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• W2022277083 abstract "Magnesium chelatase inserts Mg2+ into protoporphyrin IX in the chlorophyll and bacteriochlorophyll biosynthetic pathways. In photosynthetic bacteria, the products of three genes, bchI, bchD, andbchH, are required for magnesium chelatase activity. These genes from Rhodobacter capsulatus were cloned separately into expression plasmids pET3a and pET15b. The pET15b constructs produced NH2-terminally His6-tagged proteins. All proteins were highly expressed and were purified to near homogeneity. The BchI and BchH proteins were soluble. BchD proteins were insoluble, inactive inclusion bodies that were renatured by rapid dilution from 6 m urea. The presence of BchI in the solution into which the urea solution of BchD was diluted increased the yield of active BchD. A molar ratio of 1 BchI:1 BchD was sufficient for maximum renaturation of BchD. All of the proteins were active in the magnesium chelatase assay except His-tagged BchI, which was inactive and inhibited in incubations containing non-His-tagged BchI. Expressed BchH proteins contained tightly bound protoporphyrin IX, and they were susceptible to inactivation by light. Maximum magnesium chelatase activity per mol of BchD occurred at a stoichiometry of 4 BchI:1 BchD. The optimum reaction pH was 8.0. The reaction exhibited Michaelis-Menten kinetics with respect to protoporphyrin IX and BchH. Magnesium chelatase inserts Mg2+ into protoporphyrin IX in the chlorophyll and bacteriochlorophyll biosynthetic pathways. In photosynthetic bacteria, the products of three genes, bchI, bchD, andbchH, are required for magnesium chelatase activity. These genes from Rhodobacter capsulatus were cloned separately into expression plasmids pET3a and pET15b. The pET15b constructs produced NH2-terminally His6-tagged proteins. All proteins were highly expressed and were purified to near homogeneity. The BchI and BchH proteins were soluble. BchD proteins were insoluble, inactive inclusion bodies that were renatured by rapid dilution from 6 m urea. The presence of BchI in the solution into which the urea solution of BchD was diluted increased the yield of active BchD. A molar ratio of 1 BchI:1 BchD was sufficient for maximum renaturation of BchD. All of the proteins were active in the magnesium chelatase assay except His-tagged BchI, which was inactive and inhibited in incubations containing non-His-tagged BchI. Expressed BchH proteins contained tightly bound protoporphyrin IX, and they were susceptible to inactivation by light. Maximum magnesium chelatase activity per mol of BchD occurred at a stoichiometry of 4 BchI:1 BchD. The optimum reaction pH was 8.0. The reaction exhibited Michaelis-Menten kinetics with respect to protoporphyrin IX and BchH. polymerase chain reaction dithiothreitol N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Magnesium chelatase inserts Mg2+ into protoporphyrin IX in the chlorophyll and bacteriochlorophyll biosynthetic pathways. This enzyme is at the branch point of the (bacterio)chlorophyll and heme biosynthetic pathways and is believed to have a regulatory role in directing biosynthetic intermediates into the (bacterio)chlorophyll pathway (1Walker C.J. Willows R.D. Biochem. J. 1997; 327: 321-333Crossref PubMed Scopus (176) Google Scholar). Transposon mutagenesis studies inRhodobacter capsulatus (2Bollivar D.W. Suzuki J.Y. Beatty J.T. Dobrowolski J.M. Bauer C.E. J. Mol. Biol. 1994; 237: 622-640Crossref PubMed Scopus (161) Google Scholar) and Rhodobacter sphaeroides (3Gorchein A. Gibson L.C.D. Hunter C.N. Biochem. Soc. Trans. 