Novel mutations in 3-phosphoglycerate dehydrogenase (PHGDH) are distributed throughout the protein and result in altered enzyme kinetics.

Three-phosphoglycerate dehydrogenase (3-PGDH) deficiency is a rare recessive inborn error in the biosynthesis of the amino acid L-serine characterized clinically by congenital microcephaly, psychomotor retardation, and intractable seizures. The biochemical abnormalities associated with this disorder are low concentrations of L-serine, D-serine, and glycine in cerebrospinal fluid (CSF). Only two missense mutations (p.V425M and p.V490M) have been identified in PHGDH, the gene encoding 3-PGDH, but it is currently unclear how these mutations in the carboxy-terminal regulatory domain of the protein affect enzyme function. We now describe five novel mutations in five patients with 3-PGDH deficiency; one frameshift mutation (p.G238fsX), and four missense mutations (p.R135W, p.V261M, p.A373T, and p.G377S). The missense mutations were located in the nucleotide binding and regulatory domains of 3-PGDH and did not affect steady-state expression, protein stability, and protein degradation rates. Patients' fibroblasts displayed a significant, but incomplete, reduction in maximal enzyme activities associated with all missense mutations. In transient overexpression studies in HEK293T cells, the p.A373T, p.V425M, and p.V490M mutations resulted in almost undetectable enzyme activities. Molecular modeling of the p.R135W and p.V261M mutations onto the partial crystal structure of 3-PGDH predicted that these mutations affect substrate and cofactor binding. This prediction was confirmed by the results of kinetic measurements in fibroblasts and transiently transfected HEK293T cells, which revealed a markedly decreased V(max) and an increase in K(m) values, respectively. Taken together, these data suggest that missense mutations associated with 3-PGDH deficiency either primarily affect substrate binding or result in very low residual enzymatic activity.