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• W2116735551 abstract "Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL2 or ChlL2 of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB)2 or (ChlNB)2. Analogously, subunit BchX2 of the COR enzymes delivers electrons to subunit (BchYZ)2. Various chimeric DPOR enzymes formed between recombinant subunits (BchNB)2 and BchL2 from Chlorobaculum tepidum or (ChlNB)2 and ChlL2 from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL2 with both subunits, ChlN and ChlB, of the (ChlNB)2 tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr127 of subunit ChlL, Leu70 and Val107 of subunit ChlN, and Gly66 of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL2 or ChlL2 part of DPOR was exchanged with electron-transferring BchX2 subunits of COR and NifH2 of nitrogenase. Active chimeric DPOR was generated via a combination of BchX2 from C. tepidum or Roseobacter denitrificans with (BchNB)2 from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH2 from Azotobacter vinelandii in combination with (BchNB)2 from C. tepidum or (ChlNB)2 from P. marinus and T. elongatus, respectively. Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL2 or ChlL2 of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB)2 or (ChlNB)2. Analogously, subunit BchX2 of the COR enzymes delivers electrons to subunit (BchYZ)2. Various chimeric DPOR enzymes formed between recombinant subunits (BchNB)2 and BchL2 from Chlorobaculum tepidum or (ChlNB)2 and ChlL2 from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL2 with both subunits, ChlN and ChlB, of the (ChlNB)2 tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr127 of subunit ChlL, Leu70 and Val107 of subunit ChlN, and Gly66 of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL2 or ChlL2 part of DPOR was exchanged with electron-transferring BchX2 subunits of COR and NifH2 of nitrogenase. Active chimeric DPOR was generated via a combination of BchX2 from C. tepidum or Roseobacter denitrificans with (BchNB)2 from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH2 from Azotobacter vinelandii in combination with (BchNB)2 from C. tepidum or (ChlNB)2 from P. marinus and T. elongatus, respectively. Chlorophyll and bacteriochlorophyll biosynthesis, as well as nitrogen fixation, are essential biochemical processes developed early in the evolution of life (1.Burke D.H. Hearst J.E. Sidow A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7134-7138Crossref PubMed Scopus (129) Google Scholar). During biological fixation of nitrogen, nitrogenase catalyzes the reduction of atmospheric dinitrogen to ammonia (2.Rees D.C. Tezcan F.A. Haynes C.A. Walton M.Y. Andrade S. Einsle O. Howard J.B. Philos. Transact. A Math. Phys. Eng. Sci. 2005; 363: 971-984-1035-1040Google Scholar). Enzyme systems homologous to nitrogenase play a crucial role in the formation of the chlorin and bacteriochlorin ring system of chlorophylls (Chl) 2The abbreviations used are:ChlchlorophyllAtftetrafluorophenyl azideBchlbacteriochlorophyllBchlidebacteriochlorophyllideChlidechlorophyllideCORchlorophyllide oxidoreductaseDPORdark-operative protochlorophyllide oxidoreductaseDTTdithiothreitolGSTglutathione S-transferaseMtsmethanethiosulfonateMts-Atf-LC-biotin2-{N2-[N6-(4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl)-N6-(6- biotinamidocaproyl)-l-lysin-ylamido]}ethyl methanethiosulfonatePchlideprotochlorophyllide. and bacteriochlorophylls (Bchl) (3.Bröcker M.J. Virus S. Ganskow S. Heathcote P. Heinz D.W. Schubert W.D. Jahn D. Moser J. J. Biol. Chem. 2008; 283: 10559-10567Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 4.Burke D.H. Alberti M. Hearst J.E. J. Bacteriol. 1993; 175: 2407-2413Crossref PubMed Google Scholar) (Fig. 1a). For the synthesis of both Chl and Bchl, the stereospecific reduction of the C-17-C-18 double bond of ring D of protochlorophyllide (Pchlide) catalyzed by the nitrogenase-like enzyme light-independent (dark-operative) protochlorophyllide oxidoreductase (DPOR) results in the formation of chlorophyllide (Chlide) (Fig. 1a, left) (5.Bröcker M.J. Wätzlich D. Uliczka F. Virus S. Saggu M. Lendzian F. Scheer H. Rüdiger W. Moser J. Jahn D. J. Biol. Chem. 2008; 283: 29873-29881Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 6.Fujita Y. Matsumoto H. Takahashi Y. Matsubara H. Plant Cell Physiol. 