1993; 21: 201SCrossref PubMed Scopus (14) Google Scholar) have shown that the products of three genes,bchI, bchD, and bchH, are required for this insertion. Genes that are homologous to bchI,bchD, and bchH have been reported in plants and photosynthetic bacteria. It has been suggested that the genes be namedbchI, bchD, and bchH when referring to organisms that accumulate bacteriochlorophyll and chlI,chlD, and chlH for the respective homologous genes in organisms that accumulate chlorophyll (1Walker C.J. Willows R.D. Biochem. J. 1997; 327: 321-333Crossref PubMed Scopus (176) Google Scholar). This nomenclature will be used here. Expression of the three R. sphaeroides genes inEscherichia coli showed that all three gene products, plus ATP, Mg2+, and protoporphyrin IX, were required for magnesium chelatase activity (4Gibson L.C. Marrison J.L. Leech R.M. Jensen P.E. Bassham D.C. Gibson M. Hunter C.N. Plant Physiol. 1996; 111: 61-71Crossref PubMed Scopus (88) Google Scholar, 5Willows R.D. Gibson L.C.D. Kannangara C.G. Hunter C.N. von Wettstein D. Eur. J. Biochem. 1996; 235: 438-443Crossref PubMed Scopus (96) Google Scholar). An enzyme activation step involving the BchI and BchD subunits was observed (5Willows R.D. Gibson L.C.D. Kannangara C.G. Hunter C.N. von Wettstein D. Eur. J. Biochem. 1996; 235: 438-443Crossref PubMed Scopus (96) Google Scholar) that was similar to that reported for the pea magnesium chelatase (6Walker C.J. Weinstein J.D. Biochem. J. 1994; 299: 277-284Crossref PubMed Scopus (70) Google Scholar). However, very low expression of the BchD protein limited further characterization of magnesium chelatase (5Willows R.D. Gibson L.C.D. Kannangara C.G. Hunter C.N. von Wettstein D. Eur. J. Biochem. 1996; 235: 438-443Crossref PubMed Scopus (96) Google Scholar). Heterologous expression of individual subunits and reconstitution of magnesium chelatase activity has recently been described for tobacco (7Papenbrock J. Gräfe S. Kruse E. Hänel F. Grimm B. Plant J. 1997; 12: 981-990Crossref PubMed Scopus (76) Google Scholar), the cyanobacterium Synechocystissp. PCC 6803 (8Jensen P.E. Gibson L.C.D. Henningsen K.W. Hunter C.N. J. Biol. Chem. 1996; 271: 16662-16667Crossref PubMed Scopus (116) Google Scholar), and the green bacterium Chlorobium vibrioforme (9Petersen B.L. Jensen P.E. Gibson L.C.D. Stummann B.M. Hunter C.N. Henningsen K.W. J. Bacteriol. 1998; 180: 699-704Crossref PubMed Google Scholar) However, characterization of the reaction using recombinant proteins has been limited due to low levels of expression and poor recovery of activity from these systems. We now report the cloning and high level expression of the magnesium chelatase genes from R. capsulatus, the purification of the gene products, and characterization of the reconstituted enzyme. Portions of this work were previously published in abstract form (10Willows R.D. Beale S.I. Plant Physiol. 1997; 111: S155Google Scholar). The plasmid pRPS404 (11Marrs B. J. Bacteriol. 1981; 146: 1003-1012Crossref PubMed Google Scholar), which contains a 44-kilobase pair region of the R. capsulatus photosynthetic gene cluster, was used as a PCR1 template throughout. A modified T-vector was constructed between theEcoRI and BamHI sites of pBluescript (KS) (Stratagene, La Jolla, CA) essentially as described in Ref. 12Kwak J.-H. Kim M.-Y. Anal. Biochem. 