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Bimodular Peptide Synthetase SidE Produces Fumarylalanine in the Human Pathogen Aspergillus fumigatus

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Bimodular Peptide Synthetase SidE Produces Fumarylalanine in the Human Pathogen Aspergillus fumigatus
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    Published Ahead of Print 23 August 2013. 10.1128/AEM.02642-13. 2013, 79(21):6670. DOI: Appl. Environ. Microbiol. HoffmeisterSchrettl, Hans-Martin Dahse, Hubertus Haas and Dirk Wieland Steinchen, Gerald Lackner, Sabiha Yasmin, Markus  Pathogen Aspergillus fumigatusProduces Fumarylalanine in the Human Bimodular Peptide Synthetase SidE http://aem.asm.org/content/79/21/6670Updated information and services can be found at: These include:  SUPPLEMENTAL MATERIAL  Supplemental material REFERENCES http://aem.asm.org/content/79/21/6670#ref-list-1at: This article cites 45 articles, 13 of which can be accessed free CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:  http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:  on J  un e1  0  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om  on J  un e1  0  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   Bimodular Peptide Synthetase SidE Produces Fumarylalanine in theHuman Pathogen  Aspergillus fumigatus Wieland Steinchen, a Gerald Lackner, a Sabiha Yasmin, b Markus Schrettl, b Hans-Martin Dahse, c Hubertus Haas, b Dirk Hoffmeister a Department of Pharmaceutical Biology at the Hans-Knöll-Institute, Friedrich-Schiller-Universität, Jena, Germany a ; Division of Molecular Biology/Biocenter, Innsbruck Medical University, Innsbruck, Austria b ; Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology—Hans-Knöll-Institute, Jena,Germany c The filamentous mold  Aspergillus fumigatus  causes invasive aspergillosis, a potentially life-threatening infectious disease, inhumans. The  sidE   gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[ 32 P]pyro-phosphate exchange assay   in vitro , which identified fumarate and  L -alanine, respectively, as the preferred substrates. Using full-length  holo -SidE, fumarylalanine (FA) formation was observed  in vitro . Furthermore, FA was identified in  A. fumigatus  culturesupernatants under inducing conditions, unless  sidE   was genetically inactivated. As FA is structurally related to establishedpharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulenceof   A. fumigatus . T he fungus  Aspergillus fumigatus  is an opportunistic pathogen,particularlywithimmunocompromisedpatients.Itrepresentsthe most common etiological agent of invasive aspergillosis,whichisassociatedwithmortalityratesashighas30%to90%(1).  A. fumigatus  small molecules, such as gliotoxin, are thought of ascontributing to virulence (2). Consequently, the secondary  metabolome of this fungus has been object of intensive research,with an emphasis on nonribosomal peptide synthetases (NRPSs).These are multidomain enzymes, which include all catalytic func-tions to assemble complex metabolites from amino acids or otherbuilding blocks (3). Adenylation (A) domains are integral com-ponents of NRPSs, which select and activate the monomeric sub-strates. The function of 8 of 14 nonribosomal peptide synthetases(NRPSs) (FtmA, GliP, HasD, PesL, PesM, Pes1, SidC, SidD) in  A. fumigatus  was previously clarified (4–10). The metabolite of aninth NRPS (Pes3) has not yet been identified but is predicted toplay a structural role (11). The  A. fumigatus  peptide synthetasegene  sidE  , which encodes a bimodular NRPS, is located in thevicinity of the genetic locus that is essential for biosynthesis of thesiderophorefusarinineC.Therefore,SidEwasexpectedtoserveinsiderophoreproduction,too(12).However,unlikethatofsidero- phore-related genes, its expression depends on the presence of LaeA, a positive regulator of virulence factors and secondary me-tabolismof   A. fumigatus (13,14).Intriguingly, sidE  isupregulated16-fold in  Aspergillus  germlings growing in the neutropenic mu-rine lung and is therefore believed to play a role in virulence (15). Here, we present genetic and biochemical evidence that SidE isunrelated to siderophore biosynthesis but has fumarylalanine(FA) synthetase activity. MATERIALS AND METHODS General.  