Electrostatic Engineering of Phosphoketolase Enhances Activity on Small Nonphosphorylated Sugars and Improves Cell-Free ATP Regeneration from Inexpensive C2-Substrates.

Phosphoketolases can convert nonphosphorylated sugars to the high-energy compound acetyl phosphate and the versatile metabolic precursor acetyl-CoA. However, their application is limited by low catalytic activity toward these substrates. Here, we report the rational engineering of the phosphoketolase from Bifidobacterium adolescentis (Bad.F6Pkt) to enhance its activity and affinity toward glycolaldehyde (GA) and d-erythrulose (ERU) through reorganization of the protein electric field. Guided by predicted induced side-chain pKa shifts, visualization of electrostatic potential difference maps alongside molecular modeling and sequence variation analyses, we identified mutations that could promote in situ ring opening of the predominant cyclic GA dimer. This approach yielded the GA-specific double mutant H142N:E153D, exhibiting a 10-fold improved affinity (KM = 4.4 mM) and slightly enhanced catalytic efficiency compared to the previously reported H142N variant. In addition, the H256Y:H260Y:H548Y variant comprising long-range electrostatic mutations exhibited a 3.8-fold higher catalytic efficiency toward ERU than the wild-type. The engineered enzymes were evaluated in cell-free enzyme cascades for ATP regeneration via acetyl phosphate formation. The H142N variant enabled efficient ATP regeneration from GA and ethylene glycol, whereas H142N:E153D exhibited reduced stability under synthesis conditions. Furthermore, coupling of a d-threose aldolase and isomerase with the PKT triple mutant enabled rapid GA conversion to C4 sugar intermediates, increasing the ATP yield.