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• W3107257413 abstract "The siderophore rhizoferrin (N1,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure. The siderophore rhizoferrin (N1,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure. Almost all organisms require iron for growth (1Miethke M. Marahiel M.A. Siderophore-based iron acquisition and pathogen control.Microbiol. Mol. Biol. Rev. 2007; 71: 413-451Crossref PubMed Scopus (932) Google Scholar), and many microorganisms obtain iron from the environment by synthesizing, secreting, and retrieving small molecular weight iron chelators known as siderophores (2Neilands J.B. Siderophores: structure and function of microbial iron transport compounds.J. Biol. Chem. 1995; 270: 26723-26726Abstract Full Text Full Text PDF PubMed Scopus (1116) Google Scholar). Siderophores employ hydroxamate, catecholate, or α-hydroxycarboxylate functional groups to bind Fe3+ (3Hider R.C. Kong X. Chemistry and biology of siderophores.Nat. Prod. Rep. 2010; 27: 637-657Crossref PubMed Scopus (848) Google Scholar). Biosynthesis of siderophores employs either the intensively studied nonribosomal peptide synthetase siderophore synthetases (4Crosa J.H. Walsh C.T. Genetics and assembly line enzymology of siderophore biosynthesis in bacteria.Microbiol. Mol. Biol. Rev. 2002; 66: 223-249Crossref PubMed Scopus (574) Google Scholar) or the more recently characterized nonribosomal peptide synthetase–independent siderophore (NIS) synthetases (5Challis G.L. A widely distributed bacterial pathway for siderophore biosynthesis independent of nonribosomal peptide synthetases.Chembiochem. 2005; 6: 601-611Crossref PubMed Scopus (224) Google Scholar, 6Oves-Costales D. Kadi N. Challis G.L. The long-overlooked enzymology of a nonribosomal peptide synthetase-independent pathway for virulence-conferring siderophore biosynthesis.Chem. Commun. (Camb). 2009; : 6530-6541Crossref PubMed Scopus (69) Google Scholar). Diverse siderophores utilize primary metabolites of the polyamine family 1,3-diaminopropane, putrescine (1,4-diaminobutane), cadaverine (1,5-diaminpentane), norspermidine (N-aminopropyl-1,3-diaminopropane), spermidine (N-aminopropylputrescine), and homospermidine (N-aminobutylputrescine) (7Michael A.J. Polyamines in eukaryotes, bacteria, and archaea.J. Biol. Chem. 2016; 291: 14896-14903Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 8Burrell M. Hanfrey C.C. Kinch L.N. Elliott K.A. Michael A.J. Evolution of a novel lysine decarboxylase in siderophore biosynthesis.Mol. Microbiol. 2012; 86: 485-499Crossref PubMed Scopus (31) Google Scholar), as structural backbones onto which the iron-binding hydroxamate, catecholate, or α-hydroxycarboxylate functional groups are appended via amide linkages. Examples of spermidine-containing siderophores are the dicatecholate siderophore petrobactin from the anthrax agent Bacillus anthracis (9Abergel R.J. Wilson M.K. Arceneaux J.E. Hoette T.M. Strong R.K. Byers B.R. Raymond K.N. Anthrax pathogen evades the mammalian immune system through stealth siderophore production.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18499-18503Crossref PubMed Scopus (146) Google Scholar) and the tricatecholate siderophore agrobactin from the plant pathogen Agrobacterium tumefaciens (10Ong S.A. Peterson T. Neilands J.B. Agrobactin, a siderophore from Agrobacterium tumefaciens.J. Biol. Chem. 1979; 254: 1860-1865Abstract Full Text PDF PubMed Google Scholar). Homospermidine is found in the petrobactin structural analog rhodopetrobactin from the environmental bacterium Rhodopseudomonas palustris (11Baars O. Morel F.M.M. Zhang X. The purple non-sulfur bacterium Rhodopseudomonas palustris produces novel petrobactin-related siderophores under aerobic and anaerobic conditions.Environ. Microbiol. 