Biochemical and biophysical characterization of recombinant human 3‐phosphoglycerate dehydrogenase

Abstract

The human enzyme D‐3‐phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3‐phosphoglycerate (3PG) into 3‐phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L‐ serine. We focused on the full‐length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α‐ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L‐serine inhibited hPHGDH, nor did glycine and D‐serine, the coagonists of NMDA receptors related to L‐ serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L‐ serine levels and thus to reduce cancer progression and treat neurological disorders.