Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Cambridge Commonwealth TrustA biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not all the suggested intermediates could be detected experimentally. Here we show that a thioester that was not originally included in the pathway is an intermediate. In addition, the enzyme PigE was originally described as a transaminase but we present evidence that it also catalyses the reduction of the thioester intermediate to its aldehyde substrate

Bhalara, Hiral D

Couturier, Maxime

Leeper, Finian J

Monson, Rita E

Salmond, George P C

English

Apollo (Cambridge)

© 2021 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2021, 2, 551–555 |  551Cite this: RSC Chem. Biol., 2021,2, 551Revision in the first steps of the biosynthesis ofthe red antibiotic prodigiosin: use of a syntheticthioester to validate a new intermediate†Maxime Couturier,a Hiral D. Bhalara,a Rita E. Monson,b George P. C. Salmondb andFinian J. Leeper *aA biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not allthe suggested intermediates could be detected experimentally. Here we show that a thioester that wasnot originally included in the pathway is an intermediate. In addition, the enzyme PigE was originallydescribed as a transaminase but we present evidence that it also catalyses the reduction of the thioesterintermediate to its aldehyde substrate.IntroductionProdigiosin 11 is a tripyrrolic natural product produced by awide range of bacteria.1 It originally caught researchers’ attentionbecause of its bright red colour, but its various biological activitieshave renewed interest in this molecule in recent decades.2The biosynthetic pathway to prodigiosin was established byWilliamson et al. via cross-feeding experiments of variousknock-out mutants of Serratia ATCC sp.39006 (S39006).3 Theyshowed that prodigiosin resulted from a bifurcated pathway(Fig. 1) with the independent synthesis of monopyrrole 2-methyl-3-amylpyrrole (MAP) 7 and bipyrrole 4-methoxy-2,20-bipyrrole-5-carbaldehyde (MBC) 9 before a final condensationstep catalysed by the enzyme PigC.MBC is an intermediate in the biosynthesis of other prodigi-nines, natural products which share the tripyrrolic structure ofprodigiosin 11. In particular, the enzymes involved in thebiosynthesis of MBC in Streptomyces coelicolor A(3) have beenwell characterised.4 The biosynthesis of MAP, on the other hand,is specific to the formation of prodigiosin 11. 2-Octenal 1 wasoriginally proposed to be the starting point of the pathway.3Addition of an acetyl group from pyruvate by PigD was proposedto form 3-acetyloctanal 4, which then underwent a transamina-tion catalysed by PigE,5 leading to amino ketone 5 whichspontaneously cyclises to form dihydroMAP (H2MAP) 6. H2MAPis then oxidised to MAP 7 by PigB.6However, Dresen et al. showed that, when presented with analdehyde such as 2-octenal 1, PigD catalysed the direct additionof the acetyl group onto the aldehyde carbon, whereas, with lessreactive a,b-unsaturated ketones and thioesters, attack on theb position was preferred.7 Kasparyan et al. extended this workto some homologues of PigD and suggested that thioester3 may be the true natural product of PigD.8In this work, we show that thioester 3 is indeed an intermediatein the MAP biosynthetic pathway. In addition, we provide evidencethat PigE catalyses the reduction of the thioester intermediate toaldehyde 4 as well as catalysing the transamination of thataldehyde. As a result, we propose an alternative biosynthetic path-way for the formation of MAP which uses 2-octenoyl thioester 2,either with coenzyme A (CoA) or an acyl carrier protein (ACP), as itsstarting point (Fig. 1).Results and discussionPreparation of potential substratesCoA and ACP thioesters are common intermediates in biosynthesis.In particular, straight-chain thioesters with even numbers of car-bons are readily available from fatty acid biosynthesis (or degrada-tion). In addition, it has been shown that PigD catalyses the transferof an acetyl group from pyruvate to N-acetylcysteamine (NAC)thioester 12 to give thioester 13 (Fig. 2).7,8 Considering that NACthioesters mimic CoA or ACP thioesters, we synthesised 13 toinvestigate if it could be converted to prodigiosin by S39006.As thioesters are prone to hydrolysis and all testing of 13would be done in aqueous solution, its hydrolysis product,3-acetyloctanoic acid 14, was also synthesised, as a negativecontrol, and the known PigE substrate 3-acetyloctanal 4 wassynthesised as the positive control.a Yusuf Hamied Dept. of Chemistry, University of Cambridge, Lensfield Road,Cambridge CB2 1EW, UK. E-mail: fjl1@cam.ac.ukb Dept. of Biochemistry, University of Cambridge, Tennis Court Road,Cambridge CB2 1QW, UK† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0cb00173bReceived 30th September 2020,Accepted 5th January 