BIOSYNTHETIC-PATHWAY TO RHIZOBITOXINE IN PSEUDOMONAS-ANDROPOGONIS

When DL-[4-C-13]aspartic acid was provided at regular intervals to Pseudomonas andropogonis cultures between two and three days growth, hydroxythreonine was specifically labelled at C-4 (4-fold enrichment), and rhizobitoxine was specifically labelled at C-4 and C-1 (4-fold and 2-fold enrichments, respectively). The result shows that aspartic acid is converted directly to the 2-amino-4-oxy-but-3-enoic acid moiety of rhizobitoxine. Various L-[U-C-14]amino acids were added to P. andropogonis cultures after two days growth. [C-14]Aspartic acid was rapidly (< 30 min) incorporated into rhizobitoxine (ca 1.5%) and hydroxythreonine (ca 0.5%). Incorporation from [C-14]homoserine into rhizobitoxine and hydroxythreonine was 5.7 and 3.2% respectively, whereas from [C-14]threonine it was 0.05 and 0%. These results suggest that homoserine lies on the pathway to rhizobitoxine and that the biosynthetic pathway to rhizobitoxine branches from the usual aspartic acid --> threonine pathway prior to the formation of threonine. Whereas exogenous lysine and methionine had little effect on the biosynthesis of rhizobitoxine, both threonine and homoserine substantially reduced (2.8-fold and 11-fold respectively) the incorporation of C-14 from aspartic acid into rhizobitoxine, by P. andropogonis. In the former case this was probably due to feedback inhibition of the aspartic --> threonine pathway, and in the latter as a result of competition/dilution. The collective results suggest that the biosynthesis of rhizobitoxine follows the route aspartic acid --> homoserine --> hydroxythreonine --> rhizobitoxine. On the basis of the present results and recent publications, speculation is made concerning the final stages of the biosynthesis of rhizobitoxine, and of the biosynthesis of dihydrorhizobitoxine, a compound that is frequently produced by Bradyrhizobium japonicum.