CARBON-13 NMR STUDIES OF PROLINE BIOSYNTHESIS AND FORMALDEHYDE METABOLISM IN ESCHERICHIA COLI

General methods for growing E. coli for NMR experiments have been described. The in vivo aerobiosis of concentrated cell suspen- sions in the magnet has been demonstrated using a 30 mL/minute continuous air stream. Assimilation of the ('13)C lactate methyl res- onance produced by anaerobic growth on glucose was used to indicate aerobic conditions.; Anaerobic in vivo ('13)C NMR has been used to detect the tran- sient formation of S-(hydroxymethyl)glutathione (GSCH(,2)OH) from endogenous-glutathione and ('13)C formaldehyde, at the onset of formaldehyde metabolism by E. coli. Two dimensional ('1)H-('13) shift correlation was used to locate the chemical shift of the cysteine and formaldehyde-derived protons and carbons of the adduct. The equi- librium constant (K(,eq)) for: GSH + CH(,2)O (DBLHARR) GSCH(,2)OH was found to be 950 (+OR-) 50 M('-1) at pH = 7 by ('13)C NMR. This equilibrium constitutes the initial reaction in vivo. The adduct remains intracellular and is subsequently consumed by metabolism.; A small batch culture technique has been developed and used to demonstrate simultaneous acetate consumption and proline produc- tion in a proline overproducing E. coli strain. The metabolism during exponential growth is readily monitored by in vivo ('13)C NMR. Proline isotopomer ratios were determined by ('13)C-('13)C scalar multiplet inten- sity analysis. The ratios of proline isotopomers derived from growth on 1-('13)C acetate, 2-('13)C acetate, 1-('13)C glucose and/or 20% D(,2)O indicate normal proline biosynthesis in this strain. Growth on 1-('13)C acetate yields 60% 1,5-('13)C(,2) proline and 35% 5-('13)C proline. These isotopomers may prove useful in other NMR studies using proline. Futile cycling has been identified in this strain by observing a switch in the location of the ('13)C label in the singly ('13)C labeled acetate substrate.; Gating the proton decoupler produces a maximum in signal intensity for C-5 proline using (pi)/3 pulses and a 3 second recycle delay. Equations describing this phenomenon in terms of T1 and NOE have been presented. A Cr(acac)(,3) doped external benzene standard has been used to relate nonthermal-equilibrium ('13)C signal intensities to concentration, within 20% of authentic values.