1995; 228: 178-180Crossref PubMed Scopus (5) Google Scholar, and this vector was used to subclone the bchID andbchD genes as described below. The oligonucleotides 5′-GGATCATCTTGCGGAAACTGT-3′ and 5′-ATGACTACCGCCGTCGCTCGACTTCAACCCTCTGCT-3′ were used to amplify thebchID region of the photosynthetic gene cluster by PCR usingTaq DNA polymerase (Stratagene). The 3-kilobase pair PCR product was then cloned directly into the T-vector, yielding plasmid pKSBchID. This cloning created an NdeI restriction site at the bchI translation start site. A region containing thebchI gene and the 5′ portion of bchD was then subcloned between the NdeI and BamHI sites of either pET3a (Novagen, Madison, WI) to create pBchI or pET15b (Novagen) to create pHisBchI. The oligonucleotides 5′-ATGGACCACGAACGCCTGAAGTCGGCCCTTG-3′ and 5′-ACCCTGCGTCGCGCCGCCGCCGACCGATAGG-3′ were used to amplify thebchD gene by PCR using Pfu DNA polymerase (Stratagene). The 1.8-kilobase pair PCR product was cloned into the modified T-vector, yielding plasmid pKSBchD. This cloning created anNdeI restriction site at the bchD translation start site. This plasmid was digested with NdeI andXhoI, and the bchD fragment was subcloned between either the NdeI and BamHI sites of pET3a to create pBchD, or the NdeI and XhoI sites of pET15b to create pHisBchD. The oligonucleotides 5′-AGGCCCCATATGCACGATGAGTCGATGAGC-3′ and 5′-CCCTCCTTTTCGTAGTCGTAGATCTCATTC-3′ were used to amplifybchH by PCR using Pfu DNA polymerase. These oligonucleotides introduced NdeI and BglII restriction sites. The PCR product was then either digested and cloned directly into pET3a to create pBchH or into the pCRBlunt (Invitrogen, Carlsbad, CA) vector to create pCRBchH and then subcloned into pET15b to create pHisBchH. The magnesium chelatase genes in these plasmids were sequenced to check for errors using a Dye-Deoxy terminator kit (PE Applied Biosystems, Foster City, CA) with the Applied Biosystems model 377 sequencer. The first subclone of bchI, which was used to make the expression clones, was sequenced completely, and the finalbchI expression clones were sequenced in from the 5′-end to verify that they were in frame. The first bchD andbchH subclones were sequenced 600 base pairs in from each end, and the final bchD and bchH expression clones were sequenced in from the 5′-end to verify that they were in frame. No errors were detected. E. coli BL21 (DE3) pLysS (Novagen) strains containing the expression plasmids were grown at 25 or 37 °C in 1 liter of LB medium (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol until the A 600 of the cultures was 0.8. Protein expression was induced by the addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 1 mm. After 4 h, the cells were harvested by centrifugation. For the expressed pET3a constructs, the cells from 1 liter of culture were resuspended in 20 ml of 50 mmTricine-NaOH, pH 8.0, 15 mm MgCl2, and 4 mm DTT. The suspension was then frozen at –20 °C, thawed once, and then lysed completely by passage through a French pressure cell (SLM, Urbana, IL) at 20,000 p.s.i. For expression from pET15b constructs, the cells from 1 liter of culture were resuspended in 20 ml of 20 mm Tris-HCl, pH 7.9, 0.5 m NaCl, 15 mm MgCl2, and 5 mm imidazole. This suspension was frozen at –20 °C, thawed once, and then lysed by passage through a French pressure cell at 20,000 p.s.i. The BchH protein was purified essentially as described previously for the protein from R. sphaeroides (5Willows R.D. Gibson L.C.D. Kannangara C.G. Hunter C.N. von Wettstein D. Eur. J. Biochem. 1996; 235: 438-443Crossref PubMed Scopus (96) Google Scholar). The BchD protein was expressed as inclusion bodies, and these were purified as described in the pET system manual (Novagen). The inclusion bodies were then solubilized in 6 m urea, 50 mmTricine-NaOH, pH 8.0, 15 mm MgCl2, and 4 mm DTT. The solubilized protein was loaded onto a RESOURCE Q (Amersham Pharmacia Biotech) cation exchange chromatography column that was pre-equilibrated in the same buffer, and then the protein was eluted with this buffer containing a linear gradient of 0–1m NaCl, in 10 column volumes. The BchD protein eluted at 0.4 m NaCl. The BchI expression