Growth of Lactobacillus casei in deuterated media. Purification of deuterated thymidylate synthase and proton nuclear magnetic resonance studies.
The purification of thymidylate synthase from amethopterin-resistant Lactobacillus casei grown in a deuterated medium is described. The deuterated enzyme and the non-deuterated enzyme appear to be identical with respect to specific activity, pH optimum, electrophoretic mobility on polyacrylamide gels and ability to form ternary complexes with 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and 5,10-methylenetetrahydrofolate. The deuterated enzyme remained stable at 25 degrees C for 8 h during the acquisition of 400 MHz 1H-NMR spectra and was stored at 5 degrees C for 3 months without loss of catalytic activity. The incorporation of deuterium was essentially complete as demonstrated by a comparison of the NMR spectra of deuterated and non-deuterated enzyme. Proton NMR data obtained with deuterated thymidylate synthase and 1H-FdUMP are in agreement with the fluorine-19 NMR studies of Lewis et al. (Lewis, C.A., Jr., Ellis, P.D. and Dunlap, R.B. (1980) Biochemistry 19, 116-123) which support the formation of a binary complex where the enzyme is covalently linked to carbon 6 of 5-fluoro-5,6-dihydro-2'-deoxyuridylate.
D003903:Deuterium; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000254:growth & development; D009682:Magnetic Resonance Spectroscopy; D008780:Methyltransferases / Q000302:isolation & purification*; D013940:Thymidylate Synthase / Q000302:isolation & purification* / Q000378:metabolism
D003903:Deuterium; D008780:Methyltransferases; D013940:Thymidylate Synthase