1993; 34: 305-314PubMed Google Scholar). DPOR enzymes consist of three protein subunits which are designated BchN, BchB and BchL in Bchl-synthesizing organisms and ChlN, ChlB and ChlL in Chl-synthesizing organisms. A second reduction step at ring B (C-7-C-8) unique to the synthesis of Bchl converts the chlorin Chlide into a bacteriochlorin ring structure to form bacteriochlorophyllide (Bchlide) (Fig. 1a, right, Bchlide). This reaction is catalyzed by another nitrogenase-like enzyme, termed chlorophyllide oxidoreductase (COR) (7.Nomata J. Mizoguchi T. Tamiaki H. Fujita Y. J. Biol. Chem. 2006; 281: 15021-15028Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). COR enzymes are composed of subunits BchY, BchZ, and BchX. chlorophyll tetrafluorophenyl azide bacteriochlorophyll bacteriochlorophyllide chlorophyllide chlorophyllide oxidoreductase dark-operative protochlorophyllide oxidoreductase dithiothreitol glutathione S-transferase methanethiosulfonate 2-{N2-[N6-(4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl)-N6-(6- biotinamidocaproyl)-l-lysin-ylamido]}ethyl methanethiosulfonate protochlorophyllide. All subunits share significant amino acid sequence homology to the corresponding subunits of nitrogenase, which are designated NifD, NifK, and NifH, respectively (1.Burke D.H. Hearst J.E. Sidow A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7134-7138Crossref PubMed Scopus (129) Google Scholar) (compare Fig. 1, a and b). Whereas subunits BchL or ChlL, BchX and NifH exhibit a sequence identity at the amino acid level of ∼33%, subunits BchN or ChlN, BchY, NifD, and BchB or ChlB, BchZ, and NifK, respectively, show lower sequence identities of ∼15% (1.Burke D.H. Hearst J.E. Sidow A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7134-7138Crossref PubMed Scopus (129) Google Scholar). For all enzymes a common oligomeric protein architecture has been proposed consisting of the heterotetrameric complexes (BchNB)2 or (ChlNB)2, (BchYZ)2, and (NifD/NifK)2, which are completed by a homodimeric protein subunit BchL2 or ChlL2, BchX2, and NifH2, respectively (compare Fig. 1, a and b) (3.Bröcker M.J. Virus S. Ganskow S. Heathcote P. Heinz D.W. Schubert W.D. Jahn D. Moser J. J. Biol. Chem. 2008; 283: 10559-10567Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 7.Nomata J. Mizoguchi T. Tamiaki H. Fujita Y. J. Biol. Chem. 2006; 281: 15021-15028Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 8.Tezcan F.A. Kaiser J.T. Mustafi D. Walton M.Y. Howard J.B. Rees D.C. Science. 2005; 309: 1377-1380Crossref PubMed Scopus (175) Google Scholar). Nitrogenase is a well characterized protein complex that catalyzes the reduction of nitrogen to ammonia in a reaction that requires at least 16 molecules of MgATP (2.Rees D.C. Tezcan F.A. Haynes C.A. Walton M.Y. Andrade S. Einsle O. Howard J.B. Philos. Transact. A Math. Phys. Eng. Sci. 2005; 363: 971-984-1035-1040Google Scholar, 9.Peters J.W. Fisher K. Dean D.R. Annu. Rev. Microbiol. 1995; 49: 335-366Crossref PubMed Scopus (140) Google Scholar, 10.Dean D.R. Bolin J.T. Zheng L. J. Bacteriol. 1993; 175: 6737-6744Crossref PubMed Google Scholar). During nitrogenase catalysis, subunit NifH2 (Fe protein) associates with and dissociates from the (NifD/NifK)2 complex (MoFe protein). Binding, hydrolysis of MgATP and structural rearrangements are coupled to sequential intersubunit electron transfer. For this purpose, NifH2 contains an ATP-binding motif and an intersubunit [4Fe-4S] cluster coordinated by two cysteine residues from each NifH monomer (1.Burke D.H. Hearst J.E. Sidow A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7134-7138Crossref PubMed Scopus (129) Google Scholar, 11.Suzuki J.Y. Bollivar D.W. Bauer C.E. Annu. Rev. Genet. 