10YN

Joint X-ray/neutron structure of E53Q mutant of Thermus thermophilus serine hydroxymethyltransferase (TthSHMT)

Summary for 10YN

• Entry DOI: 10.2210/pdb10yn/pdb
• Descriptor: Serine hydroxymethyltransferase, SULFATE ION (3 entities in total)
• Functional Keywords: homodimer, pyridoxal-5'-phosphate, one-carbon metabolism, inactive mutant, transferase
• Biological source: Thermus thermophilus
• Total number of polymer chains: 2
• Total formula weight: 89546.19

Authors

Kovalevsky, A.,Drago, V.N.,Phillips, R.S. (deposition date: 2026-02-12, release date: 2026-05-20, Last modification date: 2026-06-10)

Primary citation

Drago, V.N.,Phillips, R.S.,Kovalevsky, A.
Mutation of active site glutamate in serine hydroxymethyltransferase allows trapping a reactive intermediate: a combined neutron and X-ray crystallography study.
Rsc Adv, 16:22600-22609, 2026

PubMed Abstract: Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5'-phosphate (PLP) dependent enzyme that catalyzes a chemical transformation essential for the one-carbon (1C) metabolism. SHMT reversibly converts l-Ser into Gly and transfers a 1C unit to tetrahydrofolate (THF) to give 5,10-methylene-THF (5,10-MTHF). 5,10-MTHF, a 1C-unit donor, plays a crucial role in the downstream biomolecular syntheses required for the cell homeostasis and proliferation. SHMT is a prominent target for the drug discovery to battle bacterial and parasitic infections, and to treat various types of cancer. SHMT-catalyzed chemistry is governed by the general acid-base catalysis. Knowledge of the catalytic mechanism can aid drug design but can only be achieved when the atomic details of each reaction step are mapped, including accurate determination of hydrogen atom positions. Here we utilized the inactive E53Q mutant of () SHMT to directly determine protonation states with room-temperature neutron crystallography and to capture a reactive intermediate containing the PLP-l-Ser external aldimine and THF in the enzyme active site. We observed protonation of the Schiff base nitrogen (N) in the PLP internal aldimine but no change in the protonation states of other ionizable PLP groups and active site residues compared to wild-type SHMT. X-ray structural analysis of the ternary intermediate complex E53Q-Ser-THF that eluded previous structural characterization shows the strategic positioning of the E53Q side chain in close proximity to the external aldimine and THF and reinforces the proposed role for E53 as the driver of proton transfer events along the reaction pathway.

PubMed: 42112277
DOI: 10.1039/d6ra01814a

Experimental method

NEUTRON DIFFRACTION (2.4 Å)
X-RAY DIFFRACTION (1.65 Å)