Recombinant Escherichia coli D-3-phosphoglycerate dehydrogenase (serA)

Enzyme Overview

D-3-phosphoglycerate dehydrogenase (PHGDH, EC 1.1.1.95) encoded by serA oxidizes D-3-phosphoglycerate (3PG) to 3-phosphohydroxypyruvate (PHP) using NAD$^+$/NADH as a cofactor . Key features include:

• Structure: A homotetramer with each subunit comprising 409 amino acids (excluding the cleaved initiator methionine) .

• Domains: Catalytic domain, coenzyme-binding domain, and regulatory ACT (aspartate kinase–chorismate mutase–TyrA) domain .

• Function: Drives serine synthesis by coupling with downstream enzymes (SerC and SerB) .

Primary Reaction

PHGDH dehydrogenates 3PG to PHP, a thermodynamically unfavorable reaction ($\Delta G^\circ_{\text{obs}} = +33.0 \, \text{kJ·mol}^{-1}$) . This step is enabled by coupling with subsequent reactions in the serine pathway .

Secondary Activity

SerA exhibits α-ketoglutarate (αKG) reductase activity, reducing αKG to D-2-hydroxyglutarate (D-2HG) with higher catalytic efficiency ($k_{\text{cat}} = 33.3 \, \text{s}^{-1}$) compared to its primary reaction ($k_{\text{cat}} = 27.8 \, \text{s}^{-1}$) . This activity links serine synthesis to D-2HG metabolism, a pathway implicated in cancer .

Table 1: Kinetic Parameters of SerA

Regulatory Mechanisms

PHGDH is regulated allosterically:

• Inhibition: L-serine binds the ACT domain, inducing conformational changes that reduce catalytic activity .

• Activation: L-homocysteine and other amino acids enhance activity at low concentrations (EC$_{50}$ = 0.1 mM for L-homocysteine vs. 10 mM for L-serine) .

• Mutations: Specific mutations (e.g., N364A) abolish feedback inhibition by serine, enabling sustained enzyme activity under serine-rich conditions .

Table 2: Impact of SerA Mutations

Production in E. coli

• Expression Systems: High-yield production (125 U/L culture, 96 mg protein) achieved using pETM vectors in E. coli BL21(DE3) with IPTG induction .

• Purification: Single-step chromatography yields >95% purity (specific activity = 1.3 U/mg) .

Biotechnological Relevance

• Cancer Research: PHGDH overexpression in tumors (e.g., breast cancer) drives D-2HG accumulation, promoting oncogenesis .

• Metabolic Engineering: Engineered E. coli strains (e.g., SHuffle® T7) improve disulfide bond formation for functional enzyme assembly .

Thermodynamic Coupling and Metabolic Role

PHGDH couples 3PG dehydrogenation with D-2HG synthesis ($\Delta G^\circ_{\text{obs}} = -28.5 \, \text{kJ·mol}^{-1}$ for αKG reduction), enabling flux through the serine pathway under physiological conditions . This coupling is maintained by D-2HG dehydrogenase (D2HGDH), which recycles D-2HG back to αKG via electron transfer flavoproteins .

Research Implications

• Disease Models: PHGDH mutations correlate with D-2-hydroxyglutaric aciduria, a neurometabolic disorder .

• Therapeutic Targets: Inhibitors targeting PHGDH’s coenzyme-binding domain are under investigation for cancer therapy .