[P005] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P03-371] Genetic code expansion technology reveals Sirt2-mediated reactivation of 2-hydroxyisobutyrylated G6PD

○Santhosh Paramasivam^1,2, Lior Cohen Shelach^2,3, Fang Wu^1,2, Eyal Arbely^1,2,3 (1. Department of Chemistry, Ben Gurion University (Israel), 2. The National Institute for Biotechnology in the Negev, Ben Gurion University (Israel), 3. Department of Life Sciences, Ben Gurion University (Israel))

Keywords:

Genetic code expansion,Synthetic biology,Post-translational modification,G6PD,2-hydroxyisobutyrylation,Molecular dynamics,Lysine deacylases

Lysine 2-hydroxyisobutyrylation (Hib) is a bulky acyl post-translational modification that is widespread among metabolic enzymes, yet its functional consequences remain poorly understood. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme for cytosolic NADPH production, is modified by Hib at multiple lysine residues; however, the impact of this modification on enzyme function has not been characterized. Here, using genetic code expansion, an innovative synthetic biology technique, we generated site-specifically 2-hydroxyisobutyrylated G6PD in mammalian cells by incorporating the non-canonical amino acid 2-hydroxyisobutyryl-lysine. Activity measurements revealed that Hib at active-site residues K171, K205, and K360, as well as at the dimer–dimer interface residue K288, completely abolished enzymatic activity. Interestingly, Hib of K386 at the monomer–monomer interface reduced G6PD activity by approximately 40% only in the presence of lysine deacylase inhibitors, suggesting that K386 Hib is a labile modification regulated by lysine deacylases. Consistently, we found that the deacylase Sirt2 can catalyse the hydrolysis of HibK386 in cells and in vitro, revealing a previously unidentified de-2-hydroxyisobutyrylase activity of Sirt2. In addition, we found that HibK386 renders G6PD monomeric, resulting in reduced thermal stability, increased proteolytic susceptibility, and rapid loss of activity over time. Molecular dynamics simulations further showed that HibK386 disrupts stabilizing salt-bridge interactions at the oligomerization interface and remodels the catalytic G6P and NADP⁺ binding sites, thereby providing a mechanistic basis for the lower stability and loss of activity. Together, our data show that G6PD can be inhibited by Hib of K386 that reduces enzyme activity and stability, while deacylation by Sirt2 reactivates the enzyme. These findings emphasize the advantages of genetic code expansion technology in studying site-specifically modified proteins, and highlight the role of reversible Hib in metabolic regulation and cellular redox homeostasis.