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Conference Papers Year : 2022

Computational design of the cofactor specificity of GAPN enzyme for cell-free biocatalysis


The use of enzymes for the sustainable and environmentally friendly production of biochemicals is continuously expanding and allows to bypass some of the drawbacks of the chemical production. However, these bioprocesses currently rely mainly on microbial fermentation, whose effectiveness may be limited. An appealing alternative is the use of in vitro multi-enzymatic pathways for cell-free bioproduction that may enable higher productivity while reducing production costs. Furthermore, the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) catalyzes the transformation of glyceraldehyde-3-phosphate into 3-phosphoglycerate and can hence be used as a shortcut into the glycolysis pathway. It is thus a crucial enzyme that can be involved in the regulation of ATP concentration in cell-free biocatalysis processes. However, GAPN is strictly dependent on the NADP+ cofactor [1], which prevents its use for NAD+ cofactor-dependent pathways or those based on the use of more stable and less expensive biomimetic cofactors. Therefore, we aimed to engineer this enzyme to be active with the NAD+ cofactor. In order to alter the cofactor specificity of GAPN, we developed and implemented a new rational enzyme engineering strategy based on computational protein design (CPD) methods [2-3]. These CPD approaches rely on artificial intelligence algorithms to predict mutant sequences for the design target while simultaneously considering several conformational states of the enzyme representing different steps along the catalysis reaction. We combined these CPD methods with all-atom molecular dynamics simulations and binding free energy calculations, and ranked the GAPN mutants according to cofactor binding free energy and geometric features required for catalysis. This overall rational design strategy led to the prediction and the selection of mutants for experimental testing. As a result, although the wild-type enzyme was not able to use the NAD+, we succeeded to generate several mutants exhibiting an activity with this cofactor, reaching up to about 72% of the activity of the wild-type enzyme with NADP+. References [1] D.A., Boyd; D.G., Cvitkovitch; I.R., Hamilton. J Bacteriol, 1995, 177(10), 2622–2627. [2] J., Vucinic; D., Simoncini; M., Ruffini; S., Barbe; T., Schiex. Bioinformatics, 2020, 36(1), 122-130. [3] S., Traoré; D., Allouche; I., André; S., de Givry; G., Katsirelos; T., Schiex; S., Barbe. Bioinformatics, 2013, 29(17), 2129–2136.
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hal-04223580 , version 1 (30-09-2023)


  • HAL Id : hal-04223580 , version 1


Delphine Dessaux, Sam Mallinson, Yannick Bomble, Sophie Barbe. Computational design of the cofactor specificity of GAPN enzyme for cell-free biocatalysis. 29ième colloque du Club Biocatalyse en Synthèse Organique, CBSO, Jun 2022, En ligne, France. ⟨hal-04223580⟩
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