Ebony is an unusual type of Non-Ribosomal Peptide Synthetase (NRPS) as it has been reported to function much faster than typical NRPS machineries and does not contain all catalytic domains typically required for a functional assembly line. Indeed, with a single A-domain involved in amino acid activation and a peptidyl carrier protein domain (PCP), Ebony lacks a typical condensation (C) domain that would usually be needed to catalyse the peptide bond formation between b-alanine and histamine (or dopamine) (Ebony’s substrates). In place of this missing condensation domain, the primary sequence shows an uncharacterised C-terminal extension of roughly 27kDa.
Objectives: Characterize the structure and the function of the C-terminal domain of Ebony.
Results:
We cloned, purified and determined the crystal structure of the C-terminus domain of Ebony. Crystals belonged to the P21space group and diffracted to 2.0 Å. The structure was solved by molecular replacement using a model generated by Robetta server from an arylalkylamine N-acetyltransferase (AANAT). Despite a low sequence identity (less than 18%) Ebony C-terminal domain shares a well conserved fold with other AANATs. In addition to the apo structure, we solved the structure of Ebony C-terminal domain in complex with Histamine, Dopamine, Carcinine and beta-ala-dopamine. This allowed us to understand the specificity of the enzyme for its amine-substrates. Residues important for substrate binding and catalytic activity have also been identified through mutations and condensation reaction assays.
Conclusion:
This finding unravels a new type of NRPS domain architecture and unveils how, without a standard condensation domain, insects evolved a fast, specific, and efficient way of making peptide bonds using an NRPS machinery. Understanding the diverse structure-function relationship in domains forming NRPS machineries is a crucial step to successfully redesign these medically relevant enzymes.