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Description
Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5’-phosphate (PLP) dependent enzyme that catalyzes the tetrahydrofolate (THF)-dependent cleavage of L-Ser to form glycine and 5,10-methylene-THF. This reaction is significant for its role in the biosynthesis of thymidine and purines, as well as the methyl group of methionine by providing single carbon units to one-carbon metabolism. Human mitochondrial SHMT (hSHMT2) is overexpressed in a multitude of cancers and is acknowledged as a significant target for anti-cancer therapeutics. Here, we present a 2.3 Å joint X-ray/neutron (XN) structure of the homodimeric SHMT from Thermus thermophilus (TthSHMT), whose active site is conserved compared to that of hSHTM2, in the open conformation depicting the PLP cofactor covalently bound to the catalytic lysine in the internal aldimine state and a sulfate ion occupying the substrate binding site. In addition, a second joint XN structure obtained by soaking a TthSHMT crystal with L-Ser revealed the substrate bound at the entrance of the active site in a solvent-exposed shallow pocket in a pre-Michaelis complex while the sulfate anion continues to block the active site. We further tracked the substrate through the active site by obtaining an X-ray structure of a pseudo-Michaelis complex by soaking a TthSHMT crystal with D-Ser, a non-reactive substrate enantiomer. Nuclear density maps revealed the positions of hydrogen atoms and provided the ability to accurately assign the protonation states for the amino acid residues, L-Ser substrate, and the PLP cofactor. By direct observation of the locations of hydrogen atoms and tracking substrate positions, our study provides unique atomic-level understanding of the SHMT active site that sheds new light on the enzyme’s catalytic mechanism and can be employed to advance the design of anticancer drugs targeting hSHMT2.