2021DOI: 10.1039/d0cb00173brsc.li/rsc-chembioRSCChemical BiologyPAPER552 |  RSC Chem. Biol., 2021, 2, 551–555 © 2021 The Author(s). Published by the Royal Society of ChemistryAcid 14 was obtained in three steps from 2-octanone (see ESI†):bromination on C-3 was followed by displacement of bromideby dimethyl malonate and then hydrolysis/decarboxylation.Acid 14 was then converted to 13 via thioesterification withN-acetylcysteamine. Aldehyde 4 was obtained by reduction of 14to the diol 15 followed by Swern oxidation.Chemical complementation assaysS39006 DpigD has an in-frame deletion in pigD, avoiding anydownstream polarity in the prodigiosin biosynthetic operon,and presents a white phenotype.3 However, when fed with anintermediate accepted by one of the subsequent enzymes in theMAP biosynthetic pathway this strain produces 11 and there-fore presents a red phenotype.3,6In a qualitative assay, S39006 DpigD was streaked on agarplates and spots (2 ml, 1 M) of aldehyde 4 and thioester 13 wereplaced next to the streaks. After 12 h, a red colour was visible inthe streak next to the spot in both cases (Fig. 3), suggesting thatboth compounds could restore prodigiosin production. Thecolour was less intense in the case of the thioester, perhapssuggesting less efficient uptake into the cell or less efficientutilisation of the NAC thioester than the true thioesterintermediate.These results were confirmed by a quantitative complemen-tation assay in liquid culture. After optimisation to overcomelow levels of production of 11 due to the toxicity of 4, S39006DpigD was treated with 4, 13 or 14 (1 mM) for 16 h andprodigiosin was then extracted from cell pellets with acidifiedEtOH and quantified by its absorbance at 535 nm.9 Again theresults (Fig. 4) were that pigmentation was observed withaldehyde 4 and thioester 13 but not with carboxylic acid 14.The UV spectrum of the extract of the culture treated with 13showed a maximum at 535 nm (Fig. 4c), consistent withprodigiosin.Extracts from cultures of S39006 DpigD not treated with anysubstrate have some absorption at 535 nm but this is a slopingbaseline and no peak is seen. Nevertheless, to confirm that theincrease in absorbance during the chemical complementationexperiments was due to prodigiosin and not some otherFig. 1 Biosynthesis of prodigiosin; green; formation of MAP, blue: formation of MBC, red: condensation. HBM is 4-hydroxy-2,2 0-bipyrrole-5-methanoland HBC is 4-hydroxy-2,2 0-bipyrrole-5-carbaldehyde.Fig. 2 Syntheses of potential substrates of PigE.Fig. 3 Chemical complementation of S39006 DpigD with aldehyde 4 (left)and thioester 13 (right).Paper RSC Chemical Biology© 2021 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2021, 2, 551–555 |  553metabolite, an ethanolic extract from a culture of wild-typeS39006 was analysed by HPLC. Two peaks absorbing at 535 nmwere observed and LCMS (see ESI†) showed they were prodigiosin11 and norprodigiosin 10. Norprodigiosin results from the con-densation of the unmethylated bipyrrole HBC 8 with MAP 7 (seeFig. 1). The peaks corresponding to 11 and 10 were also observedin the HPLC trace of the extract from S39006 DPigD treated with13 (Fig. 4d). This proves that thioester 13 can restore productionof prodigiosin in S39006 DpigD. Thus it is very likely that a relatedthioester is an intermediate in the biosynthesis of MAP.Bioinformatic analysis of PigEThe presence of a thioester intermediate in MAP biosynthesisimplies that a reduction step converting the thioester to analdehyde needs to be added to the original pathway. Thepossibility that PigE had thioester reductase activity as well asthe established aminotransferase activity was investigated.The sequence of PigE was analysed using the NCBI BLASTsoftware.10 This showed that the C-terminal region of PigE(residues 394 to 848) had strong similarity with pyridoxalphosphate (PLP)-dependent aminotransferases, particularlyclass III aminotransferases that generally act on or produceterminal alkylamines.11 A crystal structure of the C-terminaldomain with PLP bound has been published.5 The N-terminalregion (residues 2 to 341) showed homology with acyl-ACPreductases, which catalyse the reduction of fatty acyl ACP thioestersto the corresponding aldehydes.12 The majority of proteins thatshowed good similarity with full length PigE were homologues inother organisms known to produce prodigiosin or related com-pounds (e.g. TamH in tambjamine biosynthesis13).PHYRE2 homology modelling software14 generated a struc-tural model for the N-terminal domain (see ESI†) using therecently published structure of the acyl-ACP reductase (AAR)involved in cyanobacterial biosynthesis of hydrocarbons (26%identity).15 The second best hit was glutamyl tRNA reductase(GluTR) from Methanopyrus kandleri (17% identity), whichcatalyses the transformation of glutamyl tRNA into