| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Institute of Microbiology, Technical University of Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany
2 Division of Microbiology, Helmholtz Centre for Infection Research (HZI), Inhoffenstr. 7, D-38124 Braunschweig, Germany
3 Laboratoire de Biologie et de Génomique Structurales, IGBMC, Parc d'Innovation, 1 rue Laurent Fries, BP 10142, F-67404 Illkirch Cedex, France
Correspondence
Dieter Jahn
d.jahn{at}tu-bs.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
The structural core of tetrapyrroles implies a highly conserved biosynthetic pathway. The formation of protohaem comprises at least seven enzymic reactions starting from the common precursor molecule 
-aminolaevulinic acid (ALA) (Frankenberg et al., 2003). The three terminal steps of the haem biosynthetic pathway are executed by coproporphyrinogen III oxidase (CPO, EC 1.3.3.3), protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) and ferrochelatase (FC, EC 4.99.1.1) (Fig. 1A). CPO oxidatively decarboxylates the propionate side chains on rings A and B of coproporphyrinogen III (coprogen) to the corresponding vinyl groups (Dailey, 2002). The oxygen-dependent enzyme lacks obvious cofactors (Labbe, 1997; Medlock & Dailey, 1996). The crystal structures of the human and yeast enzymes have been solved (Phillips et al., 2003, 2004). PPO catalyses the aromatization of protoporphyrinogen IX (protogen), producing protoporphyrin IX (proto). The six-electron oxidation of proto is accomplished using a flavin cofactor. In eukaryotes PPO is associated with the outer surface of the inner mitochondrial membrane (Deybach et al., 1985), while in plants the enzyme is additionally located in chloroplasts (Jacobs & Jacobs, 1987). When PPO is inhibited, protogen is exported into the cytoplasm and oxidized to proto, where it accumulates with detrimental consequences upon light exposure. The ultimate step in haem biosynthesis is catalysed by FC, with the insertion of ferrous iron into the porphyrin macrocycle to form protohaem (Porra & Jones, 1963).
|
); their localization in plants is less well characterized (Lister et al., 2001). There is conflicting evidence regarding the inner-membrane localization of PPO and FC. Two isomers of PPO and FC have been reported to be targeted to either chloroplasts or mitochondria, where they are associated with membranes (Lermontova et al., 1997; Suzuki et al., 2002; Watanabe et al., 2001).
Kinetic studies using solubilized membrane-bound and vesicle-reconstituted enzymes from mouse mitochondria suggested possible complex formation between PPO and FC in order to protect this sensitive biosynthetic intermediate proto (Ferreira et al., 1988). Based on the crystal structures of tobacco PPO (Koch et al., 2004) and human FC (Wu et al., 2001), Koch and coworkers suggested a tight complex between these two enzymes. CPO was also associated with the in silico model complex. The proposed model allows for metabolic channelling via an overlap of the active-site channel openings of tobacco PPO and human FC. Here, we experimentally investigated the existence of protein complexes between CPO, PPO and FC in the phototrophic cyanobacterium Thermosynechococcus elongatus using co-immunoprecipitation experiments and immunoelectron microscopy.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Employed bacterial strains and constructed plasmids.
Escherichia coli strain DH10b [F– mcrA 
(mrr-hsdRMS-mcrBC) 
80lacZM15 lacX74 recA1 endA1 ara139 (ara, leu)7697 galU galK 
– rpsL (Strr) nupG] was used for cloning purposes. Strain BL21(DE3)RIL [F–ompT hsdS(
) dcm+ Tetr gal (DE3) endA Hte(argU ileY leuW Camr)] was the host for gene overexpression and protein production. Thermosynechococcus elongatus BP1 cells were cultivated in a 5 l fermenter at 55 °C under continuous illumination from fluorescent white lamps (
80 µmol photons m–2 s–1). The cells were grown in D-medium supplemented with micronutrients in a CO2-enriched (10 %) atmosphere to an OD700 of 2.0 as previously described (Schulze et al., 2006; Zouni et al., 2001). After harvesting by centrifugation, 1.4 g wet cell mass (l culture)–1 was obtained.
