FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079181465> ?p ?o ?g. }

• W2079181465 endingPage "28500" @default.
• W2079181465 startingPage "28493" @default.
• W2079181465 abstract "The molybdenum cofactor (Moco) exists in different variants in the cell and can be directly inserted into molybdoenzymes utilizing the molybdopterin (MPT) form of Moco. In bacteria such as Rhodobacter capsulatus and Escherichia coli, MPT is further modified by attachment of a GMP nucleotide, forming MPT guanine dinucleotide (MGD). In this work, we analyzed the distribution and targeting of different forms of Moco to their respective user enzymes by proteins that bind Moco and are involved in its further modification. The R. capsulatus proteins MogA, MoeA, MobA, and XdhC were purified, and their specific interactions were analyzed. Interactions between the protein pairs MogA-MoeA, MoeA-XdhC, MoeA-MobA, and XdhC-MobA were identified by surface plasmon resonance measurements. In addition, the transfer of Moco produced by the MogA-MoeA complex to XdhC was investigated. A direct competition of MobA and XdhC for Moco binding was determined. In vitro analyses showed that XdhC bound to MobA, prevented the binding of Moco to MobA, and thereby inhibited MGD biosynthesis. The data were confirmed by in vivo studies in R. capsulatus cells showing that overproduction of XdhC resulted in a 50% decrease in the activity of bis-MGD-containing Me2SO reductase. We propose that, in bacteria, the distribution of Moco in the cell and targeting to the respective user enzymes are accomplished by specific proteins involved in Moco binding and modification. The molybdenum cofactor (Moco) exists in different variants in the cell and can be directly inserted into molybdoenzymes utilizing the molybdopterin (MPT) form of Moco. In bacteria such as Rhodobacter capsulatus and Escherichia coli, MPT is further modified by attachment of a GMP nucleotide, forming MPT guanine dinucleotide (MGD). In this work, we analyzed the distribution and targeting of different forms of Moco to their respective user enzymes by proteins that bind Moco and are involved in its further modification. The R. capsulatus proteins MogA, MoeA, MobA, and XdhC were purified, and their specific interactions were analyzed. Interactions between the protein pairs MogA-MoeA, MoeA-XdhC, MoeA-MobA, and XdhC-MobA were identified by surface plasmon resonance measurements. In addition, the transfer of Moco produced by the MogA-MoeA complex to XdhC was investigated. A direct competition of MobA and XdhC for Moco binding was determined. In vitro analyses showed that XdhC bound to MobA, prevented the binding of Moco to MobA, and thereby inhibited MGD biosynthesis. The data were confirmed by in vivo studies in R. capsulatus cells showing that overproduction of XdhC resulted in a 50% decrease in the activity of bis-MGD-containing Me2SO reductase. We propose that, in bacteria, the distribution of Moco in the cell and targeting to the respective user enzymes are accomplished by specific proteins involved in Moco binding and modification. The molybdenum cofactor (Moco) 2The abbreviations used are: Mocomolybdenum cofactorMPTmolybdopterinbis-MGDbismolybdopterin guanine dinucleotideXDHxanthine dehydrogenasehSO-MDhuman sulfite oxidase Moco domainNi-NTAnickel-nitrilotriacetic acidSPRsurface plasmon resonance. 2The abbreviations used are: Mocomolybdenum cofactorMPTmolybdopterinbis-MGDbismolybdopterin guanine dinucleotideXDHxanthine dehydrogenasehSO-MDhuman sulfite oxidase Moco domainNi-NTAnickel-nitrilotriacetic acidSPRsurface plasmon resonance. is an essential component of a diverse group of enzymes involved in important redox reactions in the global carbon, nitrogen, and sulfur cycles. Moco consists of a molybdenum atom coordinated to the dithiolene group of a tricyclic pyranopterin referred to as molybdopterin (MPT) (1Rajagopalan K.V. Johnson J.L. J. Biol. Chem. 1992; 267: 10199-10202Abstract Full Text PDF PubMed Google Scholar). The biosynthesis of Moco is highly conserved in eukaryotes and prokaryotes (1Rajagopalan K.V. Johnson J.L. J. Biol. Chem. 1992; 267: 10199-10202Abstract Full Text PDF PubMed Google Scholar) and can be divided into three general steps. In the first step, GTP is converted to the meta-stable intermediate Precursor Z (2Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1995; 270: 1082-1087Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 3Hänzelmann P. Schindelin H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12870-12875Crossref PubMed Scopus (211) Google Scholar). In the second step, Precursor Z is further transformed by MPT synthase into MPT by generation of its characteristic dithiolene group (4Pitterle D.M. Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13506-13509Abstract Full Text PDF PubMed Google Scholar, 5Gutzke G. Fischer B. Mendel R. Schwarz G. J. Biol. Chem. 2001; 276: 36268-36274Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In the third step, molybdate is inserted to the MPT dithiolene sulfurs, a reaction catalyzed by MogA and MoeA in Escherichia coli (6Joshi M.S. Johnson J.L. Rajagopalan K.V. J. Bacteriol. 1996; 178: 4310-4312Crossref PubMed Google Scholar, 7Nichols J. Rajagopalan K.V. J. Biol. Chem. 2002; 277: 24995-25000Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 8Nichols J. Rajagopalan K.V. J. Biol. Chem. 2005; 280: 7817-7822Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). MoeA mediates molybdenum ligation, whereas MogA helps to facilitate this step in an ATP-dependent manner (8Nichols J. Rajagopalan K.V. J. Biol. Chem. 2005; 280: 7817-7822Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Recently, studies with the homologous Arabidopsis thaliana CNX1 protein G and E domains identified the formation of an MPT-AMP intermediate before the ligation of molybdate to the MPT moiety (9Llamas A. Mendel R.R. Schwarz G. J. Biol. Chem. 2004; 279: 55241-55246Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Llamas A. Otte T. Multhaup G. Mendel R.R. Schwarz G. J. Biol. Chem. 2006; 281: 18343-18350Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). An unexpected observation in the crystal structure of the A. thaliana CNX1 protein G domain was the identification of copper bound to the MPT-AMP dithiolene sulfurs (11Kuper J. Llamas A. Hecht H.J. Mendel R.R. Schwarz G. Nature. 2004; 430: 803-806Crossref PubMed Scopus (156) Google Scholar). Up to now, the function of this novel MPT ligand has been unknown, but it was speculated that copper might play a role in sulfur transfer to Precursor Z, in protection of the MPT dithiolene from oxidation, and/or in presentation of a suitable leaving group for molybdenum insertion (12Schwarz G. CMLS Cell. Mol. Life Sci. 2005; 62: 2792-2810Crossref PubMed Scopus (129) Google Scholar). To date, copper-MPT-AMP has not been identified as an intermediate in the biosynthesis of Moco in E. coli (8Nichols J. Rajagopalan K.V. J. Biol. Chem. 2005; 280: 7817-7822Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). molybdenum cofactor molybdopterin bismolybdopterin guanine dinucleotide xanthine dehydrogenase human sulfite oxidase Moco domain nickel-nitrilotriacetic acid surface plasmon resonance. molybdenum cofactor molybdopterin bismolybdopterin guanine dinucleotide xanthine dehydrogenase human sulfite oxidase Moco domain nickel-nitrilotriacetic acid surface plasmon resonance. After the insertion of molybdenum into MPT in E. coli, Moco either can be directly inserted into molybdoenzymes (such as YedY) binding the MPT form of Moco (13Loschi L. Brokx S.J. Hills T.L. Zhang G. Bertero M.G. Lovering A.L. Weiner J.H. Strynadka N.C. J. Biol. Chem. 2004; 279: 50391-50400Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) or is further modified by attachment of GMP (14Johnson J.L. Bastian N.R. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3190-3194Crossref PubMed Scopus (155) Google Scholar, 15Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar), forming the bis-MPT guanine dinucleotide (bis-MGD) form of Moco found in enzymes of the Me2SO reductase family (16Kisker C. Schindelin H. Rees D.C. Annu. Rev. Biochem. 1997; 66: 233-267Crossref PubMed Scopus (439) Google Scholar). In E. coli, the GMP attachment to Moco is catalyzed by the MobA and MobB proteins (17Palmer T. Vasishta A. Whitty P.W. Boxer D.H. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar). Whereas MobA was shown to be essential for this reaction (18Palmer T. Santini C.L. Iobbi-Nivol C. Eaves D.J. Boxer D.H. Giordano G. Mol. Microbiol. 