glutamate-1-semialdehyde, the first committed step of heme biosynthesisin plants, algae and most bacteria. Both these enzymes initiallytransfer the acyl group onto an active site cysteine residue andit is this thioester intermediate that is subsequently reduced byNADPH.16 This cysteine residue is conserved in PigE (Cys296)and is in the same position in the homology model as Cys294 inAAR when the two structures are overlaid. Interestingly bothAAR and GluTR have been shown to channel their productaldehydes directly to the next enzyme in the pathway via an exittunnel that is distinct from the substrate entry route. For AARthe next enzyme is a di-iron-dependent aldehyde-deformylatingoxygenase (ADO),15 but for GluTR it is a PLP-dependent amino-transferase, glutamate-1-semialdehyde aminomutase (GSAM).17It seems likely, therefore, that the reductase and aminotransferasedomains of PigE form a structure similar to the GluTR-GSAMcomplex and substrate channelling of aldehyde 4 may occur.The bioinformatic analysis, therefore, reveals the presence oftwo domains in PigE: a C-terminal aminotransferase domain,and a N-terminal thioester reductase domain. This stronglysuggests that the N-terminal domain of PigE is the protein thatcatalyses the reduction of the thioester intermediate.In vitro characterisation of PigEPlasmid pQE80-L::oriT containing pigE from S39006 was trans-formed into E. coli BL21, giving strain E. coli pPigE. PigEprotein was purified from cultures of this strain. A UV spectrumof the purified PigE showed a peak at 410 nm, consistent withPLP bound as its Schiff’s base with the active-site lysine residue.(Fig. 5).To determine the likely amino donor in the transaminasereaction PigE was incubated with various amino acids individuallyand the formation of the pyridoxamine phosphate (PMP) inter-mediate was monitored by recording the absorbance at 330 nm.The most significant increase was observed with ornithine andFig. 4 Results of the quantitative chemical complementation assays;(a) ethanolic extract of cultures of S39006 WT and S39006 DpigDincubated with no additive (f), aldehyde 4, carboxylic acid 14 or thioester13; (b) Absorbance at 535 nm normalised by OD600 for the specifiedextracts; (c) UV spectrum of the extract of S39006 DpigD inoculated withthioester 13; (d) Comparison of the HPLC traces (535 nm) of the extracts ofS39006 WT and S39006 DpigD incubated with thioester 13.Fig. 5 (a) Intermediates in PLP-catalysed transamination; (b) UV spectraof isolated PigE; (c) change in the A330 of PigE after 24 h in the presence ofvarious amino acids.RSC Chemical Biology Paper554 |  RSC Chem. Biol., 2021, 2, 551–555 © 2021 The Author(s). Published by the Royal Society of Chemistryarginine (Fig. 5c) suggesting that PigE is a transaminase dependenton these amino acids.Lysates prepared from E. coli BL21 and E. coli pPigE weresupplemented with thioester 13, ornithine and PLP and incubatedat 30 1C for 16 h. After removing cell debris, the resulting mixtureswere analysed by LCMS and compared with synthetic samples ofH2MAP 6 and thioester 13 (Fig. 6). In the extract from E. coli BL21,only the peak corresponding to the thioester 13 was visible. In theextract from E. coli pPigE, the main peak corresponded to H2MAP6 and only a very small peak corresponding to 13 was visible. Thisis a strong indication that the presence of PigE was necessary for13 to be reduced in addition to the transamination to give 6.When purified PigE was incubated with thioester 13, ornithineand NAD(P)H at 30 1C for 16 h in phosphate buffer, no peakcorresponding to H2MAP could be detected by LCMS. Similarlyexperiments with the same substrates in the presence of a variety ofmetal ions failed to show any significant change in absorbance ofNAD(P)H at 340 nm over time resulting from the presence of PigE.Taken together these experiments suggest that additional cofactorsand/or metal ions contained in the cell lysates may be necessary forrapid reaction to occur. It is noteworthy that the activity of GluTR, towhich the N-terminal domain of PigE may be related, is affected byseveral regulatory proteins and by the presence of the tRNA18 andactivity can easily be lost by oxidation of the active site cysteine.16ConclusionsWe have shown that thioester 13 can restore production ofprodigiosin in the deficient strain, S39006 DpigD. In the presenceof PigE, the thioester can be reduced, and the resulting aldehydeundergoes transamination, leading to the next biosyntheticintermediate, H2MAP.We therefore propose the revised biosynthetic pathway forprodigiosin shown in Fig. 1. Our results, along with thebioinformatic analysis, support the notion that PigE is abifunctional enzyme, with a thioester reductase domain in itsN-terminal region and a transaminase domain in its C-terminalregion. We also propose that the amino donor for the trans-aminase activity is