lysate was centrifuged at 30,000 ×g for 30 min, and then polyethylene glycol-8000 (molecular weight, 8000) was added to the soluble supernatant to a concentration of 10% (w/v). The mixture was placed on ice for 30 min and then centrifuged for 30 min at 10,000 × g, the pellet was discarded, and polyethylene glycol-8000 was added to a final concentration of 25% (w/v). After standing for 2 h on ice, the solution was centrifuged at 30,000 × g for 30 min. The supernatant was discarded, and the precipitate was washed once with 30% (w/v) polyethylene glycol-8000 and then redissolved in 50 mm Tricine-NaOH, pH 8.0, 15 mmMgCl2, and 4 mm DTT. The BchI protein was then purified by cation exchange chromatography as described above for BchD but without urea in the buffers. BchI eluted at 0.3 mNaCl. The HisBchH and HisBchI proteins were purified on a Ni2+-chelating column as described in the pET system manual, except that the eluted proteins were immediately desalted by Sephadex G-25 (Amersham Pharmacia Biotech) chromatography into 6% glycerol, 50 mm Tricine-NaOH, pH 8.0, 15 mmMgCl2, and 4 mm DTT. The HisBchD protein was expressed as inclusion bodies, and these were purified as described in the pET system manual (Novagen). The solubilized protein was then further purified on a Ni2+-chelating column under denaturing conditions in 6m urea as described in the pET system manual. After the protein was eluted, the buffer was changed to 6 m urea, 50 mm Tricine-NaOH, pH 8.0, 15 mmMgCl2, and 4 mm DTT. The assay mixture contained, in 50–1000 μl, the following: 50 mm Tricine-NaOH, pH 8.0, 15 mm MgCl2, 4 mm DTT, 4 mm ATP, 20 mm phosphocreatine, 20 units/ml of rabbit muscle creatine phosphokinase, 4 μm protoporphyrin IX, and various amounts of recombinant proteins as described in the figure and table legends. The assay mixtures were incubated for 30 or 60 min at 30 °C and stopped by the addition of 9 ml of acetone:H2O:32% (w/v) NH3 (80:20:1, v/v/v) per ml of incubation mixture and analyzed by fluorescence spectroscopy with the excitation wavelength set at 418 nm and the emission wavelength set at 596 nm. The intensity of the emission at 596 nm of standard magnesium-protoporphyrin IX in the same solution was proportional to the concentration within the range of 1 nm to 1 μm. A standard curve was used to determine the amount of magnesium-protoporphyrin IX formed in the assay. Antiserum to HisBchD was raised in a New Zealand White rabbit using standard procedures (14Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). The antiserum was cross-absorbed onto an E. colilysate column (Pierce) according to the manufacturer's instructions. Immunoblots were made using standard methods (14Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) with a 1/5000 dilution of antiserum and detection by a horseradish peroxidase-conjugated secondary antibody and a chemiluminescent substrate. SDS-polyacrylamide gel electrophoresis was done according to the procedure of Fling and Gregerson (15Fling S.P. Gregerson D.S. Anal. Biochem. 1986; 155: 83-88Crossref PubMed Scopus (773) Google Scholar) and gels were stained with colloidal Coomassie Brilliant Blue (16Neuhoff V. Arold N. Taube D. Ehrhardt W. Electrophoresis. 1988; 9: 255-262Crossref PubMed Scopus (2337) Google Scholar). R. capsulatus strains ZY6 and DB350 (2Bollivar D.W. Suzuki J.Y. Beatty J.T. Dobrowolski J.M. Bauer C.E. J. Mol. Biol. 1994; 237: 622-640Crossref PubMed Scopus (161) Google Scholar, 17Yang Z. Bauer C.E. J. Bacteriol. 