Production of an aromatic amino acid auxotroph of a trimethoprim-resistant Escherichia coli and deuteration of the aromatic amino acids in its dihydrofolate reductase.
Interpretation of the 1H-NMR spectra of Escherichia coli dihydrofolate reductase is complicated by the large number of overlapping resonances due to protonated aromatic amino acids. Deuteration of the aromatic protons of aromatic amino acid residues is one technique useful for simplifying the 1H-NMR spectra. Previous attempts to label the dihydrofolate reductase from overproducing strains of Escherichia coli were not completely successful. This labeling problem was solved by transducing via P1 phage a genetic block into the de novo biosynthetic pathway of aromatic amino acids in a trimethoprim resistant strain of E. coli, MB 3746. A new strain, MB 4065, is a very high level producer of dihydrofolate reductase and requires exogenous aromatic amino acids for growth, therefore allowing efficient labeling of its dihydrofolate reductase with exogenous deuterated aromatic amino acid.
D000596:Amino Acids / Q000032:analysis; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis*; D014295:Trimethoprim
D000596:Amino Acids; D014295:Trimethoprim; D003903:Deuterium; D013762:Tetrahydrofolate Dehydrogenase