A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release.

摘要: Human cytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains flexible "flap motif" whose function yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, ligand-binding enzyme-kinetic analyses, we studied the functional roles of flap motif by comparing WT hcSHMT with flap-deleted variant (hcSHMT/Δflap). We found that deletion results in mixture apo-dimers holo-tetramers, whereas was mostly tetrameric form. MD simulations indicated stabilizes structural compactness thereby enhances oligomerization. The hcSHMT/Δflap exhibited different catalytic properties (6S)-tetrahydrofolate (THF)-dependent reactions compared but had similar activity THF-independent aldol cleavage β-hydroxyamino acid. less sensitive to THF inhibition than (Ki 0.65 0.27 mm at pH 7.5, respectively), dissociation constant also 3-fold lower hcSHMT/Δflap, indicating important binding. did not display burst kinetics observed WT. These indicate that, upon removal flap, product release no longer rate-limiting step, implying controlling release. findings reported here improve our understanding provide fundamental insight into how loop can be involved enzymatic other enzymes.