1990; 172: 5001-5010Crossref PubMed Google Scholar) were a gift from C. E. Bauer and D. Bollivar. They were grown in RCV+ medium in the dark at 25 °C as described by Bollivar et al. (2Bollivar D.W. Suzuki J.Y. Beatty J.T. Dobrowolski J.M. Bauer C.E. J. Mol. Biol. 1994; 237: 622-640Crossref PubMed Scopus (161) Google Scholar). Protoporphyrin IX and magnesium-protoporphyrin IX were purchased from Porphyrin Products, Inc. (Logan, UT). Except where indicated otherwise, all other chemicals were from Fisher, Sigma, or Research Organics (Cleveland, OH). The individual magnesium chelatase recombinant subunits were purified as described under “Experimental Procedures.” With the exception of non-His-tagged BchH, they were homogeneous as judged by SDS-polyacrylamide gel electrophoresis (Fig. 1). BchH and HisBchH were expressed in soluble form with protoporphyrin IX noncovalently bound, as was found for the R. sphaeroidesBchH protein expressed in E. coli (5Willows R.D. Gibson L.C.D. Kannangara C.G. Hunter C.N. von Wettstein D. Eur. J. Biochem. 1996; 235: 438-443Crossref PubMed Scopus (96) Google Scholar). The absorption peaks attributed to protoporphyrin IX are visible in the absorption spectrum of purified HisBchH (Fig. 2 A). The fluorescence excitation spectrum (Fig. 2 B) shows a pronounced peak at 280 nm that is not present in the spectrum of free protoporphyrin IX, indicating that one or more protein tryptophan residues are in close proximity to the protoporphyrin IX and can transfer excitation energy to the pigment. The bound protoporphyrin IX caused BchH to become very susceptible to inactivation by light. Under normal laboratory lighting, it was inactivated in 10 min at 0 °C (data not shown). To prevent this light inactivation, all of the BchH expression and purification steps were performed in the dark or under a dim green safelight. In addition, all magnesium chelatase assays were done in the dark. BchD and HisBchD were both expressed as inclusion bodies. As described below, these proteins, after solubilization with urea, reconstituted magnesium chelatase when added to assay buffer containing BchH and BchI. In vitro magnesium chelatase activity was reconstituted only upon the addition of all three expressed proteins, BchI, BchH (or HisBchH), and BchD (or HisBchD) to the incubation mixture (Fig.3). Some product was formed in incubations without added protoporphyrin IX, because the protoporphyrin IX that is bound to purified BchH can be used as substrate. Although both BchI and HisBchI were highly expressed in soluble form, only non-His-tagged BchI was active in the magnesium chelatase assay. HisBchI was inactive, and it inhibited the reaction in incubations containing BchI (Fig. 4). For reconstitution of activity, BchD (or HisBchD), solubilized in 6m urea, was added directly to the assay mixture containing the other two protein components. BchD, therefore, refolded to an active form in the reaction mixture during the incubation, as was recently reported for C. vibrioforme BchD (9Petersen B.L. Jensen P.E. Gibson L.C.D. Stummann B.M. Hunter C.N. Henningsen K.W. J. Bacteriol. 1998; 180: 699-704Crossref PubMed Google Scholar). Although this method of assaying magnesium chelatase was useful for reconstitution experiments, it was unsatisfactory for enzyme kinetic studies because the proportion of BchD that is in the active form in the assay mixture could not be controlled. The factors that mediate renaturation of BchD were therefore examined as described below. Renatured BchD (or HisBchD) provided a consistent preparation for further study of the magnesium chelatase reaction. Efficient in vitro renaturation of BchD required DTT and ATP (Table I). Moreover, the degree of renaturation was increased by the presence of BchI. A time course of the renaturation of BchD at 0 °C with BchI, ATP, and MgCl2 indicated that BchD was maximally renatured at 90 min (data not shown). The concentration of