2018; 20: 1667-1676Crossref PubMed Scopus (6) Google Scholar). Norspermidine is found in the tricatecholate siderophore vibriobactin produced by the cholera agent Vibrio cholerae (12Griffiths G.L. Sigel S.P. Payne S.M. Neilands J.B. Vibriobactin, a siderophore from Vibrio cholerae.J. Biol. Chem. 1984; 259: 383-385Abstract Full Text PDF PubMed Google Scholar). The diamine putrescine, biosynthetic precursor of spermidine and homospermidine, is found in a range of cyclic and linear hydroxamate-based siderophores such as desferrioxamine, putrebactin, and avaroferrin (13Codd R. Richardson-Sanchez T. Telfer T.J. Gotsbacher M.P. Advances in the chemical biology of desferrioxamine B.ACS Chem. Biol. 2018; 13: 11-25Crossref PubMed Scopus (31) Google Scholar, 14Codd R. Soe C.Z. Pakchung A.A.H. Sresutharsan A. Brown C.J.M. Tieu W. The chemical biology and coordination chemistry of putrebactin, avaroferrin, bisucaberin, and alcaligin.J. Biol. Inorg. Chem. 2018; 23: 969-982Crossref PubMed Scopus (9) Google Scholar). It is also found as a monocatecholate siderophore, e.g., aminochelin (15Page W. von Tigerstrom M. Aminochelin, a catecholamine siderophore produced by Azotobacter vinelandii.J. Gen. Microbiol. 1988; 134: 453-460Google Scholar), and the dicatecholate siderophore photobactin (16Ciche T.A. Blackburn M. Carney J.R. Ensign J.C. Photobactin: a catechol siderophore produced by Photorhabdus luminescens, an entomopathogen mutually associated with Heterorhabditis bacteriophora NC1 nematodes.Appl. Environ. Microbiol. 2003; 69: 4706-4713Crossref PubMed Scopus (50) Google Scholar). Putrescine is also found in the simple polycarboxylate siderophore rhizoferrin (N1,N4-dicitrylputrescine), consisting of two citrate molecules linked to a putrescine backbone (17Thieken A. Winkelmann G. Rhizoferrin: a complexone type siderophore of the Mucorales and entomophthorales (Zygomycetes).FEMS Microbiol. Lett. 1992; 73: 37-41Crossref PubMed Google Scholar). Rhizoferrin was first characterized from the zygomycete fungus Rhizopus microspores, and the fungal rhizoferrin is produced as the R,R-rhizoferrin enantiomer (18Dreschel H. Metzger J. Freund S. Jung G. Boelaert J.R. Winkelmann G. Rhizoferrin - a novel siderophore from the fungus Rhizopus microsporus Var. Rhizopodiformis.Biometals. 1991; 4: 238-243Google Scholar, 19Drechsel M. Jung G. Winkelmann G. Stereochemical characterization of rhizoferrin and identification of its dehydration products.BioMetals. 1992; 5: 141-148Crossref Scopus (45) Google Scholar). Fungal rhizoferrin was found to be synthesized by a single NIS synthetase in Rhizopus delemar (20Carroll C.S. Grieve C.L. Murugathasan I. Bennet A.J. Czekster C.M. Liu H. Naismith J. Moore M.M. The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family.Int. J. Biochem. Cell Biol. 2017; 89: 136-146Crossref PubMed Scopus (12) Google Scholar). Subsequent to the discovery of fungally produced rhizoferrin, it was then found in the β-proteobacterium Ralstonia pickettii, as the S,S-rhizoferrin enantiomer (21Munzinger M. Taraz K. Budzikiewicz H. Dreschel H. Heymann P. Winkelmann G. Meyer J.-M. S,S-rhizoferrin (enantio-rhizoferrin) - a siderophore of Ralstonia (Pseudomonas) pickettii DSM 6297 - the optical antipode of R, R-rhizoferrin isolated from fungi.BioMetals. 