted alteration of salt bridge network can effectively regulate enzymatic pH adaptation

For a multi-enzyme cascade system, reconciling all enzymes under optimal catalytic conditions is key to improving pathway throughput. However, changing the enzymatic pH adaptation has long been a difficult challenge. To develop an effective and versatile method to alter enzymatic pH adaptation, we investigated members of the PfkB family. We found that one enzyme of this family has markedly different environmental pH requirements when phosphorylating dissimilar substrates, called pH promiscuity. Experiments proved that such promiscuity is a property of the enzyme itself rather than caused by the diversity of physicochemical properties of the substrate. Ancestral sequence reconstruction showed that the ancestral PfkB family enzymes also possessed pH promiscuity. We hypothesized that the pH of the enzyme might be an evolutionarily relevant feature and those regions directly affecting the pH could be found in the amino acid sequence. Sequences analysis revealed a region that was highly variable across genera but relatively conserved within the same genus. The results showed that direct changes in a few amino acids in this region could significantly alter the pH adaptation and optimal pH of the engineered enzymes without affecting the function. Structural analysis indicated that the optimal pH value and pH adaptation of the enzyme may be influenced by changes in the salt bridge network in the pH-affecting region. This leads to the proposal of engineering enzymatic pH adaptation by changing the salt bridge network around the active site. Finally, based on the same strategy, we successfully modified the pH adaptation of an enzyme not related to the PfkB function. Our work should provide an ingenious strategy for the engineering of enzymatic pH adaptation.