1996; 20: 875-884Crossref PubMed Scopus (145) Google Scholar), the role of MobB still remains uncertain. From the crystal structure, it was postulated that MobB is an adapter protein that acts in concert with MobA to achieve the efficient biosynthesis and utilization of MGD (19McLuskey K. Harrison J.A. Schüttelkopf A.W. Boxer D.H. Hunter W.N. J. Biol. Chem. 2003; 278: 23706-23713Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Enzymes containing the MPT form of Moco belong to either the sulfite oxidase or xanthine oxidase family, whereas enzymes binding the bis-MGD form of Moco belong to the Me2SO reductase family of molybdoenzymes. In Rhodobacter capsulatus, xanthine dehydrogenase (XDH; EC 1.17.1.4) is the only identified enzyme harboring the MPT form of the cofactor, whereas all other known molybdoenzymes bind the bis-MGD form of the cofactor (20Leimkühler S. Kern M. Solomon P.S. McEwan A.G. Schwarz G. Mendel R.R. Klipp W. Mol. Microbiol. 1998; 27: 853-869Crossref PubMed Scopus (88) Google Scholar). An essential role for the XdhC protein in the maturation of R. capsulatus XDH has been described, which entails binding of Moco and its insertion into the XdhB subunit of XDH (21Neumann M. Schulte M. Jünemann N. Stöcklein W. Leimkühler S. J. Biol. Chem. 2006; 281: 15701-15708Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). For all members of the xanthine oxidase family, the sulfurated form of Moco is essential for catalysis, where the equatorial of the two oxygen ligands of nascent Moco is replaced by sulfur (22Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1470) Google Scholar). Previous work showed that XdhC specifically promotes the exchange of this ligand by interaction with the l-cysteine desulfurase NifS4, which transfers the sulfur to Moco bound to XdhC (23Neumann M. Stöcklein W. Walburger A. Magalon A. Leimkühler S. Biochemistry. 2007; 46: 9586-9595Crossref PubMed Scopus (42) Google Scholar). It has remained so far unclear which protein of the Moco biosynthesis pathway acts as the direct Moco donor for XdhC. So far, R. capsulatus XdhC is the only protein identified in bacteria shown to be involved in the modification of Moco by exchange of an oxo ligand of Moco with sulfur. To investigate the question of targeting, distribution, and insertion of different forms of Moco into the specific molybdoenzymes in R. capsulatus, we cloned and purified the MogA, MoeA, and MobA proteins from R. capsulatus for the investigation of protein-protein interactions in the homologous system. A MobB homolog seems not to be present in R. capsulatus (24Leimkühler S. Klipp W. FEMS Microbiol. Lett. 1999; 174: 239-246Crossref PubMed Scopus (17) Google Scholar). In this study, we show for the first time that the amounts of sulfurated Moco and bis-MGD produced in the cell are regulated at the protein level by protein-protein interactions. We show that both MobA and XdhC receive Moco from MoeA; however, by binding to MobA, XdhC prevents the Moco transfer to MobA, thus inhibiting MGD formation. This regulation ensures that enough Moco is abstracted from the major route of bis-MGD biosynthesis for further modification to the sulfurated form of Moco and thus ensures that enough of the MPT form of Moco is provided to produce an active XDH, an enzyme involved in the purine degradation pathway. Bacterial Strains, Plasmids, Media, and Growth Conditions—E. coli BL21(DE3) cells were used for heterologous expression of the R. capsulatus proteins MobA, MoeA, and MogA. R. capsulatus XdhC was expressed in E. coli ER2566(DE3) cells and purified as described previously (21Neumann M. Schulte M. Jünemann N. Stöcklein W. Leimkühler S. J. Biol. Chem. 2006; 281: 15701-15708Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The human sulfite oxidase Moco domain (hSO-MD) was expressed from plasmid pTG818 (25Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar) in E. coli TP1000(ΔmobAB) cells (18Palmer T. Santini C.L. Iobbi-Nivol C. Eaves D.J. Boxer D.H. Giordano G. Mol. Microbiol. 1996; 20: 875-884Crossref PubMed Scopus (145) Google Scholar) to obtain Moco-containing hSO-MD or in E. coli RK5202(modC-) cells (26Miller J.B. Scott D.J. Amy N.K. J. Bacteriol. 