mostly likely ornithine or arginine.ExperimentalProdigiosin production and quantificationProdigiosin production from S30096 WT. Luria-Bertani broth(LB: tryptone 10 g L1, yeast extract 5 g L1, NaCl 10 g L1)supplemented with 0.25 M sorbitol was inoculated with anovernight culture of the strain. The culture was incubated at30 1C, with shaking at 250 rpm for 16 h. 25 ml of cells were thenpelleted (2219 g, 20 min, 4 1C) and the supernatant was dis-carded. The pellets were vortexed for 1 min in 5 ml of acidifiedEtOH (4% 1 M HCl). After centrifugation the supernatant gavethe desired prodiginine extract.Prodigiosin production from S39006 DpigD. The strain wasgrown in LB supplemented with 0.25 M sorbitol at 30 1C, withshaking at 250 rpm until the OD600 reached 4. Then 4, 13 or 14(1 mM final concentration) was added and the incubationextended for a further 16 h. Cells were then pelleted andprodiginine extracted as described above.Prodigiosin quantification. A direct measurement of theabsorbance at 535 nm was performed on a Cary 300-BioUV-visible spectrophotometer.Cell extract assaysE. coli BL21 or E. coli pPigE were cultured in 200 ml of LB untiland OD600 = 0.6. Cultures were then induced with 1 mM IPTG at16 1C for 14–16 h. Cells were harvested by centrifugation at4500 rpm for 20 min at 4 1C. The cells were held on ice for15 min and then resuspended in 20 ml of phosphate buffer.After a further 30 min on ice, the cell suspensions weresonicated (6  20 s with 30 s breaks). Eppendorf tubes werecharged with 13 (1 mM), and ornithine and PLP (1.25 mM finalconcentrations), then cell lysate was added to a total volume of0.5 ml. The resulting mixture was gently shaken overnight at30 1C, then centrifuged and the supernatant was analyseddirectly by LCMS.Conflicts of interestThere are no conflicts to declare.AcknowledgementsThis study was supported by the BBSRC, UK (awards BB/N008081/1and BB/K001833/1). MC was supported by the Frances andFig. 6 Extracted ion chromatograms (m/z 154.2 + 288.2) (a) and (b) afterincubation of 13 with cell lysates of (a) E. coli BL21 or (b) E. coli pPigE,(c) synthetic H2MAP 6 (m/z 154.16, 6 + H+), (d) synthetic 13 (m/z 288.16, 13 + H+).Retention times corresponding to 6 and 13 are shown.Paper RSC Chemical Biology© 2021 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2021, 2, 551–555 |  555Augustus Newman foundation, and HDB by the CambridgeCommonwealth Trust and Emmanuel College, Cambridge.Notes and references1 N. R. Williamson, P. C. Fineran, F. J. Leeper andG. P. C. Salmond, Nat. Rev. Microbiol., 2006, 4, 887–899.2 A. Fürstner, Angew. Chem., Int. Ed., 2003, 42(31), 3582–3603.3 N. R. Williamson, H. T. Simonsen, R. A. A. Ahmed,G. Goldet, H. Slater, L. Woodley, F. J. Leeper andG. P. C. Salmond, Mol. Microbiol., 2005, 56(4), 971–989.4 A. M. Cerdeño, M. J. Bibb and G. L. Challis, Chem. Biol.,2001, 8(8), 817–829.5 X. Lou, T. Ran, N. Han, Y. Gao, J. He, L. Tang, D. Xu andW. Wang,, Biochem. Biophys. Res. Commun., 2014, 447(1),178–183.6 M. Couturier, H. D. Bhalara, S. R. Chawrai, R. Monson,N. R. Williamson, G. P. C. Salmond and F. J. Leeper, ChemBio-Chem, 2020, 21(4), 523–530.7 C. Dresen, M. Richter, M. Pohl, S. Lüdeke and M. Müller,Angew. Chem., Int. Ed., 2010, 49(37), 6600–6603.8 E. Kasparyan, M. Richter, C. Dresen, L. S. Walter, G. Fuchs,F. J. Leeper, T. Wacker, S. L. A. Andrade, G. Kolter, M. Pohland M. Müller, Appl. Microbiol. Biotechnol., 2014, 98(23),9681–9690.9 H. Slater, M. Crow, L. Everson and G. P. C. Salmond, Mol.Microbiol., 2003, 47(2), 303–320.10 S. F. Altschul, W. Gish, W. Miller, E. W. Myers andD. J. Lipman, J. Mol. Biol., 1990, 215(3), 403–410.11 H. J. Cha, J. H. Jeong, C. Rojviriya and Y. G. Kim, PLoS One,2014, 9(11), e113212; D. Schiroli and A. Peracchi, Biochim.Biophys. Acta, Gen. Subj., 2015, 1854(9), 1200–1211.12 A. Schirmer, M. A. Rude, X. Li, E. Popova and S. B. delCardayre, Science, 2010, 329(5991), 559–562.13 C. Burke, T. Thomas, S. Egan and S. Kjelleberg, Environ.Microbiol., 2007, 9(3), 814–818.14 L. A. Kelley, S. Mezulis, C. M. Yates, M. N. Wass andM. J. E. Sternberg,, Nat. Protoc., 2015, 10(6), 845–858.15 Y. Gao, H. Zhang, M. Fan, C. Jia, L. Shi, X. Pan, P. Cao,X. Zhao, W. Chang and M. Li, Nat. Commun., 2020,11(1), 1525.16 J. Moser, W. D. Schubert, V. Beier, I. Bringemeier, D. Jahnand D. W. Heinz, EMBO J., 2001, 20(23), 6583–6590.17 C. Lüer, S. Schauer, K. Möbius, J. Schulze, W.-D. Schubert,D. W. Heinz, D. Jahn and J. Moser, J. Biol. Chem., 2005,280(19), 18568–18572; L. A. Nogaj and S. I. Beale, J. Biol.Chem., 2005, 280(26), 24301–24307.18 A. Zhao, Y. Fang, X. Chena, S. Zhao, W. Dong, Y. Lin,W. Gong and L. Liu, Proc. Natl. Acad. Sci. U. S. A., 2014,111(18), 6630–6635.RSC Chemical Biology Paper

Revision in the first steps of the biosynthesis of the red antibiotic prodigiosin: use of a synthetic thioester to validate a new intermediate.

A biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not all the suggested intermediates could be detected experimentally. Here we show that a thioester that was not originally included in the pathway is an intermediate. In addition, the enzyme PigE was originally described as a transaminase but we present evidence that it also catalyses the reduction of the thioester intermediate to its aldehyde substrate.BBSRC, UK, awards BB/N008081/1 and BB/K001833/1
Frances and Augustus Newman foundation
Cambridge Commonwealth Trust 
Emmanuel College, Cambridg

Salmond, George PC

© 2021 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2021, 2, 551–555 |  551Cite this: RSC Chem. Biol., 2021,2, 551Revision in the first steps of the biosynthesis ofthe red antibiotic prodigiosin: use of a syntheticthioester to validate a new intermediate†Maxime Couturier,a Hiral D. Bhalara,a Rita E. Monson,b George P. C. Salmondb andFinian J. Leeper *aA biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not allthe suggested intermediates could be detected experimentally. Here we show that a thioester that wasnot originally included in the pathway is an intermediate. In addition, the enzyme PigE was originallydescribed as a transaminase but we present evidence that it also catalyses the reduction of the thioesterintermediate to its aldehyde substrate.IntroductionProdigiosin 11 is a tripyrrolic natural product produced by awide range of bacteria.1 It originally caught researchers’ attentionbecause of its bright red colour, but its various biological activitieshave renewed interest in this molecule in recent decades.2The biosynthetic pathway to prodigiosin was established byWilliamson et al. via cross-feeding experiments of variousknock-out mutants of Serratia ATCC sp.39006 (S39006).3 Theyshowed that prodigiosin resulted from a bifurcated pathway(Fig. 1) with the independent synthesis of monopyrrole 2-methyl-3-amylpyrrole (MAP) 7 and bipyrrole 4-methoxy-2,20-bipyrrole-5-carbaldehyde (MBC) 9 before a final condensationstep catalysed by the enzyme PigC.MBC is an intermediate in the biosynthesis of other prodigi-nines, natural products which share the tripyrrolic structure ofprodigiosin 11. In particular, the enzymes involved in thebiosynthesis of MBC in Streptomyces coelicolor A(3) have beenwell characterised.4 The biosynthesis of MAP, on the other hand,is specific to the formation of prodigiosin 11. 2-Octenal 1 wasoriginally proposed to be the starting point of the pathway.3Addition of an acetyl group from pyruvate by PigD was proposedto form 3-acetyloctanal 4, which then underwent a transamina-tion catalysed by PigE,5 leading to amino ketone 5 whichspontaneously cyclises to form dihydroMAP (H2MAP) 6. H2MAPis then oxidised to MAP 7 by PigB.6However, Dresen et al. showed that, when presented with analdehyde such as 2-octenal 1, PigD catalysed the direct additionof the acetyl group onto the aldehyde carbon, whereas, with lessreactive a,b-unsaturated ketones and thioesters, attack on theb position was preferred.7 Kasparyan et al. extended this workto some homologues of PigD and suggested that thioester3 may be the true natural product of PigD.8In this work, we show that thioester 3 is indeed an intermediatein the MAP biosynthetic pathway. In addition, we provide evidencethat PigE catalyses the reduction of the thioester intermediate toaldehyde 4 as well as catalysing the transamination of thataldehyde. As a result, we propose an alternative biosynthetic path-way for the formation of MAP which uses 2-octenoyl thioester 2,either with coenzyme A (CoA) or an acyl carrier protein (ACP), as itsstarting point (Fig. 1).Results and discussionPreparation of potential substratesCoA and ACP thioesters are common intermediates in biosynthesis.In particular, straight-chain thioesters with even numbers of car-bons are readily available from fatty acid biosynthesis (or degrada-tion). In addition, it has been shown that PigD catalyses the transferof an acetyl group from pyruvate to N-acetylcysteamine (NAC)thioester 12 to give thioester 13 (Fig. 2).7,8 Considering that NACthioesters mimic CoA or ACP thioesters, we synthesised 13 toinvestigate if it could be converted to prodigiosin by S39006.As thioesters are prone to hydrolysis and all testing of 13would be done in aqueous solution, its hydrolysis product,3-acetyloctanoic acid 14, was also synthesised, as a negativecontrol, and the known PigE substrate 3-acetyloctanal 4 wassynthesised as the positive control.a Yusuf Hamied Dept. of Chemistry, University of Cambridge, Lensfield Road,Cambridge CB2 1EW, UK. E-mail: fjl1@cam.ac.ukb Dept. of Biochemistry, University of Cambridge, Tennis Court Road,Cambridge CB2 1QW, UK† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0cb00173bReceived 30th September 2020,Accepted 5th