1997; 31: 61-89Crossref PubMed Scopus (141) Google Scholar). Electrons from this [4Fe-4S] cluster are transferred via a [8Fe-7S] cluster (P-cluster) onto the [1Mo-7Fe-9S-X-homocitrate] cluster (MoFe cofactor). Both of the latter clusters are located on (NifD/NifK)2, where dinitrogen is reduced to ammonia (10.Dean D.R. Bolin J.T. Zheng L. J. Bacteriol. 1993; 175: 6737-6744Crossref PubMed Google Scholar). Three-dimensional structures of NifH2 in complex with (NifD/NifK)2 revealed a detailed picture of the dynamic interaction of both subcomplexes (8.Tezcan F.A. Kaiser J.T. Mustafi D. Walton M.Y. Howard J.B. Rees D.C. Science. 2005; 309: 1377-1380Crossref PubMed Scopus (175) Google Scholar, 12.Schindelin H. Kisker C. Schlessman J.L. Howard J.B. Rees D.C. Nature. 1997; 387: 370-376Crossref PubMed Scopus (426) Google Scholar). Based on biochemical and bioinformatic approaches, it has been proposed that the initial steps of DPOR reaction strongly resemble nitrogenase catalysis. Key amino acid residues essential for DPOR function have been identified by mutagenesis of the enzyme from Chlorobaculum tepidum (formerly denoted as Chlorobium tepidum) (3.Bröcker M.J. Virus S. Ganskow S. Heathcote P. Heinz D.W. Schubert W.D. Jahn D. Moser J. J. Biol. Chem. 2008; 283: 10559-10567Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The catalytic mechanism of DPOR includes the electron transfer from a “plant-type” [2Fe-2S] ferredoxin onto the dimeric DPOR subunit, BchL2, carrying an intersubunit [4Fe-4S] redox center coordinated by Cys97 and Cys131 in C. tepidum. Analogous to nitrogenase, Lys10 in the phosphate-binding loop (P-loop) and Leu126 in the switch II region of DPOR were found essential for DPOR catalysis. Moreover, it was shown that the BchL2 protein from C. tepidum does not form a stable complex with the catalytic (BchNB)2 subcomplex. Therefore, a transient interaction responsible for the electron transfer onto protein subunit (BchNB)2 has been proposed (3.Bröcker M.J. Virus S. Ganskow S. Heathcote P. Heinz D.W. Schubert W.D. Jahn D. Moser J. J. Biol. Chem. 2008; 283: 10559-10567Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The subsequent [Fe-S] cluster-dependent catalysis and the specific substrate recognition at the active site located on subunit (BchNB)2 are unrelated to nitrogenase. The (BchNB)2 subcomplex was shown to carry a second [4Fe-4S] cluster, which was proposed to be ligated by Cys21, Cys46, and Cys103 of the BchN subunit and Cys94 of subunit BchB (C. tepidum numbering) (3.Bröcker M.J. Virus S. Ganskow S. Heathcote P. Heinz D.W. Schubert W.D. Jahn D. Moser J. J. Biol. Chem. 2008; 283: 10559-10567Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). No evidence for any type of additional cofactor was obtained from biochemical and EPR spectroscopic analyses (5.Bröcker M.J. Wätzlich D. Uliczka F. Virus S. Saggu M. Lendzian F. Scheer H. Rüdiger W. Moser J. Jahn D. J. Biol. Chem. 2008; 283: 29873-29881Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 13.Nomata J. Ogawa T. Kitashima M. Inoue K. Fujita Y. FEBS Lett. 2008; 582: 1346-1350Crossref PubMed Scopus (39) Google Scholar). Thus, despite the same common oligomeric architecture, the catalytic subunits (BchNB)2 and (ChlNB)2 clearly differ from the corresponding nitrogenase complex, as no molybdenum-containing cofactor or P-cluster equivalent is employed (5.Bröcker M.J. Wätzlich D. Uliczka F. Virus S. Saggu M. Lendzian F. Scheer H. Rüdiger W. Moser J. Jahn D. J. Biol. Chem. 2008; 283: 29873-29881Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 14.Sarma R. Barney B.M. Hamilton T.L. Jones A. Seefeldt L.C. Peters J.W. Biochemistry. 