1987; 169: 1853-1860Crossref PubMed Google Scholar) to obtain MPT-containing hSO-MD. The hSO-MD variants were purified as described previously by Temple et al. (25Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). E. coli cultures were grown in LB medium under aerobic conditions at 30 °C. The E. coli S17-1 strain was used for conjugation of R. capsulatus KS36 (B10S(ΔnifHDK::Spc)) (27Wang G. Angermüller S. Klipp W. J. Bacteriol. 1993; 175: 3031-3042Crossref PubMed Google Scholar) with plasmid pSL160, expressing xdhC under the control of the nifH promotor from plasmid pPHU231 (28Reyes F. Roldan M.D. Klipp W. Castillo F. Moreno-Vivian C. Mol. Microbiol. 1996; 19: 1307-1318Crossref PubMed Scopus (82) Google Scholar, 29Pollock D. Bauer C.E. Scolnik P.A. Gene (Amst.). 1988; 65: 269-275Crossref PubMed Scopus (43) Google Scholar, 30Leimkühler S. Angermüller S. Schwarz G. Mendel R.R. Klipp W. J. Bacteriol. 1999; 181: 5930-5939Crossref PubMed Google Scholar). For control experiments, pSL143 harboring only the nifH promotor cloned into plasmid pPHU231 and pMN80 expressing mobA under the control of the nifH promotor were introduced into KS36 cells. R. capsulatus cells were grown in RCV minimal medium supplemented with (NH4)2SO4 as described previously (31Klipp W. Masepohl B. Pühler A. J. Bacteriol. 1988; 170: 693-699Crossref PubMed Google Scholar). For expression of Me2SO reductase, Me2SO was added to a final concentration of 30 mm, and for induction of XdhC or MobA expression from the plasmids, (NH4)2SO4 was replaced with 1 g/liter serine as the nitrogen source. R. capsulatus cells were grown under anaerobic, phototrophic conditions and harvested at late log phase after 48 h of growth at 30 °C. When required, 1 mm Na2MoO4, 150 μg/ml ampicillin, 25 μg/ml kanamycin, and 5 μg/ml tetracycline (for E. coli) or 1 μg/ml tetracycline (for R. capsulatus) were added to the medium. Cloning, Expression, and Purification of R. capsulatus MobA, MoeA, and MogA—DNA fragments containing the coding regions for R. capsulatus mobA, moeA, and mogA were amplified by PCR, and flanking restriction sites were introduced. The moeA and mogA genes were cloned into the NdeI-XhoI sites and mobA into the NdeI-SalI sites of pET28a (Novagen), resulting in plasmids pMN32, pMN53, and pMN56, respectively. For expression of MoeA, MogA, and MobA, E. coli BL21(DE3) cells were transformed with plasmids pMN32, pMN53, and pMN56, respectively, and cell growth was started with 10 ml of overnight culture/liter of LB medium. The cells were grown at 30 °C, and expression of MogA and MobA was induced at A600 = 0.3–0.5 with 100 μm isopropyl β-d-thiogalactopyranoside. Cell growth was continued for 5 h, and cells were harvested and resuspended in 50 mm NaH2PO4 and 300 mm NaCl (pH 8.0). After cell lysis, the soluble fraction was transferred onto a column with nickel-nitrilotriacetic acid (Ni-NTA; Qiagen Inc.). The resin was washed with 20 column volumes of phosphate buffer first containing 10 mm and then 20 mm imidazole. The proteins were eluted with phosphate buffer containing 250 mm imidazole and dialyzed against 100 mm Tris (pH 7.2). For MoeA, expression was induced with 300 μm isopropyl β-d-thiogalactopyranoside; the shaking rate was increased to 210 rpm; and cells were harvested 3 h after induction of gene expression. After cell lysis, the soluble fraction was transferred onto nickel-triscarboxymethylethylenediamine (Macherey-Nagel). The column was washed with 40 column volumes of phosphate buffer containing 10 mm imidazole, and MoeA was eluted with the same buffer containing 250 mm imidazole and dialyzed against 100 mm Tris (pH 7.2). MGD Formation by MobA—Moco was obtained after heat treatment of purified hSO-MD expressed in TP1000 cells, and MPT was obtained after heat treatment of purified hSO-MD expressed in RK5202 cells as described previously by Temple et al. (25Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar) and Neumann et al. (21Neumann M. Schulte M. Jünemann N. Stöcklein W. Leimkühler S. J. Biol. Chem. 2006; 281: 15701-15708Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). 100 μm MobA was incubated with 160 μm Moco or MPT before excess Moco/MPT was removed by gel filtration. 