1999; 12: 189-193Crossref Scopus (38) Google Scholar). The polyamines produced by R. pickettii are putrescine and 2-hydroxyputrescine (22Hamana K. Saito T. Okada M. Polyamine profiles within the beta subclass of the class Proteobacteria : distribution of 2-hydroxyputrescine.Microbiol. Cult. Coll. 2000; 16: 63-69Google Scholar, 23Li B. Lowe-Power T. Kurihara S. Gonzales S. Naidoo J. MacMillan J.B. Allen C. Michael A.J. Functional identification of putrescine C- and N-hydroxylases.ACS Chem. Biol. 2016; 11: 2782-2789Crossref PubMed Scopus (14) Google Scholar), but nothing is known about how rhizoferrin is produced in this species. Recently, the citrate-containing siderophore of the Legionnaires’ disease agent Legionella pneumophila was shown to be rhizoferrin (24Burnside D.M. Wu Y. Shafaie S. Cianciotto N.P. The Legionella pneumophila siderophore legiobactin is a polycarboxylate that is identical in structure to rhizoferrin.Infect. Immun. 2015; 83: 3937-3945Crossref PubMed Scopus (20) Google Scholar), and it was previously demonstrated that production of the siderophore is dependent on the NIS synthetase LbtA and major facilitator superfamily (MFS)–type transporter LbtB (25Allard K.A. Viswanathan V.K. Cianciotto N.P. lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin.J. Bacteriol. 2006; 188: 1351-1363Crossref PubMed Scopus (52) Google Scholar). Putrescine and homospermidine are produced by L. pneumophila (26Hamana K. Takeuchi M. Polyamine profiles as chemotaxonomic marker within alpha, beta, gamma, delta and epsilon subclasses of class proteobacteria: distribution of 2-hydroxyputrescine and homospermidine.Microbiol. Cult. Coll. 1998; 14: 1-14Google Scholar). Intriguingly, rhizoferrin is also produced by the LVS and SCHU S4 strains of the tularemia agent Francisella tularensis (27Sullivan J.T. Jeffery E.F. Shannon J.D. Ramakrishnan G. Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production.J. Bacteriol. 2006; 188: 3785-3795Crossref PubMed Scopus (69) Google Scholar, 28Deng K. Blick R.J. Liu W. Hansen E.J. Identification of Francisella tularensis genes affected by iron limitation.Infect. Immun. 2006; 74: 4224-4236Crossref PubMed Scopus (89) Google Scholar, 29Ramakrishnan G. Iron and virulence in Francisella tularensis.Front. Cell Infect. Microbiol. 2017; 7: 107Crossref PubMed Scopus (13) Google Scholar). The first sequenced genome of F. tularensis, the virulent SCHU S4 strain, revealed that the pathogen has a small genome of 1.89 Mbp that is undergoing decay associated with an intracellular lifestyle (30Larsson P. Oyston P.C. Chain P. Chu M.C. Duffield M. Fuxelius H.H. Garcia E. Halltorp G. Johansson D. Isherwood K.E. Karp P.D. Larsson E. Liu Y. Michell S. Prior J. et al.The complete genome sequence of Francisella tularensis, the causative agent of tularemia.Nat. Genet. 2005; 37: 153-159Crossref PubMed Scopus (353) Google Scholar). More than 10% of the coding sequences contain insertion-deletion or substitution mutations resulting in loss of metabolic pathways. Closely related species Francisella novicida does not infect humans except opportunistically and is 98% identical to F. tularensis (31Oyston P.C. Sjostedt A. Titball R.W. Tularaemia: bioterrorism defence renews interest in Francisella tularensis.Nat. Rev. Microbiol. 2004; 2: 967-978Crossref PubMed Scopus (385) Google Scholar, 32Rohmer L. Fong C. Abmayr S. Wasnick M. Larson Freeman T.J. Radey M. Guina T. Svensson K. Hayden H.S. Jacobs M. Gallagher L.A. Manoil C. Ernst R.K. Drees B. Buckley D. et al.Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains.Genome Biol. 2007; 8: R102Crossref PubMed Scopus (129) Google Scholar). The ancestor of Francisella probably synthesized putrescine via the activities of arginine decarboxylase, agmatine deiminase, and N-carbamoylputrescine amidohydrolase; however, these genes have become disrupted in the case of F. tularensis or lost in F. novicida (30Larsson P. Oyston P.C. Chain P. Chu M.C. Duffield M. Fuxelius H.H. Garcia E. Halltorp G. Johansson D. Isherwood K.E. Karp P.D. Larsson E. Liu Y. Michell S. Prior J. et al.The complete genome sequence of Francisella tularensis, the causative agent of tularemia.Nat. Genet. 