Ebony, a Novel Nonribosomal Peptide Synthetase for β-Alanine Conjugation with Biogenic Amines in Drosophila *

Using Ebony protein either expressed in Escherichia coli or in Schneider S2 cells, we provide evidence for its substrate specificity and reaction mechanism. Ebony activates β-alanine to aminoacyladenylate by an adenylation domain and covalently attaches it as a thioester to a thiolation domain in a nonribosomal peptide synthetase (NRPS) related mechanism. In a second reaction, biogenic amines act as external nucleophiles on β-alanyl-S-pantetheine-Ebony, thereby releasing in a fast reaction the dipeptide (peptidoamine) in a process that is novel in higher eucaryotes. Therefore, we define Ebony as a β-alanyl-biogenic amine synthetase. Insight into the reaction mechanism stems from mutational analysis of an invariant serine that disclosed Ebony as a multienzyme with functional analogy to the starting modules of NRPSs. In light of a putative biogenic amine-deactivating capacity, Ebony function in the nervous system must be reconsidered. We propose that in the Drosophila eye Ebony is involved in the transmission process by inactivation of histamine through β-alanyl conjugation.