Tetrasodium [ 32 P]pyrophosphate (59.9 Ci/mmol) was fromPerkinElmer. Plasmid propagation was done in  Escherichia coli  XL1 blue.Transformation of   A. fumigatus  was carried out as described previously for  A.nidulans (16).  A.fumigatus strains(seeTableS1inthesupplementalmaterial)wereroutinelyculturedat37°Conminimalmediumcontaining1%glucoseasacarbonsource,20mMglutamineasanitrogensource,and10   M FeSO 4 . The medium was supplemented with 1.5 mM FeSO 4  forhigh-iron conditions, whereas iron was omitted for iron-depleted condi-tions. Inactivation of   sidE  .  The construct to inactivate  sidE   was made by amplifying a 5.1-kb fragment of the  A. fumigatus sidE   gene by PCR using Taq polymeraseandprimersoAf538PS2-2andoAf538PS2-1(seeTableS2in the supplemental material). Conditions were as follows: 32 cycles of 95°C for 60 s, 56°C for 60 s, and 72°C for 4 min. The product was subse-quently cut with BglII and ligated to the BamHI site in pBluescript-KS(Stratagene), resulting in pPS2. A 4-kb NheI-DraI fragment containingthe hygromycin resistance cassette was removed from pAN7-1 (17) and insertedbetweentheNheIandNruIsitesofpPS2toobtainpPS2hph.The6.2-kb SmaI-SpeI fragment of pPS2hph containing the disrupted genewas purified by agarose gel electrophoresis and used for transformationafter recovery from the gel. Putative mutants were selected based on theirantibiotic resistance as confirmed by Southern blotting of MluI-digestedgenomic DNA. The  sidE  -specific hybridization probe was generated by amplifyingafragmentofpPS2hphusingaT7universalprimerandohph2andthePCRconditionsdescribedabove,exceptfortheshorterelongationtime (2 min). Complementation of   A. fumigatus   sidE  .  To complement  A. fu-migatus   sidE  , it was transformed with cosmid psidD-COS, which wasidentifiedpreviouslyandincludesthe sidE  gene(9),togetherwithplasmid pSK275 that confers pyrithiamine resistance (18). Ectopic integration of  the cosmid psidD-COS in pyrithiamine-resistant transformants was con-firmed by Southern analysis. Northern analysis.  Liquid cultures (18 h old) of   A. fumigatus  wt and  sidE   strains grown at 37°C under iron-depleted, iron-replete, and high-iron conditions were exposed to H 2 O 2  (2 mM) or increased temperature(48°C) and grown further for 2 h. Total RNA was isolated using TRI Received  7 August 2013  Accepted  19 August 2013 Published ahead of print  23 August 2013Address correspondence to Dirk Hoffmeister, dirk.hoffmeister@hki-jena.de, orHubertus Haas, hubertus.haas@i-med.ac.at.W.S., G.L., and S.Y. contributed equally to this article.Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.02642-13.Copyright © 2013, American Society for Microbiology. All Rights Reserved.doi:10.1128/AEM.02642-13 6670  aem.asm.org Applied and Environmental Microbiology p. 6670–6676 November 2013 Volume 79 Number 21   on J  un e1  0  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   reagent (Sigma-Aldrich). Total RNA (10  g) was fractionated in formal-dehyde-agarosegels,blottedontoHybondNmembranes,andhybridizedas described by the manufacturer (Amersham Biosciences). The hybrid-ization probes were generated by PCR using primers oSidE1 and oSidE2(seeTableS2inthesupplementalmaterial).RNAfromuntreatedmyceliaof the wt strain grown for 24 h under iron-depleted, iron-replete, andhigh-iron conditions served as the control to assess the expression