Coronamic acid, an intermediate in coronatine biosynthesis by Pseudomonas syringae

Abstract

Experiments were undertaken on the biosynthetic pathway to coronatine in Pseudomonas syringae pv. glycinea, using mutants blocked in coronatine synthesis, wild type strains and radioactive labelling with l-[U- 14C] isoleucine and 14C-coronamic acid. Evaluation of the kinetics of coronatine appearance in the medium revealed an initial exponential phase with a maximum rate of 0.86 μmol hr − 1 culture − 1 after 3.1 days growth. Beyond this the rate declined to a steady rate of 0.20 μmol hr − 1 culture t $ ̄ 1 . With P. syringae 4185, 8.6% Of l-[U- 14C] isoleucine added at three days growth was incorporated into coronatine during a 90 min exposure period. At the same time there was negligible incorporation (0.04%) to coronamic acid. Two mutants blocked in coronatine synthesis were shown to produce coronamic acid by isolation and purification of this product from culture supernatants. l-[U- 14C]Isoleucine was added to one of these mutants at four days growth and 1.9% of the radioactivity was incorporated into coronamic acid, these data showed that coronamic acid is a distinct biosynthetic entity. However, the coronatine-producing strain from which these mutants were derived, P. syringae 4180, showed only 0.08% incorporation into a coronamic acid fraction, although culture supernatants after five days growth did reveal a trace of coronamic acid as detected by ninhydrin after electrophoresis and chromatography on thin layer cellulose plates. When a three-day culture of P. syringae 4185 was fed coronamic acid prior to 14C-isoleucine, a 3.7-fold reduction in the incorporation of label into coronatine occurred; instead the radioactivity was diverted to extracelluar coronamic acid which showed 7% incorporation. This result demonstrates that coronamic acid is a biosynthetic intermediate of coronatine. Pseudomonas syringae 4185 very efficiently utilized 14C-coronamic acid, giving 94% incorporation into coronatine after 90 min exposure in a four-day culture, showing the specific utilization of this compound. Other experiments were undertaken to evaluate the possible involvement of coronafacoylisoleucines in the biosynthesis of coronatine, by use of a cormutant blocked in coronafacic acid synthesis, with the conclusion that this was not an operative pathway to coronatine.