BchD in the medium did not appear to have an effect on the renaturation efficiency, because approximately equal specific activity was obtained with the three concentrations tested (Table I). The slightly lower activity at higher BchD concentrations was caused by the higher concentrations of urea in the assay medium, which ranged from 30 to 120 mm in this experiment. A BchI:BchD molar ratio of 1:1 was sufficient for maximal recovery of active BchD (Table II). Neither further stimulation or inhibition of the renaturation was observed at higher BchI:BchD ratios. In the R. capsulatuschromosome, bchI is 5′ to bchD and is part of the same transcription unit. This implies that BchI is translated before BchD, and it is proposed that its presence may aid in the folding of BchD to its active form in vivo.Table IRequirements for BchD renaturationRenaturation mediumMg chelatase assay mediumMg chelatase activityaActivity values listed are means and standard deviations of duplicate (Experiments 1–3) or triplicate (Experiment 4) assays.BchIBchDBchI:BchDμgμgmol:molpmol μg−1 BchD% maximumExperiment 1Complete11.811.21.711.57 ± 0.35100+5 μm protoporphyrin IX11.811.21.712.14 ± 0.47105−BchI11.811.21.74.41 ± 0.5338Experiment 2Complete6.22.54.013.80 ± 0.50100−ARS6.22.54.016.50 ± 0.25120−ATP, −ARS6.22.54.02.80 ± 0.1520−ATP, −ARS, −MgCl26.22.54.02.40 ± 0.3017Experiment 3Complete23.622.41.710.60 ± 0.80100−ARS23.622.41.78.45 ± 0.4480−ATP, −ARS23.622.41.72.22 ± 0.3721−DTT23.622.41.70.76 ± 0.127Experiment 4Complete13.65.54.015.30 ± 1.20100Complete28.111.44.014.05 ± 1.2092Complete56.322.84.013.75 ± 1.8090BchD protein was solubilized in 6 m urea, 10 mmTricine-NaOH, pH 8.0, and 2 mm DTT and then renatured by rapid dilution into 20 volumes of renaturation medium and maintained at 0 °C for 60 min. Complete renaturation medium contained 50 mm Tricine-NaOH, pH 8.0, 15 mm MgCl2, 4 mm DTT, 4 mm ATP; an ATP regenerating system consisting of 20 mm phosphocreatine and 20 units/ml of creatine phosphokinase; and the indicated amount of BchI protein. For the magnesium chelatase assay, 10–50 μl of the renatured BchD-BchI mixture was supplemented with 4 μm protoporphyrin IX and 26 μg of BchH protein, additional BchI was added when needed to bring the final BchI content to the indicated level, the final volume was adjusted to 100 μl with complete renaturation medium, and the mixture was incubated for 30 min at 30 °C. ARS, ATP regenerating system.a Activity values listed are means and standard deviations of duplicate (Experiments 1–3) or triplicate (Experiment 4) assays. Open table in a new tab Table IIEffect of the BchI:BchD ratio on BchD renaturationBchD renaturation conditionsMg chelatase activityaActivity values listed are means and standard deviations of duplicate assays.BchIBchDBchI:BchDμgμgmol:molpmol μg−1 BchD% maximum0.011.40.03.6 ± 0.14484.911.40.76.5 ± 0.80878.411.41.26.5 ± 0.808714.011.42.06.8 ± 1.009117.511.42.57.5 ± 0.1510022.411.43.27.2 ± 0.909628.011.44.07.5 ± 0.0010038.511.45.57.1 ± 0.6095BchD protein solubilized and renatured as described in the legend to Table I. The renaturation medium contained the indicated ratio of BchI and BchD proteins. The renatured BchD-BchI mixture was then assayed for magnesium chelatase activity. All assays (100 μl) contained renaturation medium and 4 μm protoporphyrin IX, 11.2 μg of BchD, 33 μg of HisBchH protein, and additional BchI protein to bring the final amount to 42 μg. Assay mixtures were incubated for 60 min at 30 °C.a Activity values listed are means and standard deviations of duplicate assays. Open table in a new tab BchD protein was solubilized in 6 m urea, 10 mmTricine-NaOH, pH 8.0, and 2 mm DTT and then renatured by rapid dilution into 20 