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Drosophila Melanogaster Nonribosomal Peptide Synthetase Ebony Encodes an Atypical Condensation Domain

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Drosophila Melanogaster Nonribosomal Peptide Synthetase Ebony Encodes an Atypical Condensation Domain Drosophila melanogaster nonribosomal peptide synthetase Ebony encodes an atypical condensation domain Thierry Izoréa,b,c,1, Julien Tailhadesa,b,c, Mathias Henning Hansena,b,c, Joe A. Kaczmarskid, Colin J. Jacksond, and Max J. Crylea,b,c,1 aThe Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia; bDepartment of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia; cEMBL Australia, Monash University, Clayton, VIC 3800, Australia; and dResearch School of Chemistry, The Australian National University, Acton, ACT 2601, Australia Edited by Mohamed A. Marahiel, Philipps-Universität Marburg, Marburg, Germany, and accepted by Editorial Board Member Michael A. Marletta December 27, 2018 (received for review July 5, 2018) The protein Ebony from Drosophila melanogaster plays a central peptide and the PCP-bound amino acid. This then leads to the role in the regulation of histamine and dopamine in various tissues transfer of the peptide from the upstream PCP to the down- through condensation of these amines with β-alanine. Ebony is a stream PCP and extends the peptide by one residue (3). In ad- rare example of a nonribosomal peptide synthetase (NRPS) from a dition to these essential domains, most NRPSs encode a terminal higher eukaryote and contains a C-terminal sequence that does thioesterase domain (TE domain) in the last module of the as- not correspond to any previously characterized NRPS domain. sembly line to allow the release of the product from the synthesis We have structurally characterized this C-terminal domain and machinery. The TE domain also serves as a point for further di- have discovered that it adopts the aryl-alkylamine-N-acetyl trans- versification of the peptide sequence through various pathways, ferase (AANAT) fold, which is unprecedented in NRPS biology. which include macrocyclization or dimerization in addition to Through analysis of ligand-bound structures, activity assays, and hydrolysis (1). Within modules, NRPSs may also contain addi- binding measurements, we have determined how this atypical tional domains acting in cis (such as epimerization domains) or condensation domain is able to provide selectivity for both the external enzymes acting in trans that alter the structure of the BIOCHEMISTRY carrier protein-bound amino acid and the amine substrates, a sit- NRP during synthesis (1, 2). This versatility provides a vast uation that remains unclear for standard condensation domains number of possible combinations within the NRPS assembly line identified to date from NRPS assembly lines. These results demon- and an even greater number of possible NRP products. strate that the C terminus of Ebony encodes a eukaryotic example While bacterial and fungal NRPS machineries are common and of an alternative type of NRPS condensation domain; they also largely conform to a standard architecture and domain arrange- illustrate how the catalytic components of such assembly lines ment, the presence of functional NRPS assembly lines in higher are significantly more diverse than a minimal set of conserved eukaryotes is rare (4). The NRPS-like proteins that have been functional domains. identified in the latter group mostly consist of A and PCP domains nonribosomal peptide synthetase | NRPS | condensation reaction | Significance C domain | aryl-alkylamine N-acetyl transferase Nonribosomal peptide synthesis is responsible for the forma- onribosomal peptides (NRPs) are secondary metabolites tion of many important peptide natural products in bacteria Nsynthesized in many organisms, and to which they usually and fungi; it typically utilizes a modular architecture of re- confer a significant fitness advantage. The diversity of NRPs peating catalytic domains to produce these diverse peptide stems from the structure of the nonribosomal peptide synthetase structures. The protein Ebony from Drosophila melanogaster is (NRPS) assembly lines that produce them (1, 2). NRPS systems a rare example of such a nonribosomal peptide synthetase are common in bacteria and fungi, where the products they from a higher eukaryote, where it plays a central role in the synthesize include antibiotics, siderophore-sensing and bacterial regulation of amine neurotransmitters. Here, we reveal that quorum-sensing regulators, toxins, and even compounds used the C-terminal portion of Ebony encodes an atypical peptide as anticancer agents and immunosuppressants (1). As these bond-forming nonribosomal peptide synthesis domain. Struc- peptides are synthesized independently from the ribosome, tural analysis shows that this domain adopts