Polypeptide Chains Containing d-γ-Hydroxyvaline

Life has an unexplained and distinct l-homochirality. Proteins typically incorporate only l-amino acids into their sequences. In the present study, d-Val and d-γ-hydroxyvaline (d-Hyv; <u>V</u>*) have been found within ribosomally expressed polypeptide chains. Four conopeptides were initially isolated, gld-<u>V</u>*/gld-<u>V</u>*‘ from the venom of <i>Conus gladiator</i> and mus-<u>V</u>*/mus-<u>V</u>*‘ from the venom of <i>Conus</i> <i>mus</i>. Their complete sequences (gld-<u>V</u>*/gld-<u>V</u>*‘ = Ala-Hyp-Ala-Asn-Ser-d-Hyv-Trp-Ser and mus-<u>V</u>*/mus-<u>V</u>*‘ = Ser-Hyp-Ala-Asn-Ser-d-Hyv-Trp-Ser) were determined by a combination of nano/pico-NMR and MS/MS methods. The amino acid triad that contains the γ-hydroxylated residue, Ser-d-Hyv-Trp, is a novel structural motif that is stabilized by specific interactions between the d-amino acid and its neighboring l-counterparts. These interactions inhibit lactonization, a peptide backbone scission process that would normally be initiated by γ-hydroxylated residues. Conopeptides possessing the Ser-d-Hyv-Trp motif have been termed γ-hydroxyconophans. We have also isolated analogous conopeptides (gld-<u>V</u> and mus-<u>V</u>) containing d-Val instead of d-Hyv; these are termed conophans. γ-Hydroxyconophans and conophans are particularly atypical because (i) they are not constrained as most conopeptides, (ii) they are extremely short in length, (iii) they have a high content of hydroxylated residues, and (iv) their sequences have no close match with other peptides in sequence databases. Their modifications appear to be part of a novel hyperhydroxylation mechanism found within the venom of cone snails that enhances neuronal targeting. The finding of d-Val and d-Hyv within this family of peptides suggests the existence of a corresponding d-stereospecific enzyme capable of d-Val oxidation.