Radioactivity from L-phenylalanine-carboxyl-14C was incorporated specifically into the carboxyl group of cinnamamide by cultures of Streptomyces verticillatus. L-Phenylalanine ammonia-lyase (EC. 4.3.1.5) could be extracted from the mycelium. Cultures grown in defined medium produced little cinnamamide unless supplemented with L-phenylalanine; phenylalanine ammonia-lyase activity in the mycelium was directly related to the yield of cinnamamide.
The incorporation of radioactivity into fusaric acid from 14C-labeled acetate and DL-aspartate was examined using two strains of Fusarium oxysporum. Acetate-1-14C labeled the carboxyl (C-7) group and alternate carbon atoms C-8 and C-10 of the n-butyl chain. The remaining two carbon atoms (C-9 and C-11) of the n-butyl chain were labeled by acetate-2-14C, which contributed only a small amount of radioactivity to C-7. Both precursors labeled the pyridine ring. Almost two-thirds of the label in fusaric acid produced from DL-aspartate-1-14C was in C-7 with most of the remainder in the pyridine ring; DL-aspartate-4-14C gave the reverse pattern. The proportion of radioactivity in C-7 decreased only slightly when contact time between precursor and culture was reduced. The distribution of label between C-7 and the pyridine ring was similar to that between C-1 and C-4 of protein aspartic acid isolated in the same experiment. Malonate-2-14C and glycerol-1,3-14C labeled fusaric acid to approximately the same extent as DL-aspartate-4-14C, but succinate-1,4-I4C was a poor precursor. The results are consistent with the biogenesis of fusaric acid from three units of acetate and one of aspartate. Yields of the metabolite from cultures grown on a modified Richards' medium were increased by supplements of sodium acetate, but not of aspartate.
Acetate-1-13C, acetate-2-13C, and aspartate-4-13C were used as substrates to elucidate the biosynthetic pathway for fusaric acid, the phytotoxic metabolite of Fusarium oxysporum Schlecht. It was established that C-2, C-3, C-4, and C-7 were derived from acetate via aspartate or a related four-carbon dicarboxylic acid, whereas C-5, C-6, C-8, C-9, C-10, and C-11 were derived more directly from acetate. The 13C-labeling patterns were determined by measuring the relative intensities of the 13C—H satellites by proton magnetic resonance spectroscopy.Further information was derived from experiments with D- and L-isomers of aspartate-1-14C and L-aspartate-1-14C:15N. Radioactivity from both enantiomers of aspartate-1-14C was incorporated into fusaric acid, but more efficiently from the L-isomer. Furthermore, 15N was incorporated more efficiently into fusaric acid and the aspartic acid of mycelial protein than was 14C. These results suggest that aspartic acid is metabolized to fusaric acid via oxalacetate, and that L-aspartate serves as a donor of nitrogen, in an amino-transferase reaction, to a separate oxalacetate pool of primarily endogenous origin.
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