2008; 47: 13004-13015Crossref PubMed Scopus (57) Google Scholar). From these results it was concluded that electrons from the [4Fe-4S] cluster of (BchNB)2 or (ChlNB)2 are transferred directly onto the Pchlide substrate at the active site of DPOR. The second nitrogenase-like enzyme, COR, catalyzes the reduction of ring B of Chlide during the biosynthesis of Bchl (7.Nomata J. Mizoguchi T. Tamiaki H. Fujita Y. J. Biol. Chem. 2006; 281: 15021-15028Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Therefore, an accurate discrimination of the ring systems of the individual substrates is required. COR subunits share an overall amino acid sequence identity of 15–22% for BchY and BchZ and 31–35% for subunit BchX when compared with the corresponding DPOR subunits (Table 1 and supplemental Figures S2–S4). In amino acid sequence alignments of BchX proteins with the closely related BchL or ChlL subunits of DPOR, both cysteinyl ligands responsible for [4Fe-4S] cluster formation and residues for ATP binding are conserved (1.Burke D.H. Hearst J.E. Sidow A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7134-7138Crossref PubMed Scopus (129) Google Scholar). Furthermore, all cysteinyl residues characterized as ligands for a catalytic [4Fe-4S] cluster in (BchNB)2 or (ChlNB)2 are conserved in the sequences of subunits BchY and BchZ of COR (7.Nomata J. Mizoguchi T. Tamiaki H. Fujita Y. J. Biol. Chem. 2006; 281: 15021-15028Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). These findings correspond to a recent EPR study in which a characteristic signal for a [4Fe-4S] cluster was obtained for the COR subunit BchX2 as well as for subunit (BchYZ)2 (15.Kim E.J. Kim J.S. Lee I.H. Rhee H.J. Lee J.K. J. Biol. Chem. 2008; 283: 3718-3730Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). These results indicate that the catalytic mechanism of COR strongly resembles DPOR catalysis. In vitro assays for nitrogenase as well as for DPOR and COR make use of the artificial electron donor dithionite in the presence of high concentrations of ATP (7.Nomata J. Mizoguchi T. Tamiaki H. Fujita Y. J. Biol. Chem. 2006; 281: 15021-15028Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 16.Wherland S. Burgess B.K. Stiefel E.I. Newton W.E. Biochemistry. 1981; 20: 5132-5140Crossref PubMed Scopus (55) Google Scholar, 17.Fujita Y. Bauer C.E. J. Biol. Chem. 2000; 275: 23583-23588Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar).TABLE 1Amino acid sequence identities of the individual subunits of DPOR, COR, and nitrogenaseDPORCORNitrogenaseNBLYZXNifDNifKNifHDPORN37–5815–1812–20B34–6215–2214–18L51–6931–3531–38CORY35–7813–15Z39–8111–16X42–8329–36NitrogenaseNifD17–70NifK37–58NifH67–75 Open table in a new tab In this study, we investigated the transient interaction of the dimeric subunit BchL2 or ChlL2 with the heterotetrameric (BchNB)2 or (ChlNB)2 complex, which is essential for DPOR catalysis. We make use of the individually purified DPOR subunits BchL2 and (BchNB)2 from the green sulfur bacterium C. tepidum and ChlL2 and (ChlNB)2 from the prochlorophyte Prochlorococcus marinus and from the cyanobacterium Thermosynechococcus elongatus. The individual combination of (BchNB)2 or (ChlNB)2 complexes and BchL2 or ChlL2 proteins from these organisms resulted in catalytically active chimeras of DPOR. These results enabled us to propose conserved regions of the postulated docking surface, which were subsequently verified in a mutagenesis study. To elucidate the potential evolution of the electron-transferring subunit of nitrogenase and nitrogenase-like enzymes, we also analyzed chimeric enzymes consisting of DPOR subunits (BchNB)2 or (ChlNB)2 in combination with subunits BchX2 from C. tepidum and R. denitrificans of the COR enzyme and with subunit NifH2 of nitrogenase from Azotobacter vinelandii, respectively. Genes chlN and chlB from T. elongatus BP-1 were amplified from genomic DNA by PCR using the primers TechlN_for and TechlN_rev for chlN and TechlB_for and TechlB_rev for chlB (primer sequences are given in supplemental Table S1). The Escherichia coli-specific ribosomal binding site was implemented upstream of chlB. PCR fragments for chlN were digested with BamHI/XhoI and for chlB with XhoI/NotI, respectively, and cloned into the BamHI-NotI site of pGEX-6P-1 (GE Healthcare) to yield pGEX-TeNB. The chlL gene from T. elongatus was amplified using primers TechlL_for and TechlL_rev and cloned into the BamHI-XhoI sites of pGEX-6P-1 to