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D‐3‐phosphoglycerate dehydrogenase (PHGDH) catalyzes the NAD+‐dependent conversion of D‐3‐phospho‐glycerate to 3‐phosphohydroxypyruvate, the first step in the phosphorylated pathway for L‐serine (L‐Ser) biosynthesis. L‐Ser plays different relevant metabolic roles in eukaryotic cells: alterations in L‐Ser metabolism have been linked to serious neurological disorders. The human PHGDH (hPHGDH), showing a homotetrameric state in solution, is made of four domains, among which there are two regulatory domains at the C‐terminus: the aspartate kinase‐chorismate mutase‐tyrA prephenate dehydrogenase (ACT) and allosteric substrate‐binding (ASB) domains. The structure of hPHGDH was solved only for a truncated, dimeric form harboring the N‐terminal end containing the substrate and the cofactor binding domains. A model ensemble of the tetrameric hPHGDH was generated using AlphaFold coupled with molecular dynamics refinement. By analyzing the inter‐subunit interactions at the tetrameric interface, the residues F418, L478, P479, R454, and Y495 were selected and their role was studied by the alanine‐scanning mutagenesis approach. The F418A variant modifies the putative ASB, slightly alters the activity, the fraction of protein in the tetrameric state, and the protein stability; it seems relevant in dimers' recognition to yield the tetrameric oligomer. On the contrary, the R454A, L478A, P479A, and Y495A variants (ACT domain) determine a loss of the tetrameric assembly, resulting in low stability and misfolding, triggering the aggregation and hampering the activity. The predicted tetrameric interface seems mediated by residues at the ACT domain, and the tetramer formation seems crucial for proper folding of hPHGDH, which, in turn, is essential for both stability and functionality. [ABSTRACT FROM AUTHOR]

In the brain, the non‐essential amino acid L‐serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3‐phosphoglycerate: among the different roles played by this amino acid, it can be converted into D‐serine and glycine, the two main co‐agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate‐limiting step of this pathway), 3‐phosphoserine aminotransferase, and 3‐phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L‐serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single‐point variants responsible for hPHGDH deficiency and Neu‐Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3‐phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L‐serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L‐serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches. [ABSTRACT FROM AUTHOR]

D-3-phosphoglycerate dehydrogenase (PHGDH) catalyzes the NAD + -dependent conversion of D-3-phospho-glycerate to 3-phosphohydroxypyruvate, the first step in the phosphorylated pathway for L-serine (L-Ser) biosynthesis. L-Ser plays different relevant metabolic roles in eukaryotic cells: alterations in L-Ser metabolism have been linked to serious neurological disorders. The human PHGDH (hPHGDH), showing a homotetrameric state in solution, is made of four domains, among which there are two regulatory domains at the C-terminus: the aspartate kinase-chorismate mutase-tyrA prephenate dehydrogenase (ACT) and allosteric substrate-binding (ASB) domains. The structure of hPHGDH was solved only for a truncated, dimeric form harboring the N-terminal end containing the substrate and the cofactor binding domains. A model ensemble of the tetrameric hPHGDH was generated using AlphaFold coupled with molecular dynamics refinement. By analyzing the inter-subunit interactions at the tetrameric interface, the residues F418, L478, P479, R454, and Y495 were selected and their role was studied by the alanine-scanning mutagenesis approach. The F418A variant modifies the putative ASB, slightly alters the activity, the fraction of protein in the tetrameric state, and the protein stability; it seems relevant in dimers' recognition to yield the tetrameric oligomer. On the contrary, the R454A, L478A, P479A, and Y495A variants (ACT domain) determine a loss of the tetrameric assembly, resulting in low stability and misfolding, triggering the aggregation and hampering the activity. The predicted tetrameric interface seems mediated by residues at the ACT domain, and the tetramer formation seems crucial for proper folding of hPHGDH, which, in turn, is essential for both stability and functionality., (© 2024 The Protein Society.)