THE BIOSYNTHETIC GENE-CLUSTER FOR CORONAMIC ACID, AN ETHYLCYCLOPROPYLAMINO-ACID, CONTAINS GENES HOMOLOGOUS TO AMINO ACID-ACTIVATING ENZYMES AND THIOESTERASES

Coronamic acid (CMA), an ethylcyclopropyl amino acid derived from isol eucine, functions as an intermediate in the biosynthesis of coronatine , a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. The DNA required for CMA biosynthesis (6.9 kb) was s equenced, revealing three distinct open reading frames (ORFs) which sh are a common orientation for transcription. The deduced amino acid seq uence of a 2.7-kb ORF designated cmaA contained six core sequences and two conserved motifs which are present in a variety of amino acid-act ivating enzymes, including nonribosomal peptide synthetases. Furthermo re, CmaA contained a spatial arrangement of histidine, aspartate, and arginine residues which are conserved in the ferrous active site of so me nonheme iron(II) enzymes which catalyze oxidative cyclizations. The deduced amino acrid sequence of a 1.2-kb ORF designated cmaT was rela ted to thioesterases of both procaryotic and eucaryotic otic origins. These data suggest that CMA assembly is similar to the thiotemplate me chanism of nonribosomal peptide synthesis. No significant similarities between a 0.9-kb ORF designated cmaU and other database entries were found. The start sites of two transcripts required for CMA biosynthesi s were identified in the present study. pRG960sd, a vector containing a promoterless glucuronidase gene, was used to localize and study the promoter regions upstream of the two transcripts. Data obtained in the present study indicate that CMA biosynthesis is regulated at the tran scriptional level by temperature.