generate pGEX-TeL. The construction of plasmids pGEX-PmNB carrying the genes chlN and chlB from P. marinus, pGEX-PmL with the gene chlL from P. marinus, pGEX-CtNBL encoding the genes bchN, bchB, and bchL from C. tepidum, and pGEX-CtL carrying the gene bchL from C. tepidum was described previously (3.Bröcker M.J. Virus S. Ganskow S. Heathcote P. Heinz D.W. Schubert W.D. Jahn D. Moser J. J. Biol. Chem. 2008; 283: 10559-10567Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 5.Bröcker M.J. Wätzlich D. Uliczka F. Virus S. Saggu M. Lendzian F. Scheer H. Rüdiger W. Moser J. Jahn D. J. Biol. Chem. 2008; 283: 29873-29881Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). For the amplification of bchX encoding subunit BchX of COR from C. tepidum TLS primers CtbchX_for and CtbchX_rev were used. For bchX from R. denitrificans OCh 114 primers RdbchX_for and RdbchX_rev were employed. The resulting PCR fragments were digested with BamHI/SalI for bchX from C. tepidum and SacI/XhoI for bchX from R. denitrificans and cloned into the BamHI-SalI and SacI-XhoI sites of pET32a (Novagen, Darmstadt, Germany), respectively. The constructs thus obtained were designated pET-CtX and pET-RdX. For the exchange of conserved amino acids in P. marinus DPOR subunits ChlN, ChlB, and ChlL, the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used. Amino acid substitutions in ChlN were introduced using primers E66A, L70R, L70D, V107D, and K109S. Oligonucleotides for the exchange of codons in ChlB were G66D, Q101G, and Q101D. For substitutions in ChlL the following primers were used: Y127S and Y127D. Glutathione S-transferase (GST)-tagged (BchNB)2 or (ChlNB)2 and BchL2 or ChlL2 were overproduced in E. coli BL21-CodonPlus® (DE3)-RIL cells (Stratagene). The following plasmids encoding mentioned subunits were employed: pGEX-CtNB for C. tepidum (BchNB)2, pGEX-CtL for C. tepidum BchL2, pGEX-TeNB for T. elongatus (ChlNB)2, pGEX-TeL for T. elongatus ChlL2, pGEX-PmNB for P. marinus (ChlNB)2, and pGEX-PmL for P. marinus ChlL2. Cells were grown aerobically at 17 °C for the production of the C. tepidum and T. elongatus proteins or at 25 °C for the P. marinus proteins in LB medium containing 1 mm Fe(III) citrate. Protein production was induced with 50 μm isopropyl-1-thio-β-d-galactopyranoside. After cultivation for 16 h, cultures were supplemented with 1.7 mm dithionite and incubated for a further 2 h in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) without agitation. All of the following procedures were performed under anaerobic conditions (95% N2, 5% H2, <1 ppm O2), and buffers were saturated with N2 prior to use. Cells were harvested by centrifugation, suspended in buffer A (100 mm HEPES/NaOH, pH 7.5, 150 mm NaCl, 10 mm MgCl2, 10 mm DTT), and disrupted by a single passage through a French Press® (Thermo Fisher Scientific, Waltham, MA) at 1000 p.s.i. into an anaerobic bottle. After ultracentrifugation for 1 h at 110,000 × g at 4 °C, the resulting supernatant was loaded onto glutathione-Sepharose 4B (GE Healthcare) equilibrated with buffer A. Recombinant fusion protein GST-BchN or GST-ChlN in complex with subunit BchB or ChlB or, alternatively, fusion protein GST-BchL or GST-ChlL alone, was eluted with buffer A containing 15 mm glutathione. GST was cleaved off the target proteins by incubation with 2 units of PreScission Protease (GE Healthcare) for 16 h. For subsequent label transfer experiments, affinity-purified proteins (ChlNB)2 and ChlL2 from P. marinus were further purified by gel permeation chromatography under anaerobic conditions. Therefore, protein samples (∼1.5 ml) at a concentration of 5 mg/ml were subjected to a preparative gel permeation chromatography on a Superdex 200 26/60 gel filtration column (GE Healthcare) equilibrated with 100 mm HEPES/NaOH, pH 7.5, 150 mm NaCl, 10 mm MgCl2 (buffer B) at a flow rate of 1.5 ml/min. Elution fractions were pooled and concentrated by using a Microcon® centrifugal filter unit (Millipore, Schwalbach, Germany; molecular weight cutoff 10,000). His-tagged COR BchX2 