One lysine, two acyl marks: 2-hydroxyisobutyrylation and acetylation of G6PD differ in outcome and regulation

Lysine 2-hydroxyisobutyrylation is a widespread post-translational modification enriched in enzymes of central carbon metabolism, yet its site-specific biochemical consequences remain poorly defined and usually inferred from amino acid substitutions or heterogeneously modified protein preparations. Using genetic code expansion technology, we generated homogeneous human glucose-6-phosphate dehydrogenase (G6PD) carrying an authentic 2-hydroxyisobutyryl group at nine individual lysine residues. 2-hydroxyisobutyrylation affected G6PD activity in a strongly site-dependent manner: modification within or adjacent to the catalytic pocket (K171, K205, K360), or at the tetramer interface (K288), abolished activity, whereas modification of K386, near the structural NADP+ binding site, reduced catalytic efficiency and destabilized the enzyme. HibK386 G6PD was less oligomeric, while inactive HibK288 G6PD formed stable oligomers, suggesting that in HibK288 G6PD, assembly and catalysis are separable. Molecular dynamics simulations indicated that 2-hydroxyisobutyrylation of K386 reorients the side chain toward the solvent and weakens hydrogen bonding and salt-bridge across the tetramer interface. K386 is also subject to acetylation, allowing the comparison between two acyl groups at one residue; acetylation destabilized G6PD without substantially impairing catalysis, whereas 2-hydroxyisobutyrylation did both, and the two marks were differentially recognized by their erasers. SIRT2 removed both and restored the activity of the purified enzyme, whereas SIRT1 readily deacetylated AcK386 but was a poor de-2-hydroxyisobutyrylase of this position. SIRT2 also de-2-hydroxyisobutyrylated HibK288 G6PD and PKM2 HibK62, but not PKM2 HibK115 or HibK305. Together, these findings identify site-specific 2-hydroxyisobutyrylation as a potent inhibitory modification of G6PD, demonstrate that chemically distinct acylations at the same site can exert different functional effects, and establish deacylase specificity as a function of both modification chemistry and residue context.