January 2021DOI: 10.1039/d0cb00173brsc.li/rsc-chembioRSCChemical BiologyPAPEROpen Access Article. Published on 15 January 2021. Downloaded on 11/1/2021 4:00:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View Issue552 |  RSC Chem. Biol., 2021, 2, 551–555 © 2021 The Author(s). Published by the Royal Society of ChemistryAcid 14 was obtained in three steps from 2-octanone (see ESI†):bromination on C-3 was followed by displacement of bromideby dimethyl malonate and then hydrolysis/decarboxylation.Acid 14 was then converted to 13 via thioesterification withN-acetylcysteamine. Aldehyde 4 was obtained by reduction of 14to the diol 15 followed by Swern oxidation.Chemical complementation assaysS39006 DpigD has an in-frame deletion in pigD, avoiding anydownstream polarity in the prodigiosin biosynthetic operon,and presents a white phenotype.3 However, when fed with anintermediate accepted by one of the subsequent enzymes in theMAP biosynthetic pathway this strain produces 11 and there-fore presents a red phenotype.3,6In a qualitative assay, S39006 DpigD was streaked on agarplates and spots (2 ml, 1 M) of aldehyde 4 and thioester 13 wereplaced next to the streaks. After 12 h, a red colour was visible inthe streak next to the spot in both cases (Fig. 3), suggesting thatboth compounds could restore prodigiosin production. Thecolour was less intense in the case of the thioester, perhapssuggesting less efficient uptake into the cell or less efficientutilisation of the NAC thioester than the true thioesterintermediate.These results were confirmed by a quantitative complemen-tation assay in liquid culture. After optimisation to overcomelow levels of production of 11 due to the toxicity of 4, S39006DpigD was treated with 4, 13 or 14 (1 mM) for 16 h andprodigiosin was then extracted from cell pellets with acidifiedEtOH and quantified by its absorbance at 535 nm.9 Again theresults (Fig. 4) were that pigmentation was observed withaldehyde 4 and thioester 13 but not with carboxylic acid 14.The UV spectrum of the extract of the culture treated with 13showed a maximum at 535 nm (Fig. 4c), consistent withprodigiosin.Extracts from cultures of S39006 DpigD not treated with anysubstrate have some absorption at 535 nm but this is a slopingbaseline and no peak is seen. Nevertheless, to confirm that theincrease in absorbance during the chemical complementationexperiments was due to prodigiosin and not some otherFig. 1 Biosynthesis of prodigiosin; green; formation of MAP, blue: formation of MBC, red: condensation. HBM is 4-hydroxy-2,2 0-bipyrrole-5-methanoland HBC is 4-hydroxy-2,2 0-bipyrrole-5-carbaldehyde.Fig. 2 Syntheses of potential substrates of PigE.Fig. 3 Chemical complementation of S39006 DpigD with aldehyde 4 (left)and thioester 13 (right).Paper RSC Chemical BiologyOpen Access Article. Published on 15 January 2021. Downloaded on 11/1/2021 4:00:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online© 2021 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2021, 2, 551–555 |  553metabolite, an ethanolic extract from a culture of wild-typeS39006 was analysed by HPLC. Two peaks absorbing at 535 nmwere observed and LCMS (see ESI†) showed they were prodigiosin11 and norprodigiosin 10. Norprodigiosin results from the con-densation of the unmethylated bipyrrole HBC 8 with MAP 7 (seeFig. 1). The peaks corresponding to 11 and 10 were also observedin the HPLC trace of the extract from S39006 DPigD treated with13 (Fig. 4d). This proves that thioester 13 can restore productionof prodigiosin in S39006 DpigD. Thus it is very likely that a relatedthioester is an intermediate in the biosynthesis of MAP.Bioinformatic analysis of PigEThe presence of a thioester intermediate in MAP biosynthesisimplies that a reduction step converting the thioester to analdehyde needs to be added to the original pathway. Thepossibility that PigE had thioester reductase activity as well asthe established aminotransferase activity was investigated.The sequence of PigE was analysed using the NCBI BLASTsoftware.10 This showed that the C-terminal region of PigE(residues 394 to 848) had strong similarity with pyridoxalphosphate (PLP)-dependent aminotransferases, particularlyclass III aminotransferases that generally act on or produceterminal alkylamines.11 A crystal structure of the C-terminaldomain with PLP bound has been published.5 The N-terminalregion (residues 2 to 341) showed homology with acyl-ACPreductases, which catalyse the reduction of fatty acyl ACP thioestersto the corresponding aldehydes.12 The majority of proteins thatshowed good similarity with full length PigE were homologues inother organisms known to produce prodigiosin or related com-pounds (e.g. TamH in tambjamine biosynthesis13).PHYRE2 homology modelling software14 generated a struc-tural model for the N-terminal