volumes of renaturation medium and maintained at 0 °C for 60 min. Complete renaturation medium contained 50 mm Tricine-NaOH, pH 8.0, 15 mm MgCl2, 4 mm DTT, 4 mm ATP; an ATP regenerating system consisting of 20 mm phosphocreatine and 20 units/ml of creatine phosphokinase; and the indicated amount of BchI protein. For the magnesium chelatase assay, 10–50 μl of the renatured BchD-BchI mixture was supplemented with 4 μm protoporphyrin IX and 26 μg of BchH protein, additional BchI was added when needed to bring the final BchI content to the indicated level, the final volume was adjusted to 100 μl with complete renaturation medium, and the mixture was incubated for 30 min at 30 °C. ARS, ATP regenerating system. BchD protein solubilized and renatured as described in the legend to Table I. The renaturation medium contained the indicated ratio of BchI and BchD proteins. The renatured BchD-BchI mixture was then assayed for magnesium chelatase activity. All assays (100 μl) contained renaturation medium and 4 μm protoporphyrin IX, 11.2 μg of BchD, 33 μg of HisBchH protein, and additional BchI protein to bring the final amount to 42 μg. Assay mixtures were incubated for 60 min at 30 °C. Because BchD was solubilized in 6 m urea and added to assay medium by dilution from the urea solution, it was important to determine the effect of urea on the magnesium chelatase reaction. The reaction was progressively inhibited at increasing urea concentrations, with approximately 20% inhibition at 100 mm, 50% inhibition at 250 mm, and 90% inhibition at 800 mm (Fig. 5). Because the final urea concentration in the magnesium chelatase assays was usually 30–60 mm, its presence in the assay medium had only a minor effect on activity. R. capsulatus mutants ZY6 and DB350 contain insertional disruptions of the bchH and bchIgenes, respectively (2Bollivar D.W. Suzuki J.Y. Beatty J.T. Dobrowolski J.M. Bauer C.E. J. Mol. Biol. 1994; 237: 622-640Crossref PubMed Scopus (161) Google Scholar, 17Yang Z. Bauer C.E. J. Bacteriol. 1990; 172: 5001-5010Crossref PubMed Google Scholar). Magnesium chelatase activity was absent in extract of either mutant alone, but was present in a mixture of the extracts from the two mutants (TableIII). Activity was reconstituted in extract of ZY6 cells supplemented with recombinant BchH. Neither BchD, BchI, nor a combination of BchD and BchI reconstituted activity of ZY6 extract. Fractionated ZY6 extract from which BchD was removed by high speed centrifugation and ion exchange chromatography required addition of both BchH and BchD to restore activity. Similarly, ZY6 extract from which BchI was partially removed by high speed centrifugation required addition of both BchH and BchI to restore full activity.Table IIIIn vitro complementation of R. capsulatus magnesium chelatase mutantsCell extract(s) addedRecombinant protein(s) addedRelative Mg-protoporphyrin IX formationZY6DB350BchHBchDBchIμgμgμg%Experiment 10030.01.86.010025000.00.00.0003300.00.00.00250030.00.00.018025000.01.80.0025000.00.06.0025000.01.86.0040 (I) + 20 (D)030.00.00.016040 (I)030.00.00.0040 (I)030.01.80.05020 (D)030.00.00.0520 (D)030.00.06.0500Experiment 20010.01.85.21002503300.00.00.050033010.00.00.0003300.01.80.0003300.00.05.2003300.01.85.250Strains ZY6 (bchH −) and DB350 (bchI −) were grown and extracted as described under “Experimental Procedures.” Extract of strain ZY6 (3 ml) was centrifuged for 1 h at 190,000 × g, and the glassy pellet was washed once with extraction buffer and resuspended in 0.5 ml of the same buffer to yield the BchD-containing fraction ZY6 (D) (31Kannangara C.G. Vothknecht U.C. Hansson M. von Wettstein D. Mol. Gen. Genet. 