A 1059 bp fragment encoding all 352 amino acid residues of T. elongatus CPO was produced by PCR using primers 5'-GCAAGCGCATATGGACACTA-3' and 5'-GGTTAAGCTTGACTGGCCAATTGACC-3' with T. elongatus BP1 genomic DNA as template. The resulting fragment was digested with NdeI and HindIII and ligated into appropriately cut pET22b (Novagen) to generate pET22bcpo. The construction of the T. elongatus PPO-overproducing E. coli strain is previously described (Layer et al., 2005). A DNA fragment encoding the 392 amino acid residues of T. elongatus FC was generated by PCR using primers 5'-CCGGAATTCCGGGTGATTTTGGGAATGGCGAGCC-3' and 5'-CCGCTCGAGCGGACTGATGAGTTCAATCATCAAC-3' with T. elongatus BP1 genomic DNA as template. The resulting 1179 bp fragment was digested with EcoRI and XhoI and ligated with the appropriately digested vector pET32a (Novagen) to yield pET32afc. Oligonucleotides were purchased from Biomers.net GmbH.
Recombinant production and purification of active T. elongatus CPO, PPO and FC.
All three proteins were recombinantly produced in E. coli as His-tag fusion proteins and purified to apparent homogeneity using affinity chromatography.
Recombinant T. elongatus CPO was produced with E. coli BL21(DE3)RIL cells (Novagen) carrying the vector pET22bcpo, supplemented with 100 µg ampicillin ml–1 and 34 µg chloramphenicol ml–1. Protein production was induced at OD595 0.6 with the addition of 500 µM IPTG. After induction, cells were further grown at 37 °C for 4 h, harvested by centrifugation and washed in a buffer containing 20 mM Tris (pH 8.0), 350 mM NaCl, 10 mM imidazole, 0.2 % Triton X-100. Subsequently, the cells were broken by sonication (Bandelin HD 2070; 0.5 s sound, 0.5 s pause, MS73 tip, with 50 % amplitude) and cleared by centrifugation at 150 000 g at 4 °C for 45 min. The cell-free extract obtained was further filtered through a 0.2 µm sterile filter. Purification of recombinant T. elongatus CPO was performed using Ni-NTA Sepharose chromatography (GE Healthcare). Solubilized proteins were loaded onto a Ni-NTA column and bound proteins were eluted with 200 mM imidazole in chromatography buffer.
Recombinant T. elongatus PPO was produced with E. coli BL21(DE3)RIL cells carrying vector pET32appo. Protein production was induced at OD595 0.6 with the addition of 500 µM IPTG and 5 µg riboflavin l–1. After induction, cells were grown at 25 °C overnight. The cells then were washed three times in lysis buffer [50 mM Tris (pH 8.0), 10 mM MgCl2, 350 mM NaCl, 0.5 % (w/v) Triton X-100, 0.5 % (w/v) NP-40, 10 U benzonase ml–1, 10 µg RNase A ml–1, 0.05 % (w/v) Tween 80, 8 mg lysozyme ml–1]. Cells were broken by sonication (Bandelin HD 2070; 0.5 s sound, 0.5 s pause, MS73 tip, 50 % amplitude) and cleared by centrifugation at 150 000 g at 4 °C for 40 min. Purification of recombinant T. elongatus PPO was performed using the MagneHis Protein Purification System (Promega) according to the manufacturer's instructions. After purification, the protein was dialysed against 20 mM Tris (pH 8.0), 50 mM NaCl and 0.1 % (v/v) Triton X-100 and stored at 4 °C. Approximately 20 mg purified PPO protein per 1 l shake-flask culture was yielded. MALDI-TOF mass spectrometry confirmed purified PPO identity (Layer et al., 2005).
Recombinant T. elongatus FC was produced with E. coli BL21(DE3)RIL cells carrying vector pET32afc. Protein production was induced at OD595 0.6 with the addition of 500 µM IPTG, 100 µg ampicillin ml–1 and 34 µg chloramphenicol ml–1. Cells were further grown at 37 °C for 2.5 h. Growth medium was exchanged with fresh LB medium including 100 µg ampicillin ml–1, 34 µg chloramphenicol ml–1 and 10 µg tetracycline ml–1. Further cultivation was performed at 25 °C overnight. Cells were harvested, disrupted via sonication as described above and cell debris was removed by centrifugation at 12 000 g at 4 °C for 30 min. Cells were suspended in 50 mM Tris (pH 8.0), 150 mM KCl and 0.2 % Triton X-100 and subjected to three rounds of sonication and centrifugation as described above. Solubilized proteins containing recombinant T. elongatus FC were loaded onto a cobalt-Sepharose column (Chelating Sepharose Fast Flow; GE Healthcare). Bound proteins were eluted with 140 mM imidazole in chromatography buffer and dialysed against assay buffer [50 mM MES-buffer pH 6.0, 1 % (v/v) Triton X-100, 20 mM EDTA] for further analyses. The use of cobalt-Sepharose instead of Ni-NTA significantly enhanced FC solubility during chromatography and as a consequence improved the overall yield of the purified recombinant FC.