of   sidE  under these stress conditions. Constructionofgeneexpressionplasmids. TheportionencodingtheSidE A 1 T 1  didomain (codons 1 to 638) was synthesized (GenScript) as agene optimized for  E. coli  codon usage. The gene was inserted via theBamHIandHindIIIsitesintoexpressionvectorpRSETbtocreateplasmidpWS2. The portion encoding the 660-amino-acid (aa) A 2 T 2  didomain(codons 1028 to 1687) was amplified by PCR from genomic DNA, usingoligonucleotide primers sidE-F2 and sidE-R2 (see Table S2 in the supple-mental material), which introduced BamHI and HindIII sites, respec-tively. The reaction mixture (100  l) consisted of 2 mM MgSO 4 , 0.2 mM(each) deoxynucleoside triphosphate (dNTP), 20 pmol (each) primer,and 2.5 U  Pfu  DNA polymerase. The reaction was performed using thefollowingthermocyclingparameters:90sat95°C;32cyclesof95°Cfor30s,61°Cfor30s,and72°Cfor4min;andaterminalholdfor5minat72°C.To create expression plasmid pWS4, this PCR product was ligated to theBamHI and HindIII sites of pRSETb. The full-length  sidE   gene (see TableS3inthesupplementalmaterial)wasreconstitutedbyconsecutivelycom-bining gene portions as follows: (i) a 1.4-kb XhoI-HindIII PCR fragmentwas inserted into pWS2 to yield pWS22; (ii) the BstBI-HindIII fragmentof pWS4, inserted into pWS22, yielded pWS223; and (iii) a synthetic,codon-optimized 1,559-bp fragment was restricted with PstI and HindIIIandinsertedintopWS223toyieldexpressionplasmidpWSE.Itcarriesthecomplete 6,330-bp  sidE   reading frame (see Fig. S1 in the supplementalmaterial). Enzyme characterization.  Gene expression was accomplished in  E.coli BL21(DE3)transformedwithplasmidspWS2andpWS4,respectively,toheterologouslyproduceSidEA 1 T 1 andA 2 T 2 didomains,orwithpWSEto produce the complete enzyme, as  N  -terminally hexahistidine-taggedfusion proteins. Transformed strains were grown in LB broth, inducedwith 0.5 mM IPTG (isopropyl-  - D -thiogalactopyranoside) for 48 h (24 hfor didomain proteins) at 16°C, and shaken at 180 rpm. Proteins werepurified by metal affinity chromatography using nickel-nitrilotriaceticacid (Ni-NTA) (Qiagen), and the procedure was carried out according tothe manufacturer’s manual. The enzymes were desalted on PD-10 col-umns (GE Healthcare) equilibrated with the corresponding respectiveassay buffers (below). Protein concentrations were determined by Brad-ford’s method (19). TheATP-[ 32 P]pyrophosphateexchangeassaywasusedtocharacterizethe substrate specificities of both SidE adenylation domains. All reactionswererunintriplicateandinatotalvolumeof100  lat37°Cusing100mMTris-HClbuffer(pH7.5);reactionmixturesincluded5mMMgCl 2 ,5mMATP, 100 nM purified truncated SidE A 1 T 1  or A 2 T 2  didomain proteins,0.1 mM [ 32 P]pyrophosphate, and 1 mM fumarate or  L -alanine (or othersubstrates). The reactions proceeded for 30 min as described previously (20). The tested substrates included the 20 proteinogenic  L -amino acidsand  L -ornithine, pyruvic acid, oxaloacetic acid,  -ketoglutaric acid, phe-nylpyruvic acid, 4-hydroxyphenylpyruvic acid, indolyl-3-pyruvic acid,oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, tartaricacid, citric acid, benzoic acid, 4-hydroxybenzoic acid, salicylic acid, and2,3-dihydroxybenzoic acid. Productformation in vitro . apo -SidEwasconvertedtothe holo -formby the use of   A. nidulans  NpgA 4 = -phosphopantetheinyltransferase as de-scribed previously (21). Product formation was accomplished with 200 nM  holo -SidE in a reaction mixture consisting of phosphate buffer (60mM, pH 7.5), MgCl 2  (5 mM), ATP (5 mM), EDTA (60   M), 1 mMfumaricacid,and1mM L -[ 14 N]alanine.Aparallelreactionwasrunwith1mM(each) L -[ 15 N]alanineandfumaricacid.Thereactionvolumewas100  l. Incubation was performed at 37°C for 90 min and 11 h, respectively,for the two reactions. A reaction which included water instead of