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

Abstract

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. 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. The ebony mutation (1Bridges C.B. Morgan T.H. Publ. Carnegie Inst. 1923; 327: 50Google Scholar) affects cuticle tanning and sclerotization as well as nervous system functions like vision (2Hotta Y. Benzer S. Nature. 1969; 222: 354-356Crossref PubMed Scopus (191) Google Scholar) and behavior (3Kyriacou C.P. Burnett B. Connolly K. Anim. Behav. 1978; 26: 1195-1206Crossref Scopus (45) Google Scholar, 4Newby L.M. Jackson F.R. J. Neurogenet. 1991; 7: 85-101Crossref PubMed Scopus (72) Google Scholar). Previous biochemical investigations (5Perez M. Wappner P. Quesada-Allue L.A. Insect Biochem. Mol. Biol. 2002; 32: 617-625Crossref PubMed Scopus (14) Google Scholar, 6Wright T.R.F. Scandalios J.G. Caspari E.W. Advances in Genetics. Vol. 24. Academic Press, Inc., San, Diego1987: 127-209Google Scholar) led to the conclusion that Ebony has β-alanyl-dopamine synthetase function. The visual phenotype of ebony is reflected by a reduced phototaxis and optomotor response (7Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1967; 58: 1112-1119Crossref PubMed Google Scholar, 8Heisenberg M. J. Comp. Physiol. 1972; 80: 119-136Crossref Scopus (63) Google Scholar). Based on electroretinogram measurements that lack “on” and “off” transients, the visual phenotype has been attributed to altered responses in cells postsynaptic to the terminals of retinula cells R1-R6 of the lamina (2Hotta Y. Benzer S. Nature. 1969; 222: 354-356Crossref PubMed Scopus (191) Google Scholar, 9Heisenberg M. J. Exp. Biol. 1971; 55: 85-100PubMed Google Scholar). Histamine, which has been identified as neurotransmitter at these terminals, is released when retinula cells are depolarized (10Sarthy P.V. J. Neurochem. 1991; 57: 1757-1768Crossref PubMed Scopus (72) Google Scholar). Because Ebony is expressed in the lamina glia surrounding the photoreceptor terminals that lack dopamine immunoreactivity, we previously proposed that glial expression reflects histamine conjugation to β-alanine (11Richardt A. Rybak J. Stortkuhl K.F. Meinertzhagen I.A. Hovemann B.T. J. Comp. Neurol. 2002; 452: 93-102Crossref PubMed Scopus (65) Google Scholar). Recent evidence from the elucidation of histamine metabolism in fly heads (12Borycz J. Borycz J.A. Loubani M. Meinertzhagen I.A. J. Neurosci. 2002; 22: 10549-10557Crossref PubMed Google Scholar) supported our above-mentioned hypothesis. Throughout fly development, the optic neuropile is not the sole but the major site of ebony expression in the nervous system (11Richardt A. Rybak J. Stortkuhl K.F. Meinertzhagen I.A. Hovemann B.T. J. Comp. Neurol. 2002; 452: 93-102Crossref PubMed Scopus (65) Google Scholar, 13Hovemann B.T. Ryseck R.-P. Walldorf U. Störtkuhl K.F. Dietzel I.D. Dessen E. Gene (Amst.). 1998; 221: 1-9Crossref PubMed Scopus (67) Google Scholar). This finding raised the intriguing possibility that β-alanylation reflects an as yet unknown general pathway for biogenic amine neurotransmitter inactivation. A direct approach and first step to support this hypothesis therefore is to determine the biochemical activity of purified Ebony protein in vitro. Along these lines, it is important to consider that the close relationship of Ebony to the family of nonribosomal peptide synthetases (NRPSs) 1The abbreviations used are: NRPS, nonribosomal peptide synthetase; P-pant, phosphopantetheinyl; e-cDNA, ebony-cDNA; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; GABA, γ-aminobutyric acid; WT, wild type; UAS, upstream activating sequence. should be reflected by a comparable reaction mechanism of peptide bond formation (Fig. 1). NRPSs are of modular structure with each module incorporating one amino acid into the final product. Amino acid binding to the module is a rather specific two-step process, which involves selection of the cognate amino acid and activation as aminoacyladenylate in the adenylation domain followed by transfer to a 4′-phosphopantetheinyl (P-pant) group attached to an invariant serine of a neighboring thiolation domain (Fig. 2). A comparison of the Ebony amino acid sequence with that of known NRPSs reveals solely the core motifs of a putative adenylation and thiolation domain, an arrangement that is normally found in initiation modules (Fig. 3). Therefore, only