2005; 37: 153-159Crossref PubMed Scopus (353) Google Scholar, 33Sarva S.T. Waldo R.H. Belland R.J. Klose K.E. Comparative transcriptional analyses of Francisella tularensis and Francisella novicida.PLoS One. 2016; 11e0158631Crossref PubMed Scopus (3) Google Scholar). The fslA/figA-encoded NIS synthetase is required for rhizoferrin production in F. tularensis and F. novicida (27Sullivan J.T. Jeffery E.F. Shannon J.D. Ramakrishnan G. Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production.J. Bacteriol. 2006; 188: 3785-3795Crossref PubMed Scopus (69) Google Scholar, 28Deng K. Blick R.J. Liu W. Hansen E.J. Identification of Francisella tularensis genes affected by iron limitation.Infect. Immun. 2006; 74: 4224-4236Crossref PubMed Scopus (89) Google Scholar). In addition, MFS-type transporter gene figB and figC-encoded alanine racemase (AR)-fold PLP-dependent decarboxylase, homologous to ornithine decarboxylase (OD), are required for rhizoferrin production in F. novicida (34Kiss K. Liu W. Huntley J.F. Norgard M.V. Hansen E.J. Characterization of fig operon mutants of Francisella novicida U112.FEMS Microbiol. Lett. 2008; 285: 270-277Crossref PubMed Scopus (36) Google Scholar). As F. novicida has lost all putrescine biosynthetic genes, we sought to determine how rhizoferrin is produced by this species. During the course of our study, another recent study demonstrated that a figC deletion mutant of F. tularensis accumulates N-citrylornithine, indicating that figC encodes an N-citrylornithine decarboxylase (35Ramakrishnan G. Perez N.M. Carroll C. Moore M.M. Nakamoto R.K. Fox T.E. Citryl ornithine is an intermediate in a three-step biosynthetic pathway for rhizoferrin in Francisella.ACS Chem. Biol. 2019; 14: 1760-1766Crossref PubMed Scopus (1) Google Scholar). We also sought to determine how rhizoferrin is produced by R. pickettii and L. pneumophila, which synthesize putrescine. To elucidate the R. pickettii, L. pneumophila, and F. novicida biosynthetic pathways, we expressed relevant genes in Escherichia coli, and we assayed purified recombinant proteins in vitro. The X-ray crystal structure of the PLP-dependent decarboxylase FigC, which we confirmed in vitro to be an N-citrylornithine decarboxylase, was determined. The environmental β-proteobacterium R. pickettii produces rhizoferrin and encodes (Fig. 1A) a single NIS synthetase of 655 amino acids. Immediately downstream is a 414 amino acid ORF encoding a putative MFS transporter protein. To determine whether the R. pickettii NIS synthetase and MFS transporter are responsible for rhizoferrin production and sufficient to synthesize and export it when expressed heterologously in E. coli BL21, the genes were expressed individually or together from the expression vector pETDuet-1. Siderophore production resulting from growth of liquid cultures (Fig. 1B) or from solid agar plates (Fig. 1C) was detected using the Chrome Azurol S (CAS) reagent, siderophore production being visualized by formation of an intense yellow coloration. Expression of the R. pickettii NIS synthetase alone in E. coli was sufficient to produce a positive reaction with the CAS reagent, which was more intense when the R. pickettii MFS transporter or the F. novicida FigB MFS transporter was co-expressed. After partial purification of the supernatant from liquid cultures of E. coli BL21 co-expressing the R. pickettii NIS synthetase and MFS transporter and analysis by LC-MS, a mass corresponding to rhizoferrin (m/z 437.2) was detected that was absent