ATPserved as a control. Chemicalanalysis. AnalysisofFAwasperformedonaThermoAccelainstrument (for liquid chromatography [LC]) coupled to an Exactive or-bitrap spectrometer (for high-resolution mass spectrometry [HRMS]) innegative mode using electrospray ionization and equipped with a C 18 column (Grom-Sil 100 ODS-0 AB) (3-  m particle diameter; 250 by 4.6mm). The LC solvents were 0.1% formic acid in water (solvent A) andacetonitrile (solvent B). After an initial hold at 5% solvent B for 1 min, alineargradientupto98%solventBwithin15minwasrunataflowrateof 0.9 ml/min. To detect FA in  A. fumigatus  cultures, wild-type (wt),  sidE  ,and sidE  complementation( sidE  C )strainsweregrownin1literofglucoseminimal medium. The medium was inoculated with 4  10 5 conidia/mland incubated for 18 h at 37°C and then shifted to 50°C for heat stress foranother 12 h at 180 rpm. The mycelia were removed by filtration. Thesupernatant was acidified to pH 1 and extracted three times with ethylacetate. The organic phases were combined, concentrated  in vacuo  to 25ml,andtreatedwithanequalvolumeofsaturatedNaHCO 3 solution.Theaqueous phase was reacidified, extracted with ethyl acetate which wasevaporated to dryness, dissolved in methanol, and used for liquid chro-matography-mass spectrometry (LC-MS). Characterization and quanti-ficationofextracellularandintracellularsiderophoreswereperformedby reversed-phase high-performance LC (HPLC) as described previously (22). Preparation of fumaryl- L -alanine.  Fumaryl- L -alanine was synthe-sized following a published protocol (23). In brief, fumaric acid chloride monoethylester(1g)wasaddeddropwisetoanaqueoussolution(3.5ml)of   L -alanine (0.5 g) and sodium bicarbonate (0.5 g) on ice. The stirredreactionmixturewaskeptatpH7.5byaddingNaOH(1mM).Afterbeingwashed with chloroform, the product was extracted with ethyl acetate atpH 2 and crystallized from ethyl acetate at 4°C. For  13 C- and  1 H-nuclearmagnetic resonance (NMR) spectra of fumaryl- L -alanine, see Fig. S2 inthe supplemental material. Phylogenetic analysis.  Sequence alignments were performed by theMUSCLEalignmentalgorithm(24)implementedinMega5software(25). The alignment was subjected to Bayesian phylogenetic analysis usingMrBayes, program version 3.2 (26). The amino acid model with integra- tionoverapredeterminedsetoffixed-ratematrices(aamodelpr  mixed)was chosen as the evolutionary model. Two parallel chains were run untilthe average standard deviation of split frequencies reached 0.01. The re-sulting consensus tree was visualized in FigTree, developed by Andrew Rambaut and coworkers (http://tree.bio.ed.ac.uk/software/figtree/). Nucleotide sequence accession number.  The sequence of the com-plete 6,330-bp  sidE   reading frame has been deposited with GenBank un-der accession number KF544915. RESULTS AND DISCUSSION Genetic characterization of   sidE  .  The  A. fumigatus  gene  sidE  (Afu3g03350) encodes a 2,109-aa bimodular NRPS, which fea-tures the domain arrangement A 1 -T 1 -C 1 -A 2 -T 2 -C 2 . The gene islocated in close proximity to the biosynthetic genes for fusarininesiderophores (Fig. 1). To clarify the possible function of   sidE  , itwas inactivated by replacing the region encoding amino acids 437to 1332 by the hygromycin resistance marker gene. Accurate ge-netic manipulation was verified by Southern analysis of the  A. fumigatus  wild-type (wt) and  sidE   strains (see Fig. S3 in the sup-plementalmaterial).The  sidE  andwtstrainsweregrownfor24hunder iron-depleted conditions. The HPLC analysis of   sidE   cul-ture supernatants and extracts showed normal amounts of extra-cellular triacetylfusarinine C and fusarinine C and of intracellularferricrocin, respectively (see Fig. S4).These findings pointed away from a role of SidE for sidero-phore biosynthesis and prompted us to reinvestigate  