1 mm Na2MoO4 was included in incubation mixtures containing Moco. 1 mm MgCl2 and 1 mm GTP were added, and the mixtures were incubated for 60 min at room temperature in a total volume of 400 μl of 100 mm Tris (pH 7.2) before the protein was denatured and analyzed for the presence of Moco or MGD by conversion to Form A (as described below). MPT and Moco Binding by MogA, MoeA, and MobA—KD values were determined by ultrafiltration as described previously (21Neumann M. Schulte M. Jünemann N. Stöcklein W. Leimkühler S. J. Biol. Chem. 2006; 281: 15701-15708Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Samples contained 6 μm MogA, MoeA, or MobA and 0–24 μm Moco or MPT. Moco, MPT, and MGD Analysis—The Moco/MPT content of the purified proteins was quantified after conversion to Form A as described previously (21Neumann M. Schulte M. Jünemann N. Stöcklein W. Leimkühler S. J. Biol. Chem. 2006; 281: 15701-15708Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). To separate Form A obtained from Moco/MPT and Form A-GMP obtained from MGD, the protocol originally described by Joshi and Rajagopalan (32Joshi M.S. Rajagopalan K.V. Arch. Biochem. Biophys. 1994; 308: 331-334Crossref PubMed Scopus (9) Google Scholar) was used with some modifications. MGD was converted to Form A-GMP and Moco/MPT to Form A by overnight treatment with acidic iodine at room temperature (33Johnson J.L. Hainline B.E. Rajagopalan K.V. Arison B.H. J. Biol. Chem. 1984; 259: 5414-5422Abstract Full Text PDF PubMed Google Scholar). Form A was separated from Form A-GMP by chromatography on Q-Sepharose (GE Healthcare). 400 μl of Q-Sepharose was equilibrated with H2O; the oxidized samples were loaded; and Form A was eluted with 10 mm acetic acid. Form A-GMP was eluted with 50 mm HCl and converted to Form A by the addition of MgCl2, nucleotide pyrophosphatase, and alkaline phosphatase at pH 8.0. The pH of the samples was adjusted to pH 5.3 by the addition of 10 μl of 50% acetic acid before application to a C18 reversed-phase high pressure liquid chromatography column (4.6 × 250-mm Hypersil ODS, 5-μm particle size) equilibrated in 5 mm ammonium acetate and 15% methanol. In-line fluorescence was monitored by an Agilent 1100 series detector with excitation at 383 nm and emission at 450 nm. Surface Plasmon Resonance (SPR) Measurements—All binding experiments were performed with the SPR-based instrument Biacore™ 2000 on CM5 sensor chips at 25 °C and a flow rate of 10 μl/min using BiaControl 2.1 and BiaEvaluation 3.0 software (Biacore AB) as described previously (23Neumann M. Stöcklein W. Walburger A. Magalon A. Leimkühler S. Biochemistry. 2007; 46: 9586-9595Crossref PubMed Scopus (42) Google Scholar). The proteins were immobilized after dilution in 10 mm acetate buffer at pH 4 (bovine serum albumin, MoeA, and MogA) or pH 5 (XdhC, XDH, and MobA). For control experiments, the N-terminal His6 tags of MoeA, MogA, and MobA were cleaved using the thrombin CleanCleave kit (Sigma). Cleavage was controlled by SDS-PAGE. In Vitro Transfer of Moco from MoeA to XdhC—For production of Moco, 15 μm MPT obtained from hSO-MD was incubated with 30 μm MogA, 30 μm MoeA, 37.5 μm Na2MoO4, 1 mm MgCl2, and 1 mm ATP in a volume of 200 μl of 100 mm Tris (pH 7.2). After 15 min of incubation at room temperature, 8 μm XdhC was added, and the mixture was further incubated for 20 min before MoeA and MogA were removed by Ni-NTA chromatography. Single components were left out for control experiments. Free Moco in the XdhC fraction was removed by an additional gel filtration step using NICK columns (GE Healthcare). 400 μl of the XdhC fraction was treated with acidic iodine, and bound Moco was quantified as Form A fluorescence as described above. The same setup as described above was used to analyze the competition of Moco transfer from MoeA to XdhC in the presence of MobA. To avoid free Moco in the assay, these mixtures contained 15 μm XdhC and either 15 μm or 150 μm MobA in addition to 1 mm GTP. In Vitro Transfer of Moco from MobA to XdhC—25 μm MobA was incubated with 100 μm Moco and 1 mm Na2MoO4 for 10 min at room temperature, and excess Moco was removed by gel filtration. Moco bound to MobA was quantified after conversion to Form A. 10 μm XdhC was added to the Moco-loaded MobA fraction before MobA and XdhC were separated by Ni-NTA chromatography. Free Moco in the XdhC fraction was further removed by gel filtration. 