subunits were produced in E. coli BL21 (DE3)-RIL (Stratagene) carrying plasmids pET-CtX encoding C. tepidum BchX2 and pET-RdX encoding R. denitrificans BchX2. Cells were grown aerobically at 17 °C in LB medium containing 1 mm Fe(III)-citrate. Recombinant protein production was induced by addition of 50 μm isopropyl-1-thio-β-d-galactopyranoside. After further cultivation for 16 h, cultures were incubated for 2 h in an anaerobic chamber. Cells were harvested by centrifugation, suspended in buffer B, and disrupted by a single passage through a French press (SLM-Aminco) at 1000 p.s.i. under anaerobic conditions. After ultracentrifugation for 1 h at 110,000 × g at 4 °C, the resulting supernatant was applied onto a nickel-loaded chelating Sepharose FF (GE Healthcare) equilibrated with buffer B. After extensive washing with buffer C (100 mm HEPES/NaOH, pH 7.5, 500 mm NaCl, 10 mm MgCl2), proteins were eluted with buffer C containing 200 mm imidazole. The His tag was removed from the target protein by incubation with 2 units of thrombin (Novagen) for 16 h. Analytical gel permeation chromatography was performed using a Superdex 200 HR 26/60 column (GE Healthcare) under anaerobic conditions. After calibration of the column with marker proteins (molecular weight marker kit, Sigma-Aldrich) at a flow rate of 1 ml/min, 250-μl samples (∼3–5 mg/ml) of T. elongatus (ChlNB)2 and C. tepidum BchX2 were run under identical conditions. Protein elution was monitored by measuring the absorbance at 280 nm. The concentration of purified (BchNB)2 or (ChlNB)2 proteins was determined using the Bradford reagent (Sigma-Aldrich) according to the manufacturer's instructions with bovine serum albumin as a standard. For the quantification of GST fusion proteins with subunits BchL2 or ChlL2 in cell-free extracts of E. coli, a GST detection module (GE Healthcare) was used. Alternatively, protein concentration was determined densitometrically using ImageJ software (National Institutes of Health, Bethesda, MD). UV-visible light spectra of recombinantly purified (BchNB)2 or (ChlNB)2 and BchX2 proteins were recorded under anaerobic conditions using a V-550 spectrophotometer (Jasco, Gross-Umstadt, Germany). The iron content of purified (ChlNB)2 and BchX2 proteins was measured colorimetrically with bathophenantroline following acid denaturation as described previously (18.Lovenberg W. Buchanan B.B. Rabinowitz J.C. J. Biol. Chem. 1963; 238: 3899-3913Abstract Full Text PDF PubMed Google Scholar). Purified ChlL2 protein from P. marinus after preparative gel permeation chromatography was labeled with the trifunctional label transfer reagent Mts-Atf-LC-biotin (Pierce). In addition to a biotin moiety, this reagent contains a sulfhydryl-specific methane thiosulfonate (Mts) group, which can form disulfide bonds with free sulfhydryl groups of the ChlL2 protein. After exposure to UV light, the photoreactive tetrafluorophenyl azide (Atf) moiety of the cross-linker inserts into carbon hydrogen bonds and unsaturated carbon chains of the protein within a distance of 21.8 Å. Upon the addition of reducing agents, the disulfide bond is cleaved and the biotin label is transferred to the interacting protein partner. All procedures for the labeling experiments were performed with low levels of reductant (<1 mm DTT) and under subdued light to prevent premature loss of the label and activation of the Atf moiety. Purified ChlL2 from P. marinus was mixed with a 5-fold molar excess of Mts-Atf-LC-biotin in a total reaction volume of 500 μl in phosphate-buffered saline (100 mm sodium phosphate, 150 mm NaCl, pH 7.4). After incubation for 1 h at 21 °C, the unbound reagent was removed by dialysis using Slide-A-Lyzer® MINI dialysis units (Pierce). Label transfer experiments contained 25 μm (ChlNB)2 from P. marinus and the corresponding biotinylated ChlL2 protein (ChlL2-Mts-Atf-LC-biotin; 70 μm) in a volume of 100 μl of phosphate-buffered saline. Assays also contained 2 mm ATP, an ATP-regenerating system consisting of 20 mm creatine phosphate and 10 units of creatine phosphokinase, and the DPOR substrate Pchlide at a concentration of 13 μm. Complex formation was initiated for 5 min at 21 °C. Then the cross-linking reaction was induced by exposure to UV light using a CAMAG UV lamp (365 nm; 2 × 8 watts) for 30 min at a distance of 5 cm. 100 μl of SDS sample buffer (19.Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar) containing the reducing agent DTT at a concentration of 100 mm was added. Thereby, the ChlL2 protein was liberated from the cross-linked complex, resulting in the transfer of the biotin label onto the (ChlNB)2 protein. In control experiments the (ChlNB)2 complex or the ChlL2 protein was omitted. Furthermore, negative controls were performed by substituting (ChlNB)2 with bovine serum albumin. All samples were subsequently analyzed via 12% SDS-PAGE. Proteins were blotted onto a polyvinylidene difluoride membrane using a Trans-Blot apparatus semidry transfer cell (Bio-Rad) according to manufacturer's instructions. Biotin-labeled proteins were detected by using a NeutrAvidin antibody conjugated with horseradish peroxidase (Pierce). Functional DPOR enzymes from C. tepidum, P. marinus, and T. elongatus were reconstituted by supplementing 100 pmol of purified (BchNB)2 or (ChlNB)2 with 200 pmol of purified BchL2 or ChlL2, respectively, in a total volume of 125 μl in 100 mm HEPES/NaOH, pH 7.5, 150 mm NaCl, and 10 mm MgCl2. The assays contained 2 mm ATP, an ATP-regenerating system consisting of 20 mm creatine phosphate and 10 units of creatine phosphokinase, 10 mm DTT, 10 mm sodium dithionite as an electron donor, and the substrate Pchlide at 13 μm. Pchlide was isolated from the bchL-deficient Rhodobacter capsulatus mutant strain ZY5 as described previously (3.Bröcker M.J. Virus S. Ganskow S. Heathcote P. Heinz D.W. Schubert W.D. Jahn D. Moser J. J. Biol. Chem. 2008; 283: 10559-10567Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 17.Fujita Y. Bauer C.E. J. Biol. Chem. 2000; 275: 23583-23588Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Samples were incubated under strict anaerobic conditions for 15 to 60 min in the dark at varying temperatures ranging from 20 to 50 °C. Reactions were stopped by the addition of 500 μl of acetone. After centrifugation for 30 min at 12,000 × g, the supernatant was analyzed by UV-visible spectroscopy using a V-550 spectrophotometer (Jasco). The DPOR reaction product, Chlide, shows a characteristic peak at 665 nm, whereas the substrate possesses an absorption maximum at 626 nm. For the quantification of Pchlide and Chlide, extinction coefficients of ϵ626 = 30.4 mm−1 cm−1 for Pchlide and ϵ665 = 74.9 mm−1 cm−1 for Chlide were used (17.Fujita Y. Bauer C.E. J. Biol. Chem. 2000; 275: 23583-23588Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 20.McFeeters R.F. Chichester C.O. Whitaker J.R. Plant Physiol. 1971; 47: 609-618Crossref PubMed Google Scholar). The detection limit of the assay is at 15.75 nmol min−1 mg−1. All subsequent assays for the various DPOR systems were standardized at 35 °C allowing for the determination of specific activities under identical conditions. For this standard DPOR assay, 100 pmol of purified (BchNB)2 or (ChlNB)2 was combined with 1–80 μl of a cell-free E. coli extract containing 0.05–300 pmol of the overproduced BchL2 or ChlL2 protein, respectively. The specific activities obtained were set as 100%, and all other values (values of chimeric DPOR enzymes and mutant DPOR enzymes) were related to this. In all cases, assays were completed by control experiments in which the BchL2 or ChlL2 protein, the (BchNB)2 or (ChlNB)2 complex, or alternatively the co-substrate ATP was omitted. The DPOR subcomplex (BchNB)2 from C. tepidum or (ChlNB)2 from P. marinus and T. elongatus was combined with protein subunit BchX2 of the COR enzyme from C. tepidu" @default.
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• W2116735551 title "Chimeric Nitrogenase-like Enzymes of (Bacterio)chlorophyll Biosynthesis" @default.
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