sidE   expres-sionbyNorthernanalysisingreaterdetail.Asreportedpreviously   Aspergillus fumigatus  Peptide SynthetaseNovember 2013 Volume 79 Number 21 aem.asm.org  6671   on J  un e1  0  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   (13, 27), expression of   sidE   is somewhat upregulated by (i) ironstarvation in comparison to iron-replete conditions (10   MFeSO 4 ) and (ii) under high-iron conditions (1.5 mM FeSO 4 ; Fig.2). Since excessive amounts of iron result in oxidative stress, wehypothesized that  sidE   expression may be responsive to oxidizingagents. To address this issue,  A. fumigatus  was grown in iron-depletedandiron-repletemediumaswellasunderhigh-ironcon-ditions. The cultures were then separately exposed either to heatstress (48°C) or to oxidative stress (2 mM H 2 O 2 ) for 2 h. Bothtypesofstressresultedinasignificantinductionof  sidE  expression(Fig. 2). While heat shock induced  sidE   expression in both thepresence and the absence of iron, H 2 O 2 -mediated stimulation re-quired the presence of iron. In silico  prediction of substrate specificity.  Crystallography and biochemical evidence have established 10 key amino acid res-idues within the primary amino acid sequence of A domains thatgovern substrate selection (28–30). This set of signature residues, at times referred to as nonribosomal code, helps anticipate NRPSsubstrates and, thus, predict (partial) structures of the final NRPSproduct. NRPSpredictor2 (31) software was used to analyze theSidE adenylation domains and to extract the individual nonribo-somal specificity signatures. NRPSpredictor2 identified the key amino acid residues as S-A-R-G-T-V-S-Q-L-K, which is an un-precedented result, and yet the residues were predicted to repre-sent substrates related to hydroxybenzoic acid by this software.The first position/residue within the code (32) is particularly in- dicative of the general type of substrate (Fig. 1): in the case of an amino acid substrate, the first position is occupied by asparticacid, which was shown to stabilize the  -amino group of the sub-strate, whereas an asparagine reflects selectivity for an aryl acidsubstrate (30). Distinctive specificity signatures for aromatic  -keto acids (valine and alanine at positions 1 and 2) and anthra-nilic acid (a glycine residue at positions 1 and 8) have been veri-fied, too (33, 34). Thus, the presence of a serine in code position 1 oftheSidEA 1 domainpointedtoanon-amino-acidsubstrate.TheA 2  domain showed the signature D-V-Y-F-T-G-G-V-L-K, whichmay reflect an aliphatic, hydrophobic side-chain substrate.  A 1  T 1  C 1  A 2 T 2  C 2 SidE-A 1  (fumaric acid) AnaPS (anthranilic acid)TdiA (indole-3-acetic acid) AtrA (4-hydroxyphenylpyruvic acid)DhbE (2,3-dihydroxybenzoic acid)GliP (L-serine) SidE-A 2  2  (L-alanine) SidE SidDSidF SidH   A   f  u   3  G   0   3   3   7   0  A   f  u   3  G   0   3   3   8   0  A   f  u   3  G   0   3   3   9   0  A   f  u   3  G   0   3  4   3   0  A   f  u   3  G   0   3  4  4   0  A   f  u   3  G   0   3   3   3   0  A   f  u   3  G   0   3   3   2   0  AFUA12080 (anthranilic acid) FIG 1  (Top panel) Schematic map of   sidE   (black) and adjacent genes.  sidE   is intron free; other introns are not shown. Gray arrows represent genes which havebeen shown or are expected to function during siderophore biosynthesis; open arrows represent genes probably unrelated to siderophore production. Unchar-acterized genes are designated according to the  Aspergillus  Genome Database (ASPGD) (45). Products encoded by   sid   genes are as follows: SidE, FA synthetase;SidF,transacylase;SidH,mevalonyl-CoAhydratase;SidD,fusarinineCsynthetase(9,46).