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.

L‐serine biosynthesis in the human central nervous system: Structure and function of phosphoserine aminotransferase

“…The K m,3‐PHP and K m,OPS are significantly higher than the in vivo substrate concentration (Table 1), suggesting that PSAT works under k cat / K m regime in the cell and is thus a sensor for the levels of 3‐PHP, regulating its activity based on the availability of the glycolysis‐derived substrate. OPS formation by PSAT is 300‐fold more efficient than the reverse reaction and, considering a K eq of about 10, the equilibrium of the whole pathway is shifted towards product formation, counteracting the unfavorable equilibrium of the first reaction catalyzed by PHGDH in the PP (Murtas et al, 2020; Murtas et al, 2021). Notably, human PSAT activity is strongly stimulated by increasing the ionic strength, that is, by 200 mM KCl.…”

“…In the mammalian central nervous system (CNS), the PP is crucial for maintaining the levels of L‐Ser, as this nonessential amino acid is transported inefficiently through the blood–brain barrier. The first step of the PP is catalyzed by 3‐PG dehydrogenase (PHGDH; Murtas et al, 2021; Unterlass et al, 2017) which oxidizes 3‐PG to 3‐phosphohydroxypyruvate (3‐PHP) using NAD + as the electron acceptor. The equilibrium of this reaction is strongly shifted in favor of 3‐PG (Grant, 2012; Murtas et al, 2020); therefore, the subsequent reactions of the pathway must provide a thermodynamic drive to synthesize L‐Ser.…”

Insulin and serine metabolism as sex-specific hallmarks of Alzheimer’s disease in the human hippocampus

“…A recent study reported that extracellular levels of L-and D-Ser were reduced at an early stage of AD, before b-amyloid plaque was deposited in the hippocampus of a mouse model that displays lower glycolytic flux in hippocampal astrocytes (Le Douce et al, 2020). The glycolytic flux may control brain L-Ser synthesis (Figure S5); phosphoglycerate kinase (PGK) and phosphoglycerate mutase (PGM1) are mainly responsible for the relatively high levels of cellular L-Ser (0.18-0.55 mM) (Jin et al, 2020) because the K M of human PHGDH for 3PG (0.36 mM) is larger than its cellular level (Murtas et al, 2021). Although human PHGDH activity is not affected by L-Ser (Murtas et al, 2021), the pyruvate kinase M2 isoform (PKM2, mainly expressed in tumor cells and in astrocytes) is activated by L-Ser binding (Chaneton et al, 2012), supporting glycolysis and lactate production.…”

“…The glycolytic flux may control brain L-Ser synthesis (Figure S5); phosphoglycerate kinase (PGK) and phosphoglycerate mutase (PGM1) are mainly responsible for the relatively high levels of cellular L-Ser (0.18-0.55 mM) (Jin et al, 2020) because the K M of human PHGDH for 3PG (0.36 mM) is larger than its cellular level (Murtas et al, 2021). Although human PHGDH activity is not affected by L-Ser (Murtas et al, 2021), the pyruvate kinase M2 isoform (PKM2, mainly expressed in tumor cells and in astrocytes) is activated by L-Ser binding (Chaneton et al, 2012), supporting glycolysis and lactate production. Conversely, at low L-Ser levels, the glycolytic flux to lactate is transiently reduced, favoring accumulation (doubling) of 3PG, which can be rerouted to produce L-Ser (Ye et al, 2012); a bidirectional control between PP and glycolysis is active (Figure S5).…”

“…NMDAR neurotransmission can be controlled by acting on metabolism and availability of D-Ser; brain-produced L-Ser is converted into the D-enantiomer by Ser racemase (SR or SRR) mainly in neurons and shuttled to astrocytes, where it is degraded by SR and D-amino acid oxidase (DAAO or DAO) (Pollegioni and Sacchi, 2010;Wolosker, 2011). NMDAR activation by D-Ser and Gly might also be regulated by the PP (Neame et al, 2019), and various Ser synthesis disorders because of mutations in the enzymes of the PP have been identified in individuals with neurological symptoms (Shaheen et al, 2014;Murtas et al, 2021). PHGDH is the only gene found to be consistently upregulated in transcriptomes from five brain regions (temporal cortex, dorsolateral prefrontal cortex, superior temporal gyrus, parahippocampal gyrus, and inferior frontal gyrus) of individuals with AD (Yan et al, 2020).…”