Fig 1.
(a) The MTHFD2 reaction scheme. (b) The dehydrogenase (D) activity. Note that the QM region indicates the EVB atoms used for our empirical valence bond simulation of the D activity. R = p-aminobenzoyl-glutamate, R1 = Ribose-ADP.
Fig 2.
Overview of modeled MTHFD2 system, with THF and NAD+ binding pockets. (a) Homodimer overview (b) THF binding scenario (c) NAD+ binding scenario.
Fig 3.
(a) NAD+ and Pi binding site from the X-ray structure (6KG2.pdb). Each monomer is labeled as subunit A (green) and B (cyan), respectively. Asp168, Asp216 and Asp225 from subunit B are colored as cyan for C atoms and shown as sticks. The minimum distance between Asp168 and Asp225 is 7.9Å. (b) The MgA binding pocket. (c) The MgB binding pocket.
Fig 4.
Catalytic cycle for the dehydrogenase (D) and cyclohydrolase (C) activities.
Fig 5.
The left column shows the resonance states (Φ1D, Φ2D, and Φ3D) used to describe the dehydrogenase activity. The middle column shows snapshots of the reactant, intermediate and product state, which are depicted as stick and colored by elements (C atoms: white for THF and orange for NAD+/NADH). The hydroxide is shown as a sphere and colored in salmon red. In the optimized reactant geometry, the distance between the reactive C atom of CH2-THF and the reactive C atom of NAD+ is 3.1 Å, with the transferring hydrogen at 2.1 Å from the acceptor C atom. The last column shows snapshots representing the water reorganization from the reactant state to product state. The water molecules within 7Å from the reactive C atom of THF are shown as both stick and sphere and colored red for oxygen atoms, while for the water molecules beyond 7Å are only shown as blue spheres to distinguish them.
Table 1.
Calculated binding energy and free energy (kcal/mol) for the dehydrogenase activity of MTHFD2.
The is calculated based on the experimental value kcat = 12.4 ± 0.71s-1 using transition-state theory.10
Fig 6.
Proposed cyclohydrolase reaction mechanism.
‡ represents transition state.
Fig 7.
(a) Superimposition of the NAD(P)+ binding pockets with regards to the dehydrogenase activity. The C atoms of MTHFD1 (PDB: 6ECQ) are colored in brown, and those of MTHFD2 (PDB: 6KG2) are colored in white. Switching from Ser197 to Arg233 defines a key force in binding 2′-phosphate of NADP+ rather than phosphate ion (Pi). (b) Superimposition of the MTHFD2 and MTHFD2L NAD+ binding pockets. Key residues involved in binding are shown as sticks and colored red for MTHFD2 and pink for MTHFD2L.