400 μl of the XdhC fraction was treated with acidic iodine, and released Moco was converted to Form A and quantified as described above. Formation of MGD by MobA in the Presence of XdhC—To analyze the influence of XdhC on Moco binding to MobA, 10 μm MobA was incubated with 0, 1, 10, and 100 μm XdhC for 10 min at room temperature before the addition of 1 mm GTP, 1 mm MgCl2, 1 mm Na2MoO4, and 80 μm Moco. In another set of experiments, XdhC was added after incubation of 115 μm MobA with 190 μm Moco and 1 mm Na2MoO4 for 10 min at room temperature. Unbound Moco was removed by gel filtration, and 50 μl of the Moco-loaded MobA fraction was incubated with 1 mm Na2MoO4, 1 mm GTP, and increasing amounts of XdhC (0, 1.16, 11.6, and 116 μm) in a total volume of 400 μl in 100 mm Tris (pH 7.2). The incubation mixtures were incubated for 90 min under anaerobic conditions, and the MGD content of all samples was quantified as described above. Analysis of XDH and Me2SO Reductase Activities in R. capsulatus Crude Extracts—Plasmids were mobilized from E. coli S17-1 into R. capsulatus KS36 by filter mating as described previously (31Klipp W. Masepohl B. Pühler A. J. Bacteriol. 1988; 170: 693-699Crossref PubMed Google Scholar). Crude extracts were obtained after cell lysis by sonification and subsequent removal of cell debris by centrifugation. Protein concentrations were determined following the method of Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). XDH activity was measured as described previously (24Leimkühler S. Klipp W. FEMS Microbiol. Lett. 1999; 174: 239-246Crossref PubMed Scopus (17) Google Scholar). The specific XDH activity (units/mg) is defined as the reduction of 1 μmol of NAD+/min/mg of enzyme. Me2SO reductase activity was measured as described by McEwan et al. (35McEwan A.G. Wetzstein H.G. Ferguson S.J. Jackson J.B. Biochim. Biophys. Acta. 1985; 806: 410-417Crossref Scopus (75) Google Scholar) with dithionite-reduced benzyl viologen as the electron donor. Me2SO reductase activity (units/mg) is defined as the reduction of 1 μmol of Me2SO/min/mg of protein. Purification and Analysis of the Functional Activities of R. capsulatus MobA, MoeA, and MogA—For purification of R. capsulatus MobA, MoeA, and MogA, fusion proteins were generated each containing an N-terminal His6 tag (see “Experimental Procedures”). After heterologous expression in E. coli BL21(DE3) cells, the soluble fractions of MobA, MoeA, and MogA were purified by affinity chromatography (see “Experimental Procedures”). After elution, one major band was displayed for MobA, MoeA, and MogA on Coomassie Brilliant Blue R-stained SDS-polyacrylamide gels, corresponding to molecular masses of 22.1, 44.6, and 20.3 kDa, respectively (Fig. 1A). This procedure yielded ∼6.5 mg of MobA/liter of E. coli culture, 1.5 mg of MoeA/liter of E. coli culture, and 13.3 mg of MogA/liter of E. coli culture. To show the functionality of R. capsulatus MoeA and MogA, Moco was produced from MPT in vitro and inserted into human aposulfite oxidase following the procedure described by Nichols and Rajagopalan (8Nichols J. Rajagopalan K.V. J. Biol. Chem. 2005; 280: 7817-7822Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) for E. coli MoeA and MogA. In contrast to the E. coli proteins, for which maximum human sulfite oxidase reconstitution was observed using a MogA/MoeA ratio of 1:10, the best human sulfite oxidase reconstitution was obtained when R. capsulatus MogA and MoeA where used in a ratio of at least 1:1.4 (data not shown). Thus, for all further assays, R. capsulatus MogA and MoeA were mixed in a 1:1 ratio. To test the functionality of R. capsulatus MobA, its ability to produce MGD from either MPT or Moco in the presence of MgGTP was analyzed. Moco was extracted from hSO-MD expressed in E. coli TP1000 cells (25Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar), whereas MPT was obtained from hSO-MD expressed in RK5202(modC-) cells (26Miller J.B. Scott D.J. Amy N.K. J. Bacteriol. 