domain (see ESI†) using therecently published structure of the acyl-ACP reductase (AAR)involved in cyanobacterial biosynthesis of hydrocarbons (26%identity).15 The second best hit was glutamyl tRNA reductase(GluTR) from Methanopyrus kandleri (17% identity), whichcatalyses the transformation of glutamyl tRNA into glutamate-1-semialdehyde, the first committed step of heme biosynthesisin plants, algae and most bacteria. Both these enzymes initiallytransfer the acyl group onto an active site cysteine residue andit is this thioester intermediate that is subsequently reduced byNADPH.16 This cysteine residue is conserved in PigE (Cys296)and is in the same position in the homology model as Cys294 inAAR when the two structures are overlaid. Interestingly bothAAR and GluTR have been shown to channel their productaldehydes directly to the next enzyme in the pathway via an exittunnel that is distinct from the substrate entry route. For AARthe next enzyme is a di-iron-dependent aldehyde-deformylatingoxygenase (ADO),15 but for GluTR it is a PLP-dependent amino-transferase, glutamate-1-semialdehyde aminomutase (GSAM).17It seems likely, therefore, that the reductase and aminotransferasedomains of PigE form a structure similar to the GluTR-GSAMcomplex and substrate channelling of aldehyde 4 may occur.The bioinformatic analysis, therefore, reveals the presence oftwo domains in PigE: a C-terminal aminotransferase domain,and a N-terminal thioester reductase domain. This stronglysuggests that the N-terminal domain of PigE is the protein thatcatalyses the reduction of the thioester intermediate.In vitro characterisation of PigEPlasmid pQE80-L::oriT containing pigE from S39006 was trans-formed into E. coli BL21, giving strain E. coli pPigE. PigEprotein was purified from cultures of this strain. A UV spectrumof the purified PigE showed a peak at 410 nm, consistent withPLP bound as its Schiff’s base with the active-site lysine residue.(Fig. 5).To determine the likely amino donor in the transaminasereaction PigE was incubated with various amino acids individuallyand the formation of the pyridoxamine phosphate (PMP) inter-mediate was monitored by recording the absorbance at 330 nm.The most significant increase was observed with ornithine andFig. 4 Results of the quantitative chemical complementation assays;(a) ethanolic extract of cultures of S39006 WT and S39006 DpigDincubated with no additive (f), aldehyde 4, carboxylic acid 14 or thioester13; (b) Absorbance at 535 nm normalised by OD600 for the specifiedextracts; (c) UV spectrum of the extract of S39006 DpigD inoculated withthioester 13; (d) Comparison of the HPLC traces (535 nm) of the extracts ofS39006 WT and S39006 DpigD incubated with thioester 13.Fig. 5 (a) Intermediates in PLP-catalysed transamination; (b) UV spectraof isolated PigE; (c) change in the A330 of PigE after 24 h in the presence ofvarious amino acids.RSC Chemical Biology PaperOpen Access Article. Published on 15 January 2021. Downloaded on 11/1/2021 4:00:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online554 |  RSC Chem. Biol., 2021, 2, 551–555 © 2021 The Author(s). Published by the Royal Society of Chemistryarginine (Fig. 5c) suggesting that PigE is a transaminase dependenton these amino acids.Lysates prepared from E. coli BL21 and E. coli pPigE weresupplemented with thioester 13, ornithine and PLP and incubatedat 30 1C for 16 h. After removing cell debris, the resulting mixtureswere analysed by LCMS and compared with synthetic samples ofH2MAP 6 and thioester 13 (Fig. 6). In the extract from E. coli BL21,only the peak corresponding to the thioester 13 was visible. In theextract from E. coli pPigE, the main peak corresponded to H2MAP6 and only a very small peak corresponding to 13 was visible. Thisis a strong indication that the presence of PigE was necessary for13 to be reduced in addition to the transamination to give 6.When purified PigE was incubated with thioester 13, ornithineand NAD(P)H at 30 1C for 16 h in phosphate buffer, no peakcorresponding to H2MAP could be detected by LCMS. Similarlyexperiments with the same substrates in the presence of a variety ofmetal ions failed to show any significant change in absorbance ofNAD(P)H at 340 nm over time resulting from the presence of PigE.Taken together these experiments suggest that additional cofactorsand/or metal ions contained in the cell lysates may be necessary forrapid reaction to occur. It is noteworthy that the activity of GluTR, towhich the N-terminal domain of PigE may be related, is affected byseveral regulatory proteins and by the presence of the tRNA18 andactivity can easily be lost by oxidation of the active site cysteine.16ConclusionsWe have shown that thioester 13 can restore production ofprodigiosin in the deficient strain, S39006 DpigD. In the presenceof