in the control culture containing the empty plasmid (Fig. 1D). The presence of CAS-positive siderophore in the external medium, when E. coli BL21 expressing only the NIS synthetase was grown, indicates that rhizoferrin is exported by an E. coli efflux transporter. A more intense CAS reaction when the R. pickettii or F. novicida FigB MFS was co-expressed with the NIS synthetase may be because of greater affinity of the R. picketti or F. novicida transporters for rhizoferrin, or it may be because of their potentially higher expression levels compared with the native E. coli transporter(s). An alignment of the only close E. coli homolog, the MFS transporter MdtG (NP_415571), with the R. pickettii MFS, F. novicida FigB, and L. pneumophila LbtB transporters is shown in Figure S1A. A maximum likelihood phylogenetic tree indicates that F. novicida FigB and L. pneumophila LbtB transporters are more closely related compared with the R. pickettii MFS or E. coli MdtG proteins (Fig. S1B). The E. coli MdtG efflux transporter is involved in fluoroquinolone resistance (36Fabrega A. Martin R.G. Rosner J.L. Tavio M.M. Vila J. Constitutive SoxS expression in a fluoroquinolone-resistant strain with a truncated SoxR protein and identification of a new member of the marA-soxS-rob regulon, mdtG.Antimicrob. Agents Chemother. 2010; 54: 1218-1225Crossref PubMed Scopus (31) Google Scholar), and it is notable (Fig. S1C) that there are two internal amine groups separated by three carbons in the structure of quinolone antibiotics such as ciprofloxacin (37Wise R. Andrews J.M. Edwards L.J. In vitro activity of Bay 09867, a new quinoline derivative, compared with those of other antimicrobial agents.Antimicrob. Agents Chemother. 1983; 23: 559-564Crossref PubMed Scopus (514) Google Scholar). To assess the substrate specificity of the R. pickettii NIS synthetase, the recombinant his-tagged purified protein was assayed in vitro with citrate and different diamine and amino acid cosubstrates and siderophore production then detected with the CAS reagent (Fig. 1E). Putrescine and cadaverine produced a strong CAS reaction, a less intense reaction was detected with 1,3-diaminopropane, but no reaction was detected with L-ornithine or L-2,3-diaminopropionic acid. Analysis by LC-MS (Fig. S2) of the in vitro reaction products produced with putrescine or cadaverine revealed masses for rhizoferrin (m/z 437.2) or homorhizoferrin (m/z 451.2), respectively (Fig. 1E). As a dedicated efflux transporter did not appear to be required for excretion of heterologously produced rhizoferrin from E. coli BL21, we expressed in E. coli the L. pneumophila gene encoding the LbtA NIS synthetase that is required for rhizoferrin biosynthesis in that species. Expression of the L. pneumophila subsp. pneumophila str. Philadelphia 1 lbtA NIS synthetase (599 amino acids) alone from pETDuet-1 in E. coli did not result in siderophore production, as detected by the CAS reagent after growth on solid agar plates. We then noted that the lbtA ORF from the 130b strain of L. pneumophila used in the original study (25Allard K.A. Viswanathan V.K. Cianciotto N.P. lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin.J. Bacteriol. 2006; 188: 1351-1363Crossref PubMed Scopus (52) Google Scholar) lacked 19 amino acids from the N-terminus relative to the Philadelphia 1 strain. After removal of the first 19 amino acids from the Philadelphia 1 LbtA NIS synthetase, which consisted of the region up to the first internal ATG codon, expression of the shortened version in E. coli BL21 then resulted in siderophore production (Fig. 1F). It is noteworthy that R. pickettii likely synthesizes putrescine from ornithine using an aspartate aminotransferase-fold ODC (ACS63448, 759 a.a.) that is a close homolog (95% amino acid identity) of the characterized (38Lowe-Power T.M. Hendrich C.G. von Roepenack-Lahaye E. Li B. Wu D. Mitra R. Dalsing B.L. Ricca P. Naidoo J. Cook D. Jancewicz A. Masson P. Thomma B. Lahaye T. Michael A.J. et al.Metabolomics of tomato xylem sap during bacterial wilt reveals Ralstonia solanacearum produces abundant putrescine, a metabolite that accelerates wilt disease.Environ. Microbiol. 