the initial step of the reaction, namely amino acid activation, can follow the thiotemplate mechanism described for NRPSs (Fig. 2). Interestingly, the C-terminal 230 amino acids of the Ebony protein do not share homology with any known NRPSs and might represent a novel type of domain responsible for the selection of dopamine. In this report, we provide evidence by mutational analysis and in vitro assays that Ebony exclusively activates one substrate amino acid, β-alanine, in a two-step process hitherto only known from microbial peptide synthetases. However, nucleophilic release of β-alanine is not restricted to dopamine alone but instead Ebony accepts a variety of biogenic amines.Fig. 3Ebony gene organization and NRPS sequence homology. In 15 kb of genomic ebony DNA, SalI (S), XhoI (X), and BamHI (B) restriction sites are shown. Filled boxes indicate the seven ebony gene exons. The lower bar delineates the genomic DNA cloned in front of gal4 to facilitate ebony-like cuticle expression in an ebony-gal4 transformant line. In the blow up, a bar comprising the ebony reading frame and the sites of homology to NRPS consensus core sequences A1–A10 and T (29Marahiel M.A. Stachelhaus T. Mootz H.D. Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (910) Google Scholar) are indicated. The degree of amino acid conservation in Ebony as compared with the NRPS consensus sequence is listed below.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fly Stocks—Drosophila melanogaster were cultured on standard corn meal medium. The wild type strain examined was Canton-S. w1118 ; In(3R)eAFA (14Hazelrigg T. Levis R. Rubin G.M. Cell. 1984; 36: 469-481Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 15Caizzi R. Ritossa F. Ryseck R.-P. Richter S. Hovemann B. Mol. Gen. Genet. 1987; 206: 66-70Crossref Scopus (20) Google Scholar) served as ebony null mutant in white background for rescue transformation. EbonyHis-6 Production in S2 Cells—The complete ebony reading frame was PCR-amplified from the previously described cDNA clone (13Hovemann B.T. Ryseck R.-P. Walldorf U. Störtkuhl K.F. Dietzel I.D. Dessen E. Gene (Amst.). 1998; 221: 1-9Crossref PubMed Scopus (67) Google Scholar) by using a 5′-KpnI primer 5′-GGTACC ATGGGTTCGCTGCCACAATTGTCG-3′ and a 3′-AgeI primer 5′-ACCGGT TTTGCCCACCTCCTTCCAATGGAC-3′. The product was cloned via KpnI and AgeI into the pMT-V5-HisA vector (Invitrogen), yielding pMT-ebony-HisA, which codes for the Ebony protein with a tag of six C-terminal histidines attached by a threonine-glycine bridge. A stable cell line was obtained by selection with hygromycin B according to the manufacturer's directions. EbonyHis-6 expression was induced by treatment with 0.5 mm CuSO4 for 24 h. Cells were lysed, and the protein was affinity-purified on nickel-nitrilotriacetic acid-agarose (Qiagen, Hilden, Germany) according to the manufacturer's instructions. EbonyHis-6 Production in E. coli BL21(pREP4-gsp)—Ebony cDNA was PCR-amplified and cloned in the BamHI site of pQE60 (Qiagen). E. coli BL21(pREP4-gsp) cells were transformed with the construct. Expression was induced from cells in early log phase with 0.1–0.4 mm isopropyl-1-thio-β-d-galactopyranoside overnight at 30 °C. The bacterial cell pellet was lysed in 50 mm Hepes, 300 mm NaCl, pH 8.0, with three rounds of French press treatment (1000 p.s.i.). From the cell lysate, Ebony was purified by affinity chromatography on nickel-nitrilotriacetic acid-agarose. Pooled enzyme fractions were dialyzed against 50 mm Na3PO4, 100 mm NaCl, pH 7.0. Protein concentration was determined according to Bradford (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). ATP-pyrophosphate Exchange Assay—The ATP-pyrophosphate exchange assay was carried out according to Stachelhaus et al. (17Stachelhaus T. Mootz H.D. Bergendahl V. Marahiel M.A. J. Biol. Chem. 1998; 273: 22773-22781Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar) with modifications. In a final volume of 100 μl, the reaction mixture contained 500 nm affinity-purified enzyme, 1 mm amino acid, 50 mm sodium phosphate buffer, pH 7.0, 25 mm MgCl2, 1.0 mm ATP, 50 μm tetra-sodium pyrophosphate, and 0.5 μCi (30 Ci/mmol) of tetra-[32P]sodium pyrophosphate (PerkinElmer Life Sciences). After incubation at 37 °C for 15 min, the reaction was quenched by the addition of cold stop mixture containing 0.1 m sodium pyrophosphate, 0.56 m perchloric