Tetrahydrofolate thf

Methylene-THF activity was measured photometrically at 604 nm under anoxic conditions in 1.8 mL anoxic cuvettes (Glasgerätebau Ochs, Bovenden, Germany) under a N₂ atmosphere filled with buffer (50 mM KPO₄, 5 mM MgCl₂, 2 mM DTE, 4 μM resazurin, pH 7.5). The oxidation of 10 mM methyl viologen (prereduced with sodium dithionite) was observed over time after addition of 1.5 mM formaldehyde + 0.5 mM tetrahydrofolate (THF) (Sigma-Aldrich, St. Louis, MO, USA), resulting in a racemic mixture containing 0.25 mM methylene-THF [19 (link),20 (link)], and protein from the respective purification step of the methylene-THF reductase.

All chemicals used in this study were of analytical grade or higher quality. Pyridoxal 5‐phosphate monohydrate (PLP), dithiothreitol (DTT), NADH, and NAD⁺ were purchased from Yuanye Bio‐Technology (Shanghai, China). β‐Mercaptoethanol (β‐ME) and lipoic acid were obtained from Aladdin (Shanghai, China). Tetrahydrofolate (THF) was obtained from Sigma‐Aldrich (St. Louis, MO, USA). Phusion high‐fidelity DNA polymerase and FastDigest restriction enzymes were purchased from Thermo‐Fisher Scientific (Pittsburgh, PA, USA). T4 DNA ligase was purchased from New England Biolabs (Ipswich, MA, USA). DNA extraction kit and gel extraction kit were purchased from Promega (Madison, WI, USA). The pET‐22b(+) and pET28a(+) vectors were purchased from Novagen (Darmstadt, Germany). All synthetic oligonucleotides were ordered from GeneWiz (Suzhou, China). Competent cells of E. coli TOP10 and E. coli BL21 (DE3) were purchased from WEIDI Ltd. (Shanghai, China). Ni‐NTA resin was purchased from Genscript (Nanjing, China). BCA protein assay kit was purchased from SolarBio (Beijing, China).