Structural basis of the transhydrogenase and allosteric regulation mechanisms of Ser33, the first rate‐limiting enzyme of the de novo L-serine biosynthesis pathway#

Authors#

Sebastian Perrone, Javier O. Cifuente, Leonardo Mastrella, Alberto Marina, Beatriz Trastoy, Julia Becker-Kettern, Jean-François Conrotte, Marcelo E. Guerin, Carole Linster

Abstract#

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 the 3-phosphoglycerate dehydrogenase (PHGDH) into 3-phosphohydroxypyruvate (PHP). PHGDH expression levels are associated with cancer progression, dissemination, and metastasis. PHGDH deficiencies lead to severe neurometabolic diseases. 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. In combination with extensive alanine scanning mutagenesis, enzymatic assays and biophysical methods, we unveil the molecular basis of the substrate recognition, transhydrogenase and allosteric inhibition mechanisms of Ser33.