2018; 20: 1330-1349Crossref PubMed Scopus (40) Google Scholar) Ralstonia solanacearum ODC (CAD16072, 759 a.a.). In contrast, L. pneumophila likely synthesizes putrescine from arginine using an AR-fold arginine decarboxylase, agmatine deiminase, and N-carbamoylputrescine amidohydrolase (see correction to [39Nasrallah G.K. Riveroll A.L. Chong A. Murray L.E. Lewis P.J. Garduno R.A. Legionella pneumophila requires polyamines for optimal intracellular growth.J. Bacteriol. 2011; 193: 4346-4360Crossref PubMed Scopus (32) Google Scholar]). In contrast to rhizoferrin biosynthesis in R. pickettii and L. pneumophila, F. novicida requires not only the encoded NIS synthetase FigA and MFS transporter FigB but also a homolog (FigC) of the AR-fold PLP-dependent decarboxylase family that includes L-ornithine decarboxylase (L-ODC), an enzyme that produces putrescine from L-ornithine (34Kiss K. Liu W. Huntley J.F. Norgard M.V. Hansen E.J. Characterization of fig operon mutants of Francisella novicida U112.FEMS Microbiol. Lett. 2008; 285: 270-277Crossref PubMed Scopus (36) Google Scholar). We expressed separately either the F. novicida figA NIS synthetase gene or the figC decarboxylase, from pETDuet-1 in E. coli BL21 grown in liquid culture or on solid agar plates, but no siderophore production was detected using the CAS reagent (Fig. 2, A–B). However, co-expression of figA and figC resulted in a strong positive CAS reaction comparable to the reaction produced by co-expression of the R. pickettii NIS synthetase and MFS transporter. Partial purification of the supernatant from the E. coli liquid culture co-expressing the F. novicida figA and figC genes and analysis by LC-MS (Fig. 2C) revealed a mass corresponding to rhizoferrin (m/z 437.2). To determine whether co-expression of the F. novicida figB or R. pickettii MFS transporters together with figA and figC would increase siderophore production from E. coli, we co-expressed figA and figC from pETDuet-1 and figB or the R. pickettii MFS transporter from pACYCDuet-1 (Fig. 2D). Co-expression of either transporter noticeably increased the intensity of the CAS reagent positive reaction. We reasoned that if FigC was an L-ODC producing putrescine from ornithine, then figC would not be required for heterologous rhizoferrin biosynthesis in E. coli, as there is already abundant putrescine present. Therefore, the most likely explanation for the requirement for figC would be that FigA conjugates L-ornithine to citrate to form N-citrylornithine, which is then decarboxylated by FigC to form N-citrylputrescine. Recombinant FigA and FigC proteins were purified and assayed in vitro with the CAS reagent, and assay reactions contained 2 mM Na-citrate and 2 mM L-ornithine (Fig. 2E). A slight positive reaction was observed with FigA alone, but an intense reaction was observed with FigA and FigC together, comparable with the reaction produced by the R. pickettii-purified recombinant NIS synthetase (Fig. 2E). Although FigA and FigC together produced a positive reaction with L-ornithine, no reaction was seen with D-ornithine, L-lysine, putrescine, cadaverine, 1,3-diaminopropane, L-2,4-diaminobutyrate, or L-2,3-diaminopropionate (Fig. 2F). When the reaction