Catalytic activity for all three recombinant proteins was verified for CPO activity testing as follows. Coprogen was generated as described previously (Layer et al., 2002). Proto was detected via its typical fluorescence at 633 nm (Breckau et al., 2003; Heinemann et al., 2007). The oxygen-dependent PPO assay was originally developed by Brenner and Bloomer for the mammalian enzyme and modified as previously described (Brenner & Bloomer, 1980; Heinemann et al., 2007). Specific FC activity was monitored by recording the decrease of proto, which was detected fluorimetrically at 633 nm (data not shown).
Co-immunoprecipitation assays using whole-cell T. elongatus extracts.
Approximately 1 mg purified recombinant CPO, PPO and FC was used for polyclonal antibody generation in rabbits by Eurogentec. In order to solubilize hydrophobic, membrane-bound or associated proteins, 5.5 g T. elongatus cells was suspended in 6 ml HEPES buffer (pH 8.0) containing 5 mM MgCl2, 300 mM NaCl, 0.5 % Triton X-100 and 1 % n-dodecyl-β-D-maltoside. Cells were disrupted by sonication (Bandelin HD 2070, 0.5 s sound, 0.5 s pause, 70 % amplitude) using a sonotrode (MS73 tip, Bandelin). The disrupted cells were treated in a Mikro-Dismembrator (Braun Biotech) for 30 min. Cell debris was removed by centrifugation at 4000 g at 4 °C for 15 min. The detergent-containing supernatant was again subjected to the dismembrator treatment for 40 min. Cell debris was removed by ultracentrifugation. After incubation at 4 °C for 60 min, cell debris was removed by centrifugation at 4 °C and 100 000 g for 45 min. About 300 µl Protein A-Sepharose CL 4B was pre-equilibrated with cell suspension buffer under gentle shaking at 4 °C overnight according to the manufacturer's instructions (Seize X Protein A Immunoprecipitation Kit; Pierce). Then 2.5 µl of either anti-CPO, anti-PPO or anti-FC antibodies at dilution of 1/100 000 was added and adsorbed to the Protein A-Sepharose at 4 °C for 12 h. About 500 µl of the clear T. elongatus cell extract (the equivalent of 500 mg wet cells) was incubated with the immobilized antibodies at 4 °C for 1–2 h. The immuno-adsorbant was recovered by centrifugation at 10 000 g for 1 min and washed four times by resuspension in a buffer containing 150 mM NaCl, 8 mM Na3PO4, 2 mM K3PO4 (pH 7.4), 10 mM KCl and subsequent centrifugation. Unfortunately, precipitated antibody heavy chains resulted in a diffuse protein pattern at a molecular mass of 50 000 Da in SDS-PAGE and subsequent Western blot analyses. Thus, they prevented the detection of signals for immunoprecipitated PPO with a calculated molecular mass of 50 199 Da and anti-PPO (data not shown). This problem was solved by using the Seize X Protein A Immunoprecipitation Kit (Pierce), which successfully removed most of the antibody heavy chains. Protein samples eluted from Protein A-Sepharose were heated for 5 min at 95 °C and subjected to 8 % SDS-PAGE using standard techniques. The electrophoretically separated proteins were transferred onto PVDF membranes (Millipore) using a Trans-Blot apparatus (semi-dry transfer cell, Bio-Rad) according to the manufacturer's instructions. The membrane was incubated with anti-CPO, anti-PPO and anti-FC rabbit antibodies, respectively (1 : 5000 in PBS with 3 % skim milk powder from Fluka,), washed three times with PBS, and subsequently incubated with alkaline phosphatase-conjugated goat anti-rabbit antibodies (1 : 20 000 in PBS with 3 % skim milk powder) from Pierce. The colorimetric detection of immunoreactive bands was performed using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium from Promega according to the manufacturer's instructions.
Double-immunogold labelling.