a fold not pre- this eliminates the requirement to utilize the “standard” pro- dicted by its primary sequence, and indicates how this domain teinogenic pool of amino acids, and to date over 500 different maintains its high degree of substrate specificity. monomers have been identified as being incorporated by NRPS machinery (1). However, further modification of the peptides is Author contributions: T.I., J.T., and M.J.C. designed research; T.I., J.T., M.H.H., and J.A.K. made possible via the mechanism of synthesis employed by typ- performed research; T.I., J.T., J.A.K., C.J.J., and M.J.C. analyzed data; and T.I. and M.J.C. ical NRPS assembly lines, which relies on a repeating modular wrote the paper. architecture built from varying catalytic domains (1, 2). Each The authors declare no conflict of interest. module is responsible for the incorporation of a single amino This article is a PNAS Direct Submission. M.A.M. is a guest editor invited by the Editorial Board. acid into the growing peptide; the core domains required for a minimal peptide extension module are an adenylation domain Published under the PNAS license. (A domain), a peptidyl carrier protein domain (PCP domain), Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 6DYM, 6DYN, 6DYO, 6DYR, and a condensation domain (C domain) (1). Catalytic activity and 6DYS). begins with the A domain selecting the desired monomer, which 1To whom correspondence may be addressed: Email: [email protected] or max. is then activated using ATP before attachment to the phospho- [email protected]. pantetheine moiety of the neighboring PCP domain. As a thio- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ester, this residue is then delivered to the C domain, where a 1073/pnas.1811194116/-/DCSupplemental. peptide bond is formed between the upstream PCP-bound Published online January 31, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1811194116 PNAS | February 19, 2019 | vol. 116 | no. 8 | 2913–2918 Downloaded by guest on September 27, 2021 followed by sequences usually not found in archetypical NRPS for both the PCP-bound amino acid and aromatic amine sub- assembly lines (4). Examples of such NRPS enzymes are mostly strates that fits the biological functions of Ebony. those involved in mammalian lysine metabolism (5): AASDH, a 2- aminoadipic 6-semialdehyde dehydrogenase harboring an unusual Results A-PCP-PQQ arrangement (where PQQ represents a sequence As the condensation function of Ebony appeared to rely on the containing seven binding motifs for pyrroloquinoline quinone); unusual C-terminal portion of this enzyme, we concentrated on and Lys2 (6), an α-aminoadipate reductase with an A-PCP- the characterization of this atypical condensation-like domain. NADPH–binding domain architecture. In addition to these well- Previous work has shown that Ebony is highly prone to degra- characterized mammalian enzymes, Ebony from D. melanogaster dation during E. coli expression, and we used this fact to our contains an A-PCP di-domain followed by an uncharacterized advantage to identify an optimal C-terminal construct based on sequence with no sequence homology to any known proteins proteolysis of the full-length protein during overexpression. This based on standard search using domain prediction servers (Fig. 1). region, encompassing the residues from Leu666 to the C-terminal Ebony is an 879-residue protein (98.5 kDa) expressed in both glial residue Lys879 (referred to here as CN, for the amine-selecting C and cuticular cells (7, 8). In glial cells, Ebony is involved in his- domain; Fig. 2), was well behaved and highly soluble when tamine regulation (the main neurotransmitter in the optical expressed with a C-terminal 6xHis tag. With the ability to access nerve system) and plays an essential role in neurotransmitter significant amounts of highly pure protein, we then turned to the inactivation through conversion to carcinine [β-alanyl-histamine; structural characterization of this domain. Fig. 1 (9)]. Similarly, in cuticular cells Ebony catalyzes the con- densation of β-alanine with dopamine to form β-alanyl-dopamine, Structural Characterization and Substrate Binding of the Ebony a metabolite involved in the pigmentation and sclerotization of the Condensation-Like Domain. The optimized Ebony condensation- insect cuticle. Mutants display strong phenotypes, with alteration like domain (CN) crystallized readily, forming numerous needle of vision (10), circadian regulation of locomotor activity (11), and clusters in a wide range of conditions. To obtain diffraction- cuticle sclerotization in affected flies. quality crystals, several rounds of condition optimization com- Ebony is an unusually fast NRPS enzyme (12), which can bined with crystal seeding were required, which yielded crystals achieve a condensation reaction
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