(Bottompanel)DomainsetupofSidEandthenonribosomalsubstrate specificity signature residues. Domain abbreviations are as follows: A, adenylation; T, thiolation; C, condensation. Data represent the deduced specificity codesofbothSidEAdomainscomparedtothecodesofselectedAdomainswhichadenylateaminoacids,  -ketoacids,anthranilicacid,or2,3-dihydroxybenzoicacid,based on alignment with the MUSCLE software. NRPS signature positions 1 to 10 are numbered according to the PheA sequence (32). Fe - Fe + Fe h Fe - Fe + Fe h A B48 °CH 2 O 2 rRNAunstressed FIG 2  Expression of   sidE   is transcriptionally upregulated by oxidative stressand increased temperature. (A) Total RNA was extracted from  A. fumigatus wild-type and   sidE   mycelia which were grown under iron-depleted (Fe  ),iron-replete (Fe  ), and high-iron (Fe h ) conditions for 18 h and then left un-stressed or shifted to oxidative stress (H 2 O 2 ) or heat stress (48°C). The RNAwas subjected to Northern analysis to assess the expression of   sidE  . Ethidiumbromide-stained rRNA is shown as a control for loading and the quality of theRNA. (B) An overexposed Northern blot of RNAs isolated from unstressedmycelia. Steinchen et al. 6672  aem.asm.org Applied and Environmental Microbiology   on J  un e1  0  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   Biochemical characterization of SidE substrate specificities. For a more profound insight into the substrates of SidE, weturned to heterologous production of the A 1 -T 1  didomain(72.8 kDa, 671 aa) and A 2 -T 2  didomain (75.7 kDa, 693 aa) as  N  -terminal hexahistidine fusion proteins in  E. coli  BL21(DE3).The respective coding sequences were cloned into expressionvector pRSETb. The produced  apo  proteins were subsequently profiled with regard to their specificities by examining the sub-strate-dependent exchange of the radiolabel from [ 32 P]pyro-phosphate (PP i ) to ATP. The substrates tested included L -amino acids,   -keto acids, and aryl as well as di- and tricar-boxylic acids, covering a total of 38 potential substrates. In-triguingly, the A 1  domain displayed a marked preference forfour-carbon dicarboxylic acids (Fig. 3): fumaric acid was ad- enylated best (ca. 401,000 cpm). Succinic acid and maleic acidwere turned over to a lesser degree (109,000 and 114,000 cpm,respectively), while the tested dicarboxylic acids with carbonchains shorter or longer than C4 failed to serve as substrates(  10,000 cpm). When the A 2  domain was tested with the samesubstrates as A 1 ,  L -alanine turned out to be the preferred sub-strate. Additionally, we included  D -alanine and   -alanine, asthe latter two occur, too, as monomers of microbial metabo-lites, e.g., bleomycin and cyclosporine, respectively (Fig. 3).When  L -alanine turnover was set at 100%, glycine,  L -valine,and  D -alanine showed levels of 28%, 16%, and 11%, respec-tively. All other compounds resulted in   10% ATP-PP i  radio-label exchange. Invitro fumaryl- L -alanineformation. Intriguingly, fumaryl- L -alanine (FA, Fig. 4), a natural product composed of thepreferred SidE substrates, has previously been isolated from  Aspergillus indicus  (35) and, as a racemic compound, from  Pen-icillium resticulosum  (36). Consequently, we heterologously produced hexahistidine-tagged full-length enzyme (237.0 kDa,2,142 aa; see Fig. S5 in the supplemental material) and the  A.nidulans  phosphopantetheinyltransferase NpgA to convert the apo  protein into  holo -SidE. It was then incubated with  L -ala-nine and fumaric acid, with each substrate added to reach a 1mM final concentration. Product formation was monitored by LC–high-resolution electrospray ionization–mass spectrome-try (LC-HRESIMS) and revealed a signal whose (i) retentiontime (6.5 min), (ii) accurate mass ( m / z   186.0407 [  M  -H]  ), (iii)mass fragmentation pattern (Fig. 4), and (iv) UV light-visiblelight (UV/Vis) spectrum (  max     218 nm) were identical tothose of synthetic FA. The found mass is in agreement with thetheoretical