products from FigA alone assayed with citrate and L-ornithine assay were analyzed by LC-MS, a mass for N-citrylornithine was detected (m/z 307.1). With FigA and FigC together, a mass for rhizoferrin (m/z 437.1) was detected (Fig. S3). The requirement for F. novicida figC in heterologous rhizoferrin biosynthesis in E. coli, the fact that FigA can use only L-ornithine and not putrescine in vitro for rhizoferrin biosynthesis, and the need for FigC in the conversion of N-citrylornithine to rhizoferrin in vitro confirms that FigC is an N-citrylornithine decarboxylase and not an L-ornithine decarboxylase. On the basis of these findings, we propose the two alternative bacterial rhizoferrin biosynthetic pathways depicted in Figure 3. The R. pickettii NIS synthetase, and by extension the L. pneumonphila LbtA NIS synthetase, is alone sufficient for rhizoferrin biosynthesis via N-citrylputrescine (Fig. 3A). In contrast, the F. novicida FigA NIS synthetase produces N-citrylornithine, which is then decarboxylated to N-citrylputrescine by FigC, and another citrate condensed to N-citrylputrescine by FigA to produce rhizoferrin (Fig. 3B). Recently, Ramakrishnan et al. (35Ramakrishnan G. Perez N.M. Carroll C. Moore M.M. Nakamoto R.K. Fox T.E. Citryl ornithine is an intermediate in a three-step biosynthetic pathway for rhizoferrin in Francisella.ACS Chem. Biol. 2019; 14: 1760-1766Crossref PubMed Scopus (1) Google Scholar) have shown that a figC deletion mutant of F. tularensis LVS accumulates N-citrylornithine. Given the novel substrate preference of the FigC decarboxylase for N-citrylornithine, we solved the X-ray crystal structure of FigC and compared it with other AR-fold PLP-dependent decarboxylases. The X-ray crystal structure was solved to 2.1 Å resolution by single-wavelength anomalous diffraction using incorporated selenomethionine to phase the structure (Table S1). Two functional dimers were found in the asymmetric unit (four monomers), and all four active sites showed good density for the cofactor PLP (Fig. 4). FigC is composed of an N-terminal β/α-barrel domain and a C-terminal β-barrel domain (Fig. 5A), typical of the AR-fold PLP-dependent decarboxylases. The FigC structure exhibits strong similarity to other structures within the family representing diverse substrate specificities, including meso-diaminopimelate decarboxylase (DAPDC) from the euryarchaeote Methanocaldococcus jannaschii, carboxyspermidine decarboxylase (CASDC) from Campylobacter jejuni, lysine/ornithine decarboxylase (L/ODC) from Vibrio vulnificus, and arginine decarboxylase (ADC) from V. vulnificus (Fig. 6). An internal insertion and C-terminal extension in ADC confers a tetrameric rather than dimeric structure (47Deng X. Lee J. Michael A.J. Tomchick D.R. Goldsmith E.J. Phillips M.A. Evolution of substrate specificity within a diverse family of beta/alpha-barrel-fold basic amino acid decarboxylases: X-ray structure determination of enzymes with specificity for L-arginine and carboxynorspermidine.J. Biol. Chem. 2010; 285: 25708-25719Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar).Figure 5Structure of N-citrylornithine decarboxylase FigC solved to 2.1 Å resolution. A, cartoon diagram showing the dimeric structure with subunit (a) colored in pink and subunit (b) colored in green. Bound PLP is shown as balls colored with carbons in yellow, oxygen in red, and nitrogen in blue. B, FigC shared active site structure with subunits and PLP colored as in (A). The specificity helix is drawn as a cartoon.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Structural alignment of FigC with other PLP-dependent enzymes from" @default.
• W3107257413 created "2020-12-07" @default.
