Novel flax orbitide derived from genetic deletion

Background Flaxseed orbitides are homodetic plant cyclic peptides arising from ribosomal synthesis and post-translation modification (N to C cyclization), and lacking cysteine double bonds (Nat Prod Rep 30:108-160, 2013). Screening for orbitide composition was conducted on the flax core collection (FCC) grown at both Saskatoon, Saskatchewan and Morden, Manitoba over three growing seasons (2009-2011). Two flax ( Linum usitatissimum L.) accessions ‘Hollandia’ (CN 98056) and ‘Z 11637’ (CN 98150) produce neither [1−9-N α C]-linusorb B2 ( 3 ) nor [1−9-N α C]-linusorb B3 ( 1 ). Mass spectrometry was used to identify novel compounds and elucidate their structure. NMR spectroscopy was used to corroborate structural information. Results Experimental findings indicated that these accessions produce a novel orbitide, identified in three oxidation states having quasimolecular ion peaks at m/z 1072.6 ( 18 ), 1088.6 ( 19 ), and 1104.6 ( 20 ) [M + H] + corresponding to molecular formulae C 57 H 86 N 9 O 9 S, C 57 H 86 N 9 O 10 S, and C 57 H 86 N 9 O 11 S, respectively. The structure of 19 was confirmed unequivocally as [1−9-N α C]- O LIPPFFLI. PCR amplification and sequencing of the gene coding for 18 , using primers developed for 3 and 1 , identified the putative linear precursor protein of 18 as being comprised of the first three amino acid residues of 3 (MLI), four conserved amino acid residues of 3 and/or 1 (PPFF), and the last two residues of 1 (LI). Conclusion Comparison of gene sequencing data revealed that a 117 base pair deletion had occurred that resulted in truncation of both 3 and 1 to produce a sequence encoding for the novel orbitide precursor of 18 . This observation suggests that repeat units of flax orbitide genes are conserved and suggests a novel mechanism for evolution of orbitide gene diversity. Orbitides 19 and 20 contain MetO and MetO 2 , respectively, and are not directly encoded, but are products of post-translation modification of Met present in 18 ([1−9-N α C]-MLIPPFFLI).