mass for FA ( m / z   186.0402 for C 7 H 8 NO 5 ). Theproduct formation assay was performed in parallel with fu-maric acid and  15 N- L -alanine as substrates. Consistent with theexpected activity and the isotope-labeled amino acid substrate,a signal of   m / z   187.0378 [  M  -H]  for C 7 H 8 [ 15 N]O 5  was de-tected, which is perfectly consistent with the theoretical mass.The FA signal was absent in a control reaction to which ATPhad not been added (Fig. 4). Apparently, the SidE reactionterminates in hydrolytic release of a linear product, which con-trasts with the function of other terminal C domains as cycliz-ing enzymes (37). Identification of   A. fumigatus  as an FA producer.  To testwhether FA is a natural product of   A. fumigatus , the metaboliteprofiles of   A. fumigatus  wt,  sidE  , and  sidE  C strains were reexam-ined under inducing conditions (heat stress, 50°C). Given the low pK a ofFA,thepHoftheculturesupernatantwasadjustedtopH1before extraction of the metabolites. The extracts were used forLC-MS measurements. We detected FA in the wt extracts (Fig. 4), and in accordance with our  in vitro  data, dramatically decreasedproductionofFAbythe  sidE  strainwasfound.Traceamountsof FA remained detectable by LC-MS. As  A. fumigatus  codes for aSidEparalog(NPS7),thesetracescouldbederivedfromtheactiv-ity of the latter. This would imply a redundantly encoded biosyn-thesis,asrecentlydescribedfor  Aspergillus flavus piperazines(38). ThecomplementationstrainproducedevenlargeramountsofFAthan the wt strain, most likely because of   sidE   overexpression.These results clearly demonstrate that  sidE   is involved in FA pro-duction  in vivo .Very little precedence for enzymatic fumaric acid adenylationin secondary metabolism exists, and such evidence as exists in-cludes DdaG (39), a bacterial coenzyme A (CoA)-ligase-type en- zyme for dapdiamide biosynthesis in  Pantoea agglomerans . SidEcontrasts with this enzyme in that the particular adenylating ac-tivity resides within a multidomain NRPS. Therefore, this work may help relate multiple uncharacterized fungal NRPSs to suchsecondarymetabolitesthatfeatureafumaratemoiety.Itshouldbenotedthatfumaricacidrepresentsabuildingblockinotherfungalpeptidicnaturalproducts;amongthosearethesorbicillactonesof a marine  Penicillium chrysogenum  strain (40), xylariamide A of a  Xylaria  species (41), and the thrombine inhibitor Ro09-1679 (fu- maryl- L -arginyl- L -leucyl-arginal,isolatedfrom  Mortierella alpina ;42). Strikingly, the sorbicillactones from  Penicillium chrysogenum 04080120160   g    l  y  c   i  n  e   L -  a   l  a  n   i  n  e   D -  a   l  a  n   i  n  e  ß -  a   l  a  n   i  n  e   L -  c  y  s   t  e   i  n  e   L -  s  e  r   i  n  e   L -   t   h  r  e  o  n   i  n  e   L -  v  a   l   i  n  e   L -   i  s  o   l  e  u  c   i  n  e  w  a   t  e  r   L -   l  e  u  c   i  n  e 0100200300400500600   o  x  a   l   i  c   a  c   i  d   m  a   l  o  n   i  c   a  c   i  d   s  u  c  c   i  n   i  c   a  c   i  d    f  u  m  a  r   i  c   a  c   i  d   m  a   l  e   i  c   a  c   i  d   L -   t  a  r   t  a  r   i  c   a  c   i  d  c   i   t  r   i  c   a  c   i  d   w  a   t  e  r  A 1  domainA 2  domain   r  a   d   i  o   l  a   b  e   l  e  x  c   h  a  n  g  e   [  c  p  m   x   1   0   0   0   ] FIG 3  Substrate specificity of SidE. The A domains were separately investigated (left panel, A 1  domain; right panel, A 2  domain) using the substrate-dependentATP-[ 32 P]pyrophosphate exchange assay. Diagrams represent assays with individual substrates. Error bars indicate the standard deviations.  Aspergillus fumigatus  Peptide SynthetaseNovember 2013 Volume 79 Number 21 aem.asm.org  6673   on J  un e1  0  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om 
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