1997; 254: 85-92Crossref PubMed Scopus (58) Google Scholar). The supernatant was purified by cation exchange chromatography as described under “Experimental Procedures” to yield the BchI-containing fraction ZY6 (I). Recombinant BchD was dissolved in 6 m urea, and 2 μl was added to the reactions where indicated. Incubations were 60 min at 30 °C in 100 μl of complete reaction medium containing the indicated amounts of proteins from cell extracts and purified recombinant proteins. Because the various proteins added to the incubation mixture were of different degrees of purification, the tabulated relative product formation values have not been normalized to protein concentration. Open table in a new tab Strains ZY6 (bchH −) and DB350 (bchI −) were grown and extracted as described under “Experimental Procedures.” Extract of strain ZY6 (3 ml) was centrifuged for 1 h at 190,000 × g, and the glassy pellet was washed once with extraction buffer and resuspended in 0.5 ml of the same buffer to yield the BchD-containing fraction ZY6 (D) (31Kannangara C.G. Vothknecht U.C. Hansson M. von Wettstein D. Mol. Gen. Genet. 1997; 254: 85-92Crossref PubMed Scopus (58) Google Scholar). The supernatant was purified by cation exchange chromatography as described under “Experimental Procedures” to yield the BchI-containing fraction ZY6 (I). Recombinant BchD was dissolved in 6 m urea, and 2 μl was added to the reactions where indicated. Incubations were 60 min at 30 °C in 100 μl of complete reaction medium containing the indicated amounts of proteins from cell extracts and purified recombinant proteins. Because the various proteins added to the incubation mixture were of different degrees of purification, the tabulated relative product formation values have not been normalized to protein concentration. Magnesium chelatase activity was not restored to extract of strain DB350 by the addition of any single recombinant protein, but activity was partially restored by the addition of both BchD and BchI. The requirement for both proteins is consistent with the absence of the BchD in extracts of strain DB350 (Fig.6). These results suggest that BchI is needed to stabilize BchD in vivo and are consistent with the ability of BchI to enhance the renaturation of urea-denatured BchDin vitro. The pH optimum for magnesium chelatase was approximately 8.0 (Fig.7). Activity dropped to near zero below pH 6 and above pH 10.5. The time course for product accumulation exhibited a small lag phase, followed by approximately constant activity for about 100 min, and lower activity at later times (Fig.8). Preincubation of BchD and BchI in assay mixture for 10 min at 30 °C before starting the reaction by the addition of BchH increased the initial reaction rate but did not eliminate the lag phase.Figure 8Reaction time course. Assays (1.0 ml) contained 110 μg/ml BchD, 140 μg/ml BchI, and 660 μg/ml HisBchH.Closed circles are for an experiment in which all ingredients were added in quick succession at the start of the reaction. Open circles are for an experiment in which all ingredients except BchH were preincubated for 10 min at 30 °C before BchH was added to start the reaction.View Large Image Figure ViewerDownload (PPT) A double reciprocal plot of reaction rate versusprotoporphyrin IX concentration indicates Michaelis-Menten kinetics with respect to protoporphyrin IX (Fig.9 A). The apparentK m for protoporphyrin IX was calculated to be 1.23 μm. A double reciprocal plot" @default.
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• W2022277083 title "Heterologous Expression of the Rhodobacter capsulatus BchI, -D, and -H Genes That Encode Magnesium Chelatase Subunits and Characterization of the Reconstituted Enzyme" @default.
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