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• W3107257413 date "2021-01-01" @default.
• W3107257413 modified "2023-09-30" @default.
• W3107257413 title "Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin" @default.
• W3107257413 cites W146458909 @default.
• W3107257413 cites W1525790461 @default.
• W3107257413 cites W1532422753 @default.
• W3107257413 cites W1539796472 @default.
• W3107257413 cites W1587329961 @default.
• W3107257413 cites W1971955262 @default.
• W3107257413 cites W1974861151 @default.
• W3107257413 cites W1995687577 @default.
• W3107257413 cites W1997867228 @default.
• W3107257413 cites W1997955858 @default.
• W3107257413 cites W2005091504 @default.
• W3107257413 cites W2006151417 @default.
• W3107257413 cites W2011482609 @default.
• W3107257413 cites W2012980277 @default.
• W3107257413 cites W2013740990 @default.
• W3107257413 cites W2014096376 @default.
• W3107257413 cites W2016911133 @default.
• W3107257413 cites W2018545062 @default.
• W3107257413 cites W2020594474 @default.
• W3107257413 cites W2022017052 @default.
• W3107257413 cites W2028207395 @default.
• W3107257413 cites W2028255595 @default.
• W3107257413 cites W2032247032 @default.
• W3107257413 cites W2032974626 @default.
• W3107257413 cites W2035034686 @default.
• W3107257413 cites W2053412997 @default.
• W3107257413 cites W2057188740 @default.
• W3107257413 cites W2067170848 @default.
• W3107257413 cites W2086683699 @default.
• W3107257413 cites W2088798172 @default.
• W3107257413 cites W2094823810 @default.
• W3107257413 cites W2099143902 @default.
• W3107257413 cites W2101787865 @default.
• W3107257413 cites W2102789902 @default.
• W3107257413 cites W2104929909 @default.
• W3107257413 cites W2105668062 @default.
• W3107257413 cites W2108279937 @default.
• W3107257413 cites W2114543923 @default.
• W3107257413 cites W2118351789 @default.
• W3107257413 cites W2123595241 @default.
• W3107257413 cites W2124967403 @default.
• W3107257413 cites W2126061614 @default.
• W3107257413 cites W2127594598 @default.
• W3107257413 cites W2127947770 @default.
• W3107257413 cites W2128022667 @default.
• W3107257413 cites W2132926880 @default.
• W3107257413 cites W2134967712 @default.
• W3107257413 cites W2140165894 @default.
• W3107257413 cites W2143815085 @default.
• W3107257413 cites W2144081223 @default.
• W3107257413 cites W2145251715 @default.
• W3107257413 cites W2148099246 @default.
• W3107257413 cites W2158068445 @default.
• W3107257413 cites W2169678694 @default.
• W3107257413 cites W2180229411 @default.
• W3107257413 cites W2191795945 @default.
• W3107257413 cites W2320168982 @default.
• W3107257413 cites W2336400828 @default.
• W3107257413 cites W2414062173 @default.
• W3107257413 cites W2502106085 @default.
• W3107257413 cites W2509637828 @default.
• W3107257413 cites W2516932902 @default.
• W3107257413 cites W2605019312 @default.
• W3107257413 cites W2622492575 @default.
• W3107257413 cites W2727014570 @default.
• W3107257413 cites W2763616786 @default.
• W3107257413 cites W2769891178 @default.
• W3107257413 cites W2782233064 @default.
• W3107257413 cites W2788233758 @default.
• W3107257413 cites W2809913031 @default.
• W3107257413 cites W2895685301 @default.
• W3107257413 cites W2911608134 @default.
• W3107257413 cites W2938574745 @default.
• W3107257413 cites W2953760796 @default.
• W3107257413 cites W2971186504 @default.
• W3107257413 cites W2980889426 @default.
• W3107257413 cites W4233761318 @default.
• W3107257413 cites W67239401 @default.
• W3107257413 doi "https://doi.org/10.1074/jbc.ra120.016738" @default.
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