Dimerization of PHGDH via the catalytic unit is essential for its enzymatic function

Abstract

Human D-3-phosphoglycerate dehydrogenase (PHGDH), a key enzyme in de novo serine biosynthesis, is amplified in various cancers and serves as a potential target for anticancer drug development. To facilitate this process, more information is needed on the basic biochemistry of this enzyme. For example, PHGDH was found to form tetramers in solution and the structure of its catalytic unit (sPHGDH) was solved as a dimer. However, how the oligomeric states affect PHGDH enzyme activity remains elusive. We studied the dependence of PHGDH enzymatic activity on its oligomeric states. We found that sPHGDH forms a mixture of monomers and dimers in solution with a dimer dissociation constant of ∼0.58 μM, with the enzyme activity depending on the dimer content. We computationally identified hotspot residues at the sPHGDH dimer interface. Single-point mutants at these sites disrupt dimer formation and abolish enzyme activity. Molecular dynamics simulations showed that dimer formation facilitates substrate binding and maintains the correct conformation required for enzyme catalysis. We further showed that the full-length PHGDH exists as a dynamic mixture of monomers, dimers, and tetramers in solution with enzyme concentration-dependent activity. Mutations that can completely disrupt the sPHGDH dimer show different abilities to interrupt the full-length PHGDH tetramer. Among them, E108A and I121A can also disrupt the oligomeric structures of the full-length PHGDH and abolish its enzyme activity. Our study indicates that disrupting the oligomeric structure of PHGDH serves as a novel strategy for PHGDH drug design and the hotspot residues identified can guide the design process.

Central carbon metabolism exhibits unique characteristics during the handling of fungal patterns by monocyte-derived dendritic cells

“…PHGDH protein was expressed in resting and stimulated MDDCs. Consistent with the dimerization of PHGDH in the presence of substrate [ 37 ], Western blot carried out in native conditions in a non-denaturing and non-reducing buffer showed the presence of the 55-kD PHGDH band as well as a band compatible with dimerization and constitutive 3-phosphoglycerate flux ( Fig. 6 L).…”

The human phosphorylated pathway: a multienzyme metabolic assembly for l‐serine biosynthesis

“…6 in Ref. [40]. Next, experimental traces and simulations of NADH and L-Ser production demonstrated that the former is produced slightly faster at the beginning of the reaction while the concentrations of both products are very close at times ≥ 500 s (Fig.…”

“…While the latter change is of small amplitude (2‐fold), the rate of 3PHP production by PHGDH is significantly different between the simulation and the published values. The explanation is provided by recent work [40] demonstrating that the oligomerisation state and the apparent k cat of PHGDH are both altered by the enzyme concentration: The homotetrameric state and the maximal figure of k cat are observed at ≥ 1 μ m enzyme concentration. Actually, the PHGDH kinetic parameters have been previously determined at 0.007 μ m enzyme concentration [31], while present investigations were performed at 0.82 μ m .…”

Biochemical and cellular studies of three human 3‐phosphoglycerate dehydrogenase variants responsible for pathological reduced L‐serine levels

“…On the other hand, V425M and V490M substitutions are predicted to disrupt core hydrophobic interactions that are important to stabilize the fold of the ASB and ACT domains, respectively. These domains seem essential to regulate the tetrameric assembly of the wild‐type enzyme 36 …”