Molecular mechanisms of transhydrogenase activity and allosteric regulation in eukaryotic type II PHGDH Ser33

L-serine is a critical structural constituent of proteins and membrane phospholipids, playing major roles in cell signaling, metabolism and development. L-Serine is synthesized through a conserved de novo pathway starting from the glycolytic intermediate 3-phosphoglycerate (PGA), being oxidized by 3-phosphoglycerate dehydrogenase (PHGDH) into 3-phosphohydroxypyruvate (PHP). In certain organisms, PHGDH operates as a transhydrogenase using α-ketoglutarate rather than NAD⁺ as the final electron acceptor and producing both PHP and D-2-hydroxyglutarate (2HG). We provide high-resolution X-ray crystal structures of the transhydrogenase Ser33 from Saccharomyces cerevisiae, in complex with the cofactor NADH, and with PGA, PHP, 2HG and the negative allosteric regulator L-serine. Combining extensive alanine scanning mutagenesis, enzyme activity assays and kinetics, molecular dynamics simulations, biophysical methods, and phylogenetic analysis, we establish the molecular basis of substrate recognition, transhydrogenase activity, and allosteric inhibition mechanisms, including the role of an N-terminal extension in the regulation of eukaryotic Type II PHGDHs.