Edited by Ruma BanerjeeHuman cytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains a flexible "flap motif" whose function is yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, and ligand-binding and enzyme-kinetic analyses, we studied the functional roles of the flap motif by comparing WT hcSHMT with a flap-deleted variant (hcSHMT/⌬flap). We found that deletion of the flap results in a mixture of apo-dimers and holo-tetramers, whereas the WT was mostly in the tetrameric form. MD simulations indicated that the flap stabilizes structural compactness and thereby enhances oligomerization. The hcSHMT/⌬flap variant exhibited different catalytic properties in (6S)-tetrahydrofolate (THF)-dependent reactions compared with the WT but had similar activity in THF-independent aldol cleavage of ␤-hydroxyamino acid. hcSHMT/⌬flap was less sensitive to THF inhibition than the WT (K i of 0.65 and 0.27 mM THF at pH 7.5, respectively), and the THF dissociation constant of the WT was also 3-fold lower than that of hcSHMT/⌬flap, indicating that the flap is important for THF binding. hcSHMT/ ⌬flap did not display the burst kinetics observed in the WT. These results indicate that, upon removal of the flap, product release is no longer the rate-limiting step, implying that the flap is important for controlling product release. The findings reported here improve our understanding of the functional roles of the flap motif in hcSHMT and provide fundamental insight into how a flexible loop can be involved in controlling the enzymatic reactions of hcSHMT and other enzymes.Serine hydroxymethyltransferase (SHMT 2 ; EC 2.1.2.1.) is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme in which the PLP cofactor facilitates the reversible transfer of a hydroxymethyl group from L-serine to (6S)-tetrahydrofolate (THF) to yield glycine and 5,10-methylenetetrahydrofolate (5,10-CH 2 -THF) as products (1-3). SHMT is one of the enzymes in the deoxythymidylate (dTMP) synthesis cycle in which the other two enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), also take part in the recycling of folate compounds and production of 5,10-CH 2 -THF via the SHMT reaction. 5,10-CH 2 -THF serves as a methyl donor for the TS reaction to convert dUMP to dTMP, which is a requisite precursor for DNA biosynthesis (4). Due to its important role in DNA biosynthesis, cell proliferation, and cell survival, SHMT is one of the attractive targets for antimalarial (5-12) and anticancer chemotherapy (10,(13)(14)(15)(16).

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Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate (PLP)-dependent enzyme, is involved in one-carbon metabolism in multiple biochemical pathways, including the biosynthesis of purine and thymidine. SHMT1 is the enzyme to be studied clinically as a target for cancer chemotherapy. Therefore, the binding mechanism of this enzyme would be investigated. In this study, molecular dynamics simulations for 500 ns was applied on SHMT1 tetramer in six systems with different ligand cofactors (PLP-Lys, L-ser, PLS, PLG, THF, and MTHF) in order to understand its structural dynamics properties. The key residues Y’73, S53, H231, K257, R263 and R402 were found in all ligand cofactor/SHMT1 systems. All ligand cofactors of each SHMT1 system were stabilized by a strong hydrogen bond with S119 and G120 residues. In addition, the PLS and PLG systems showed strong stabilization with SHMT1 rather than THF and MTHF. Among systems, the MTHF/PLG ligands in system 5 showed the highest binding affinity with SHMT1. Our results could be used as theoretical guidance for inhibitor developments toward SHMT1, which targeting anti-cancer inhibitors