Effect of Changes in the Pool of Acetate on the Incorporation and Distribution of 13C- and 14C-Labeled Acetate into Showdomycin by Streptomyces showdoensis

The effect of concentration of 13C- and 14C-labeled acetate in the soluble pool on the distribution of 13C or 14C in the maleimide ring of showdomycin by Streptomyces showdoensis has been studied. 13C incorporation was observed by means of proton-decoupled 13C Fourier transform NMR spectroscopy. 13C NMR spectra confirmed earlier findings that the carboxyl carbon of [1-14C]acetate incorporates exclusively in carbon 5 of the maleimide ring. When the concentration of acetate is 0.2 µm, all four carbons of the maleimide ring are labeled by 14C from [2-14C]acetate. At high concentration of [2-13C]acetate (1800 µm), carbons 2, 3, and 4 are labeled, but carbon 5 is not. These observations can be explained by the combined action of the Krebs cycle and the malic enzyme. The malic enzyme would give rise to [1,2-14C]acetate from [2-14C]acetate. At 1800 µm, the [1,2-13C]acetate formed via the malic enzyme is diluted by the large amounts of [2-13C]acetate in the acid-soluble pool. The result is a negligible 13C incorporation into carbon 5. The 13C NMR findings were confirmed by using high and low concentrations of [2-14C]acetate. The percentage of 14C in carbon 5 decreased from 22% (low acetate) to 4% (high acetate). While it is very difficult to make comparisons of enzyme activities concerned with the biosynthesis of a compound such as showdomycin, it is now possible to study the actual physiological effects of high and low concentrations of acetate in intact cells. This type of study provides an accurate picture of the biochemistry of those compounds as determined by the Krebs cycle and the malic enzyme.