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

Sam Mallinson, Delphine Dessaux, Sophie Barbe, Yannick Bomble

Research output: Contribution to journalArticlepeer-review

1 Scopus Citations


Redox cofactor utilization is one of the major barriers to the realization of efficient and cost-competitive cell-free biocatalysis, especially where multiple redox steps are concerned. The design of versatile, 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-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN) from Streptococcus mutans for enabling an NADH linked efficient cell-free glycolytic pathway with a net zero ATP usage. This rational design approach combines molecular dynamics simulations with a multistate computational design method that allowed us to consider different conformational states encountered along the GapN enzyme catalytic cycle. In particular, the cofactor flip, characteristic of this enzyme family and occurring before product hydrolysis, was taken into account to redesign the cofactor binding pocket for NAD+ utilization. While GapN exhibits only trace activity with NAD+, a ∼10,000-fold enhancement of this activity was achieved, corresponding to a recovery of ∼72% of the catalytic efficiency of the wild-type enzyme on NADP+, with a GapN enzyme harboring only 5 mutations.

Original languageAmerican English
Pages (from-to)11781-11797
Number of pages17
JournalACS Catalysis
Issue number17
StatePublished - 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

NREL Publication Number

  • NREL/JA-2700-86843


  • cell-free biocatalysis
  • cofactor affinity
  • computational protein design
  • glyceraldehyde-3-phosphate dehydrogenase
  • molecular dynamics
  • multistate enzyme design
  • protein engineering


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