Computer-Aided Engineering of a Non-Phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase to Enable Cell-Free Biocatalysis

Redox cofactor utilization is one of the major barriersto therealization of efficient and cost-competitive cell-free biocatalysis,especially where multiple redox steps are concerned. The design ofversatile, cofactor balanced modules for canonical metabolic pathways,such as glycolysis, is one route to overcoming such barriers. Here,we set up a computer-aided design framework to engineer the non-phosphorylatingglyceraldehyde-3-phosphate dehydrogenase (GapN) from Streptococcus mutans for enabling an NADH linkedefficient cell-free glycolytic pathway with a net zero ATP usage.This rational design approach combines molecular dynamics simulationswith a multistate computational design method that allowed us to considerdifferent conformational states encountered along the GapN enzymecatalytic cycle. In particular, the cofactor flip, characteristicof this enzyme family and occurring before product hydrolysis, wastaken into account to redesign the cofactor binding pocket for NAD(+) utilization. While GapN exhibits only trace activity withNAD(+), a & SIM;10,000-fold enhancement of this activitywas achieved, corresponding to a recovery of & SIM;72% of the catalyticefficiency of the wild-type enzyme on NADP(+), with a GapNenzyme harboring only 5 mutations.