acid, and 1.2% (w/v) activated charcoal (Norit A, Sigma). The charcoal was pelleted by centrifugation, washed twice with 1 ml of water, and resuspended in 0.5 ml of water. After addition of ml of the was determined by of reaction mixture contained 500 nm enzyme, 1 mm ATP, and μCi of Ci/mmol) and 25 mm in 50 mm sodium phosphate buffer, pH 7.0. After a incubation at 37 the reaction was by the addition of 15 of (w/v) and of (w/v) After 30 on the was pelleted by and washed twice with 1 ml of The pellet was in of was in ml of of Ebony with its cognate amino acid, a was carried out for 15 assay The reaction was by the addition of the amino group containing reaction in a final concentration of 1 addition of the second 30 1 min, and min, were for acid and as described reaction product for the reaction mixture was 0.5 mm enzyme, 1 mm β-alanine, 1 mm biogenic 25 mm MgCl2, and 1 mm in 50 mm sodium phosphate buffer, pH 7.0, in a final volume of 1 ml were for at 37 °C. the the reaction was by the addition of After transfer to a the was The pellet was in 1 ml of and the were for 15 to the were into an for a to Ebony a activation process, of the of an invariant in the thiolation domain of was to the function of mutation should Ebony activity as previously in mutational analysis of synthetase B. P. J. PubMed Scopus Google Scholar). on functional activity of can be in Drosophila in by cuticle of Expression of a should to wild type function in an ebony mutant should this we of Drosophila transformant in w1118 ; In(3R)eAFA mutant was transformed to provide the at the sites of Ebony In transformant were that contained either a mutant or a wild type for In with ebony rescue we a genomic DNA as that with the upstream SalI site kb upstream of the ebony and into (Fig. (13Hovemann B.T. Ryseck R.-P. Walldorf U. Störtkuhl K.F. Dietzel I.D. Dessen E. Gene (Amst.). 1998; 221: 1-9Crossref PubMed Scopus (67) Google Scholar). cuticle activity of in the transformant line was in a with a line. activity as by to the at sites of Ebony expression the (Fig. Ebony was PCR-amplified and cloned into using previously cDNA (13Hovemann B.T. Ryseck R.-P. Walldorf U. Störtkuhl K.F. Dietzel I.D. Dessen E. Gene (Amst.). 1998; 221: 1-9Crossref PubMed Scopus (67) Google Scholar, PubMed Google Scholar). A second was a mutation in the frame was by from to into w1118 ; In(3R)eAFA in the wild type as well as in with the construct. the ebony line was with these to the the of the expressed Ebony we to that each of the transformed cDNA was of Ebony was with an raised against the C-terminal of the protein (11Richardt A. Rybak J. Stortkuhl K.F. Meinertzhagen I.A. Hovemann B.T. J. Comp. Neurol. 2002; 452: 93-102Crossref PubMed Scopus (65) Google Scholar). in protein from fly heads of wild type the line w1118 ; In(3R)eAFA and the lines, w1118 ; In(3R)eAFA and w1118 ; In(3R)eAFA that Ebony was in but the w1118 ; In(3R)eAFA mutant line (Fig. 2). After of Ebony protein we the for In(3R)eAFA mutant cuticle rescue (Fig. of wild type Ebony in an to a rescue of the mutant phenotype as by of the cuticle in fly the a cuticle to that of the ebony mutant line (Fig. fly We that the mutant Ebony protein was not in the mutant Because the mutation is the thiolation T (Fig. this is a to amino acid binding of Ebony by thioester Expression of Ebony evidence has to be by biochemical of Ebony Ebony its amino acid substrate by the two-step process that is known from one has to the of an enzyme activation step the transfer of a to (Fig. 2). of activated Ebony we of ebony We used Schneider S2 cells J. Exp. 1972; Google Scholar) that the expression system might provide the activation activity and in was cloned into Schneider S2 cells were transformed with this and a transformed cell line was and affinity of the protein in a protein according to assays on amino acid selection and activation as aminoacyladenylate activity for one amino acid the of Ebony was (Fig. 2). an for the of we first S2 Ebony was with the The from M. M.M. P. 1998; PubMed Scopus Google was therefore used for in vitro of S2 Ebony into the A was as substrate for Ebony a of was into Ebony not This that been to Ebony by activity in vitro. the and the of activated Ebony our Amino and by activated enzyme in we transformed wild type as mutant ebony cDNA cloned in pQE60 into E. coli This strain the from S. Stachelhaus T. Marahiel M.A. J. PubMed