Drosophila melanogaster nonribosomal peptide synthetase Ebony encodes an atypical condensation domain

Abstract

The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with β-alanine. Ebony is a rare example of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine-N-acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic example of an alternative type of NRPS condensation domain; they also illustrate how the catalytic components of such assembly lines are significantly more diverse than a minimal set of conserved functional domains.

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity

“…One possibility is that this process operates by charge repulsion: when an aminoacyl-PPant approaches the acceptor channel, the ammonium group of the substrate triggers the rotation of the Arg side chain due to charge repulsion, which opens the channel, allowing the aminoacyl-PPant to enter it. This would explain our inability to crystallize the wild-type PCP 2 -C 3 construct loaded with PPant derivatives lacking an amino group (such as propionyl and propan-1,3-dioyl 33 ), due to interactions that interfere with crystallization when the substrate is not bound in the acceptor channel of the C domain. To further explore this mechanism, we next turned to the characterization of the Structure of the amino acid acceptor bound substrate.…”

Understanding condensation domain selectivity in non-ribosomal peptide biosynthesis: structural characterization of the acceptor bound state

“…One possibility is that this process operates by charge repulsion: when an aminoacyl-PPant approaches the acceptor channel, the ammonium group of the substrate triggers the rotation of the Arg side chain due to charge repulsion, which opens the channel, allowing the aminoacyl-PPant to enter it. This would explain our inability to crystallize the wild type PCP 2 -C 3 construct loaded with PPant derivatives lacking an amino group (such as propionyl and propan-1,3-dioyl, 33 data not shown), due to interactions that interfere with crystallization when the substrate is not bound in the acceptor channel of the C domain. To further explore this mechanism, we next turned to the characterization of the PCP 2 -C 3 construct with an aminoacyl group appended to the PPant thiol group.…”

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

“…Based on many studies of the chemical and kinetic mechanisms of GNAT enzymes, it is clear this superfamily has evolved to utilize a diversity of chemistries and active site residues for more complex and targeted modifications of acceptor substrates. Identification of additional non-acyl transfer reactions for GNATs, including decarboxylation, methylcarbamoyl transfer, and condensation ( Gu et al, 2007 ; Izoré et al, 2019 ; Karambelkar et al, 2020 ; Skiba et al, 2020 ) highlight the shear multitude of chemistries that enzymes from this superfamily can accomplish. Thus, GNATs appear to have a highly tunable scaffold that has evolved to modify a diverse range of substrates.…”