1987; 169: 1853-1860Crossref PubMed Google Scholar). After the addition of MgGTP to the incubation mixture, MGD was formed only from Moco (Fig. 1B, bars II and IV). Bound MPT and the remaining Moco eluted in the Moco/MPT fraction (bars I and III). These results show that molybdenum insertion into MPT has to precede MGD formation. Thus, for all further experiments, Moco was used as a precursor for the formation of MGD. Analysis of Protein-Protein Interactions by SPR Measurements—To identify possible protein-protein interactions between XdhC, MogA, MoeA, and MobA, SPR measurements were employed for real-time detection of specific interactions using the purified proteins. XdhC, MoeA, MogA, and MobA were immobilized via amine coupling to a CM5 chip, and interactions were analyzed with each protein partner. The results obtained by SPR measurements for the protein pairs listed in Table 1 showed the tightest interaction between MoeA and XdhC, with KD values of 1.45 and 1.15 μm depending on the immobilized protein. In contrast, no significant interaction between XdhC and MogA was obtained with either immobilized protein partner, whereas in another set of experiments, MoeA bound to MogA with KD values of 6.2 and 6.0 μm (Table 1). However, this also shows that XdhC preferentially interacts with MoeA and not with MogA. The interaction between MoeA and MobA was also investigated. When MoeA was immobilized, MobA interacted with a KD of 3.5 μm, whereas when MobA was immobilized, the KD was significantly higher (27 μm) (Table 1). The second value for immobilized MobA shows that the dissociation constant between both proteins was negatively influenced by the immobilization of MobA. Because both XdhC and MobA interacted with MoeA and might compete for the same binding site, a possible interaction between XdhC and MobA was also investigated. As shown in Table 1, an interaction between both proteins was identified with KD values of 1.75 and 1.04 μm for immobilized XdhC and MobA, respectively. In contrast to the MoeA-MobA interaction, the dissociation constant for this protein pair was not influenced by the immobilization of MobA, showing that MobA was immobilized in a functional form, partly impairing the binding site for MoeA.TABLE 1Analysis of protein-protein interactions between MobA, MoeA, MogA, and XdhC by SPR measurementsImmobilized proteinaProteins were immobilized via amine coupling (see “Experimental Procedures”)RUbRU, resonance units; ND, none detectableProtein partnercProteins were injected using the KINJECT protocol, injecting samples in a concentration range of 0.1–6.4 μm. Cells were regenerated by injection of 20 mm HClKDdKD values were obtained by global fitting procedures for a 1:1 bindingχ2μmXdhC570MoeA1.450.374MoeA9260XdhC1.151.83XdhC570MogANDMogA2010XdhCNDXdhC570MobA1.750.283MobA1130XdhC1.041.06XDH2810MoeANDMoeA2600MobA3.50.339MobA2025MoeA270.281MoeA9260MogA6.00.168MogA2180MoeA6.20.247a Proteins were immobilized via amine coupling (see “Experimental Procedures”)b RU, resonance units; ND, none detectablec Proteins were injected using the KINJECT protocol, injecting samples in a concentration range of 0.1–6.4 μm. Cells were regenerated by injection of 20 mm HCld KD values were obtained by global fitting procedures for a 1:1 binding Open table in a new tab Control experiments showed that MoeA did not interact with immobilized XDH, supporting the idea that Moco transfer to XDH is mediated by XdhC, rather than a direct transfer of Moco from MoeA to XDH. Additional control experiments were performed to exclude a possible influence of the N-terminal His6 tags fused to MoeA, MogA, and MobA on the dissociation constants. For this purpose, the His6 tags of MogA, MoeA, and MobA were cleaved by thrombin treatment, and the interaction of these proteins with immobilized XdhC was compared with the sensograms obtained for the His6-tagged proteins. Although untagged MogA also showed no interaction with XdhC (data not shown), comparison of the binding curves of His6-tagg" @default.
• W2079181465 created "2016-06-24" @default.
• W2079181465 creator A5027745690 @default.
• W2079181465 creator A5031263688 @default.
• W2079181465 creator A5049387695 @default.
• W2079181465 date "2007-09-01" @default.
• W2079181465 modified "2023-10-03" @default.
• W2079181465 title "Transfer of the Molybdenum Cofactor Synthesized by Rhodobacter capsulatus MoeA to XdhC and MobA" @default.
• W2079181465 cites W1488651464 @default.
• W2079181465 cites W1522815859 @default.
• W2079181465 cites W1631792726 @default.
• W2079181465 cites W1719008040 @default.
• W2079181465 cites W1801351858 @default.
• W2079181465 cites W1902544956 @default.
• W2079181465 cites W1967968024 @default.
• W2079181465 cites W1969540040 @default.
• W2079181465 cites W197993986 @default.
• W2079181465 cites W1981763864 @default.