Google Scholar) and is used to activated NRPSs by expression in E. that Ebony NRPS amino acid activation and binding as thioester of affinity-purified protein was followed the in specificity of amino acid selection by exchange (17Stachelhaus T. Mootz H.D. Bergendahl V. Marahiel M.A. J. Biol. Chem. 1998; 273: 22773-22781Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). This which the reaction of the formation of from aminoacyladenylate and was used to the reaction specific amino acid selection and activation as In of the the formation of was determined an incubation of 15 putative we the acids in of three to six as well as β-alanine, γ-aminobutyric acid and acid as Because this assay only activation of the cognate amino acid as aminoacyladenylate of binding as it be with of the three enzyme The obtained were with S2 E. coli expressed wild and E. coli expressed mutant Ebony not which that amino acid activation and are The were using either E. coli expressed wild type or mutant Ebony The of into was obtained when β-alanine was used as a The from at three measurements with enzyme was as (Fig. of the acids that were used in of were of this related to β-alanine as GABA, and acid not as substrate for the adenylation process of However, β-alanine was to one of the amino acid or to formation to (Fig. activity of mutant Ebony was from wild type enzyme (Fig. of the only β-alanine was as substrate and was as aminoacyladenylate by amino acid binding as thioester was determined by incubation of E. coli wild type Ebony with from at three were as (Fig. with E. coli Ebony not as well as the of from the reaction mixture containing functional wild type Ebony (Fig. led to a This direct biochemical evidence that Ebony the amino acid β-alanine, activates it as and it as thioester of the 4′-phosphopantetheinyl group to from reaction step of peptide bond formation and product release is reflected by the of from Ebony with This is by complete of the bond formation that are to a β-alanine we Ebony with putative (Fig. to of a that an complete into product. We examined be We first containing a structure to an or the and the activity in the β-alanine release assay to of the starting (Fig. We to out the structure from a to an dopamine by the of to was comparable with the previously (Fig. Because was in a and a structure to an we used the containing as comparable activity to the release of was (Fig. The by using was when biogenic amines were by related and and with the of release of the activity in the release assay when was used as substrate that the structure not peptide activity (Fig. However, when the amino group was close to the structure as in for releasing activity was We from these that a of an or group with a structure is to bond formation with We a amino group to an β-alanine in only as of the as found in and a of was we examined a contained in a putative substrate release we used and to and β-alanine release was not (Fig. the group these from as at the amino group be the major for the acids β-alanine, GABA, and acid (Fig. should a release activity with of the function from the amino A comparison of the obtained with and acid, that a group in product the structure for β-alanine release is an or group to an the in these only the biogenic amines and can be to a major in Drosophila of from Ebony is an of activity but direct of product of the and we product assays with β-alanine and the biogenic were by a assays were and with the mutant wild type using wild type the of the were the were when wild type Ebony was by the are in analysis using Ebony an Amino to NRPSs to the family of K. M. Marahiel M.A. Mol. PubMed Scopus Google Scholar). A to the process, is in addition to NRPSs only in the group of and acid T. A. Marahiel M.A. Chem. Biol. Full Text PDF PubMed Scopus Google Scholar, P. M. Marahiel M.A. R. Chem. Biol. Full Text PDF PubMed Scopus Google Scholar, E. M. PubMed Scopus Google Scholar). The homology and acid and Ebony is to the core sequence of the thiolation domain, which the invariant the and Ebony to be activated by which a for amino acid sequence motifs in previously P. M. Marahiel M.A. R. Chem. Biol. Full Text PDF PubMed Scopus Google we identified a reading frame in the Drosophila that a homology to this Expression of the putative cDNA in E. coli to a protein that in vitro S2 Ebony activity on the of comparable with the