PigE, the thioester can be reduced, and the resulting aldehydeundergoes transamination, leading to the next biosyntheticintermediate, H2MAP.We therefore propose the revised biosynthetic pathway forprodigiosin shown in Fig. 1. Our results, along with thebioinformatic analysis, support the notion that PigE is abifunctional enzyme, with a thioester reductase domain in itsN-terminal region and a transaminase domain in its C-terminalregion. We also propose that the amino donor for the trans-aminase activity is mostly likely ornithine or arginine.ExperimentalProdigiosin production and quantificationProdigiosin production from S30096 WT. Luria-Bertani broth(LB: tryptone 10 g L1, yeast extract 5 g L1, NaCl 10 g L1)supplemented with 0.25 M sorbitol was inoculated with anovernight culture of the strain. The culture was incubated at30 1C, with shaking at 250 rpm for 16 h. 25 ml of cells were thenpelleted (2219 g, 20 min, 4 1C) and the supernatant was dis-carded. The pellets were vortexed for 1 min in 5 ml of acidifiedEtOH (4% 1 M HCl). After centrifugation the supernatant gavethe desired prodiginine extract.Prodigiosin production from S39006 DpigD. The strain wasgrown in LB supplemented with 0.25 M sorbitol at 30 1C, withshaking at 250 rpm until the OD600 reached 4. Then 4, 13 or 14(1 mM final concentration) was added and the incubationextended for a further 16 h. Cells were then pelleted andprodiginine extracted as described above.Prodigiosin quantification. A direct measurement of theabsorbance at 535 nm was performed on a Cary 300-BioUV-visible spectrophotometer.Cell extract assaysE. coli BL21 or E. coli pPigE were cultured in 200 ml of LB untiland OD600 = 0.6. Cultures were then induced with 1 mM IPTG at16 1C for 14–16 h. Cells were harvested by centrifugation at4500 rpm for 20 min at 4 1C. The cells were held on ice for15 min and then resuspended in 20 ml of phosphate buffer.After a further 30 min on ice, the cell suspensions weresonicated (6  20 s with 30 s breaks). Eppendorf tubes werecharged with 13 (1 mM), and ornithine and PLP (1.25 mM finalconcentrations), then cell lysate was added to a total volume of0.5 ml. The resulting mixture was gently shaken overnight at30 1C, then centrifuged and the supernatant was analyseddirectly by LCMS.Conflicts of interestThere are no conflicts to declare.AcknowledgementsThis study was supported by the BBSRC, UK (awards BB/N008081/1and BB/K001833/1). MC was supported by the Frances andFig. 6 Extracted ion chromatograms (m/z 154.2 + 288.2) (a) and (b) afterincubation of 13 with cell lysates of (a) E. coli BL21 or (b) E. coli pPigE,(c) synthetic H2MAP 6 (m/z 154.16, 6 + H+), (d) synthetic 13 (m/z 288.16, 13 + H+).Retention times corresponding to 6 and 13 are shown.Paper RSC Chemical BiologyOpen Access Article. Published on 15 January 2021. Downloaded on 11/1/2021 4:00:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online© 2021 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2021, 2, 551–555 |  555Augustus Newman foundation, and HDB by the CambridgeCommonwealth Trust and Emmanuel College, Cambridge.Notes and references1 N. R. Williamson, P. C. Fineran, F. J. Leeper andG. P. C. Salmond, Nat. Rev. Microbiol., 2006, 4, 887–899.2 A. Fürstner, Angew. Chem., Int. Ed., 2003, 42(31), 3582–3603.3 N. R. Williamson, H. T. Simonsen, R. A. A. Ahmed,G. Goldet, H. Slater, L. Woodley, F. J. Leeper andG. P. C. Salmond, Mol. Microbiol., 2005, 56(4), 971–989.4 A. M. Cerdeño, M. J. Bibb and G. L. Challis, Chem. Biol.,2001, 8(8), 817–829.5 X. Lou, T. Ran, N. Han, Y. Gao, J. He, L. Tang, D. Xu andW. Wang,, Biochem. Biophys. Res. Commun., 2014, 447(1),178–183.6 M. Couturier, H. D. Bhalara, S. R. Chawrai, R. Monson,N. R. Williamson, G. P. C. Salmond and F. J. Leeper, ChemBio-Chem, 2020, 21(4), 523–530.7 C. Dresen, M. Richter, M. Pohl, S. Lüdeke and M. Müller,Angew. Chem., Int. Ed., 2010, 49(37), 6600–6603.8 E. Kasparyan, M. Richter, C. Dresen, L. S. Walter, G. 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Jahnand D. W. Heinz, EMBO J., 2001, 20(23), 6583–6590.17 C. Lüer, S. Schauer, K. Möbius, J. Schulze, W.-D. Schubert,D. W. Heinz, D. Jahn and J. Moser, J. Biol. Chem., 2005,280(19), 18568–18572; L. A. Nogaj and S. I. Beale, J. Biol.Chem., 2005, 280(26), 24301–24307.18 A. Zhao, Y. Fang, X. Chena, S. Zhao, W. Dong, Y. Lin,W. Gong and L. Liu, Proc. Natl. Acad. Sci. U. S. A., 2014,111(18), 6630–6635.RSC Chemical Biology PaperOpen Access Article. Published on 15 January 2021. Downloaded on 11/1/2021 4:00:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

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Revision in the first steps of the biosynthesis of the red antibiotic prodigiosin: use of a synthetic thioester to validate a new intermediate.

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