Biosynthetic pathway to rhizobitoxine in Pseudomonas andropognis

When DL-[4-13C]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-14C]amino acids were added to P. andropogonis cultures after two days growth. [14C]Aspartic acid was rapidly (< 30 min) incorporated into rhizobitoxine (ca 1.5%) and hydroxythreonine (ca 0.5%). Incorporation from [14C]homoserine into rhizobitoxine and hydroxythreonine was 5.7 and 3.2% respectively, whereas from [14C]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 leads to 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 14C from aspartic acid into rhizobitoxine, by P. andropogonis. In the former case this was probably due to feedback inhibition of the aspartic leads to 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 leads to homoserine leads to hydroxythreonine leads to 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.