THREE DIMENSIONAL STRUCT: IMPERATOXIN A, PEPTIDE LIGAND OF CA2+ RELEASE CHANNELS

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Trifolitoxin (TFX) is a peptide bacteriocin produced by Rhizobium leguminosarum bv. trifoli T24 which is bacteriostatic toward most strains of R. leguminosarum and R. fredii. Sequence analysis of the TFX production and resistance genes strongly suggests that this peptide is produced ribosomally (Breil, et al. 1993, J. Bacteriol., in press). The DNA sequence of the tfxA structural gene and Edman degradation of the purified peptide show that the amino acid sequence of the peptide precursor of TFX is DIGGSRQGCVA. This peptide sequence is post-translationally modified by the tfx genes such that the glutamine residue is cyclized to a UV absorbing chromophore. The purpose of this work was to determine the structure of this chromophore. Two isomers of TFX were isolated by reverse phase HPLC. The mass of the molecules was confirmed by low resolution FAB-MS to be 1037.4 for both molecules. The structure of the chromophore was determined by a combination of modern NMR techniques, such as COSY, TOCSY and HMQC, to be a 2-pyridone derivative which tautomerizes to give TFX isomers. Both isomers of TFX also contain a thiazoline ring which is the result of ring closure between the sulfhydryl group of cysteine and the carbonyl of glycine.