• W2079181465 cites W1982117816 @default.
• W2079181465 cites W1987064733 @default.
• W2079181465 cites W1989809910 @default.
• W2079181465 cites W2002502265 @default.
• W2079181465 cites W2006879511 @default.
• W2079181465 cites W2018058209 @default.
• W2079181465 cites W2019965297 @default.
• W2079181465 cites W2024110365 @default.
• W2079181465 cites W2046609183 @default.
• W2079181465 cites W2047876516 @default.
• W2079181465 cites W2049720097 @default.
• W2079181465 cites W2059928250 @default.
• W2079181465 cites W2066667251 @default.
• W2079181465 cites W2073020904 @default.
• W2079181465 cites W2084250862 @default.
• W2079181465 cites W2086663615 @default.
• W2079181465 cites W2092584046 @default.
• W2079181465 cites W2101943125 @default.
• W2079181465 cites W2104594638 @default.
• W2079181465 cites W2106031973 @default.
• W2079181465 cites W2106671813 @default.
• W2079181465 cites W2107846233 @default.
• W2079181465 cites W2116446688 @default.
• W2079181465 cites W2117998384 @default.
• W2079181465 cites W2119073376 @default.
• W2079181465 cites W2145262563 @default.
• W2079181465 cites W2148900757 @default.
• W2079181465 cites W2156981649 @default.
• W2079181465 cites W2336018257 @default.
• W2079181465 cites W4245399940 @default.
• W2079181465 cites W4251817171 @default.
• W2079181465 cites W4293247451 @default.
• W2079181465 doi "https://doi.org/10.1074/jbc.m704020200" @default.
• W2079181465 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17686778" @default.
• W2079181465 hasPublicationYear "2007" @default.
• W2079181465 type Work @default.
• W2079181465 sameAs 2079181465 @default.
• W2079181465 citedByCount "34" @default.
• W2079181465 countsByYear W20791814652013 @default.
• W2079181465 countsByYear W20791814652014 @default.
• W2079181465 countsByYear W20791814652015 @default.
• W2079181465 countsByYear W20791814652016 @default.
• W2079181465 countsByYear W20791814652017 @default.
• W2079181465 countsByYear W20791814652018 @default.
• W2079181465 countsByYear W20791814652020 @default.
• W2079181465 countsByYear W20791814652021 @default.
• W2079181465 countsByYear W20791814652022 @default.
• W2079181465 crossrefType "journal-article" @default.
• W2079181465 hasAuthorship W2079181465A5027745690 @default.
• W2079181465 hasAuthorship W2079181465A5031263688 @default.
• W2079181465 hasAuthorship W2079181465A5049387695 @default.
• W2079181465 hasBestOaLocation W20791814651 @default.
• W2079181465 hasConcept C104317684 @default.
• W2079181465 hasConcept C143065580 @default.
• W2079181465 hasConcept C179104552 @default.
• W2079181465 hasConcept C181199279 @default.
• W2079181465 hasConcept C185592680 @default.
• W2079181465 hasConcept C197957613 @default.
• W2079181465 hasConcept C2777850835 @default.
• W2079181465 hasConcept C549387045 @default.
• W2079181465 hasConcept C55493867 @default.
• W2079181465 hasConceptScore W2079181465C104317684 @default.
• W2079181465 hasConceptScore W2079181465C143065580 @default.
• W2079181465 hasConceptScore W2079181465C179104552 @default.
• W2079181465 hasConceptScore W2079181465C181199279 @default.
• W2079181465 hasConceptScore W2079181465C185592680 @default.
• W2079181465 hasConceptScore W2079181465C197957613 @default.
• W2079181465 hasConceptScore W2079181465C2777850835 @default.
• W2079181465 hasConceptScore W2079181465C549387045 @default.
• W2079181465 hasConceptScore W2079181465C55493867 @default.
• W2079181465 hasIssue "39" @default.
• W2079181465 hasLocation W20791814651 @default.
• W2079181465 hasOpenAccess W2079181465 @default.
• W2079181465 hasPrimaryLocation W20791814651 @default.
• W2079181465 hasRelatedWork W2014092843 @default.
• W2079181465 hasRelatedWork W2040107302 @default.
• W2079181465 hasRelatedWork W2053593675 @default.
• W2079181465 hasRelatedWork W2348692476 @default.
• W2079181465 hasRelatedWork W2366692166 @default.
• W2079181465 hasRelatedWork W2950968619 @default.

• next