that the higher Drosophila has an amino acid activation mechanism that was to be specific for microbial NRPSs T. J. V. A. B. P. M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Ebony this with a functional domain that peptide bond formation with a group of However, a or modules that the activation of amino acids and the formation of has not yet been in higher as been to in T. E. Life Sci. PubMed Scopus (45) Google Scholar). the of a activation domain in nonribosomal of in higher be However, it amino acid activation modules in addition to a functional domain for of the activated amino acids as well as a activity for peptide release (29Marahiel M.A. Stachelhaus T. Mootz H.D. Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (910) Google Scholar). that this structure of NRPS activity has been through to higher is that Ebony a novel two-step reaction mechanism amino acid activation and binding followed by peptide bond The of amino acid activation and binding that of NRPSs. bond formation and product release a nucleophilic of an amine that must the This is in NRPSs in The of peptide bond amino acid is by the specificity of a second adenylation domain the enzyme, and in a domain the modules is for peptide bond a domain is in a C-terminal domain (Fig. with a yet unknown function to be responsible for the nucleophilic of the amines substrate on the activated thioester group of The mechanism of this reaction and that of dipeptide product release are Ebony in Ebony is expressed in at (11Richardt A. Rybak J. Stortkuhl K.F. Meinertzhagen I.A. Hovemann B.T. J. Comp. Neurol. 2002; 452: 93-102Crossref PubMed Scopus (65) Google Scholar). system activity has been from the and visual phenotype of the mutant but was only by activity of an gene transformant and by (11Richardt A. Rybak J. Stortkuhl K.F. Meinertzhagen I.A. Hovemann B.T. J. Comp. Neurol. 2002; 452: 93-102Crossref PubMed Scopus (65) Google Scholar, 13Hovemann B.T. Ryseck R.-P. Walldorf U. Störtkuhl K.F. Dietzel I.D. Dessen E. Gene (Amst.). 1998; 221: 1-9Crossref PubMed Scopus (67) Google Scholar). The that evidence for in the lamina was K. J. Chem. Google Scholar) led to that that Ebony is involved in formation in the eye (12Borycz J. Borycz J.A. Loubani M. Meinertzhagen I.A. J. Neurosci. 2002; 22: 10549-10557Crossref PubMed Google Scholar). The of the biogenic amines and that functions in Drosophila to β-alanine might a function of Ebony at specific sites of the T.R.F. Scandalios J.G. Caspari E.W. Advances in Genetics. Vol. 24. Academic Press, Inc., San, Diego1987: 127-209Google Scholar, A. Rybak J. Stortkuhl K.F. Meinertzhagen I.A. Hovemann B.T. J. Comp. Neurol. 2002; 452: 93-102Crossref PubMed Scopus (65) Google Scholar). we provide evidence that Ebony is of binding biogenic amines histamine to Therefore, it is to to Ebony a function in histamine neurotransmitter metabolism at the photoreceptor of the Because histamine J. Neurosci. PubMed Google Scholar) as well as (10Sarthy P.V. J. Neurochem. 1991; 57: 1757-1768Crossref PubMed Scopus (72) Google Scholar) in the eye is the must be by a fast system J. M. M. E. J. Neurosci. 1998; PubMed Google Scholar, Morgan E. J. Neurosci. PubMed Google Scholar). Therefore, at the the postsynaptic cell by a mechanism of fast of histamine from the is Interestingly, in photoreceptor was the photoreceptor terminals, incubation in the the was found at the glia Morgan E. J. Neurosci. PubMed Google Scholar). This support to the that at a fast of out of the be by of histamine into the surrounding glia it be by Ebony via β-alanine The that β-alanine is in the glia to Ebony for histamine and a biochemical pathway that the of the histamine in the histamine into photoreceptor as well as into glia has been previously J. M. M. E. J. Neurosci. 1998; PubMed Google Scholar, Morgan E. J. Neurosci. PubMed Google it to be a mechanism that and of histamine release glia followed by inactivation by β-alanine histamine from the is for the function of photoreceptor that with release of In vitro product formation from Ebony with histamine or any of the biogenic amine was the that be determined standard assay This is from the reaction for a function in neurotransmitter inactivation. this reaction the biogenic amines as of and of biogenic amines specific in the Ebony can the for neurotransmitter inactivation. We K. Dietzel and for reading of the We for out the