T. elongatus cells were fixed in a fixation solution of 0.5 % formaldehyde and 0.2 % glutaraldehyde in cacodylate buffer [100 mM M cacodylate (pH 6.9), 90 mM sucrose, 10 mM MgCl2, 10 mM CaCl2], for 1 h on ice. Fixed cells were washed with cacodylate buffer containing 10 mM glycine and embedded into 1.75 % aqueous agar and after solidification cut into small cubes. Samples were then dehydrated following the progressive lowering of temperature (PLT) method. For this purpose 10 % and 30 % ethanol treatments were carried out on ice for 15 min, followed by the 50 % step at –20 °C for 30 min. The subsequent ethanol steps (70, 90 and 100 %) were carried out at –35 °C, each step for 30 min, and the 100 % step was repeated twice. The Lowicryl resin K4M was infiltered in several steps. First, 1 part K4M/1 part 100 % ethanol was applied overnight, followed by 2 parts K4M/1 part ethanol for 24 h and finally pure resin for 36 h with several changes. The resin was polymerized at –35 °C with UV-light treatment at 366 nm for 1 day and further polymerized at room temperature for another 2 days. Ultrathin sections were cut with a diamond knife and collected onto Formvar-coated 300 mesh nickel grids. Sections were incubated in a 1 : 25 dilution of Protein A-Sepharose-purified anti-PPO (stock solution 1.9 mg IgG protein ml–1) at 4 °C for overnight. Sections were then washed with PBS and incubated in a 1 : 200 dilution of protein A/G gold (15 nm in diameter) for 30 min at room temperature. After washing with PBS containing 0.01 % Tween 20, sections were incubated with 100 µg protein A ml–1 for 15 min at room temperature and washed again with PBS. Sections were then incubated with a 1 : 25 dilution of purified anti-FC (1.9 mg stock solution ml–1) and incubated at room temperature for 3 h. After washing with PBS, sections were incubated with a 1 : 75 dilution of protein A/G gold (10 nm in diameter) at room temperature for 30 min. After washing with PBS containing 0.01 % Tween 20, sections were washed with TE, distilled water and air-dried. Sections were counterstained with 4 % aqueous uranyl acetate for 3 min and washed with water. After air-drying, samples were examined in a Zeiss EM910 transmission electron microscope with an acceleration voltage of 80 kV. Images were recorded onto negative film (Kodak SO-163). Prints were generated on Ilford Multigrade paper and then scanned and processed using Adobe Photoshop 6.0.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Based on the crystal structures of the yeast CPO, tobacco PPO and human FC, Koch and coworkers first suggested a tetrameric complex between the PPO and FC dimers (Fig. 1B
). The part of the complex that is supposed to span the inner mitochondrial membrane has a length of 28 Å (2.8 nm), corresponding well to the assumed 30 Å for the lipidic part of a membrane. One appealing feature of the model is the overlap of the channel openings of PPO and FC that could facilitate substrate channelling between the active sites of the two proteins without solvent exposure. Koch and coworkers additionally proposed that the PPO substrate protogen could be taken up directly from CPO at the periplasmic side of the membrane, most likely with CPO as a third participant in the multienzyme complex. Here, we sought biochemical evidence for the existence of the protein complex between CPO, PPO and FC in the cyanobacterium T. elongatus. We expected potential complexes to be associated with the cytoplasmic side of the thylakoid membrane as the thylakoids of cyanobacteria represent invaginations of the cytoplasmic membrane and the cytoplasmically synthesized enzymes do not carry obvious export signals. Co-immunoprecipitation analysis was combined with electron microscopy based localization of protein complexes in T. elongatus cells. These two methods provided independent evidence for formed complexes and their cellular localization. However, the exact positioning of these complexes, i.e. transmembrane or membrane-associated, requires additional biochemical approaches.
Recombinant CPO, PPO and FC production and antibody generation
The proteins of interest were produced and purified to apparent homogeneity as detailed in Methods. All purified recombinant proteins showed specific enzymic activity when tested with their natural substrates. Solutions containing 1 mg purified recombinant CPO, PPO and FC were supplied to Eurogentec for the generation of polyclonal rabbit antibodies. The specificity of antigen detection was evaluated by Western blot analysis. A dilution of 1/100 000 of anti-CPO, anti-PPO and anti-FC specifically detected 100 ng purified recombinant CPO, PPO and FC (data not shown). The results of the following immunoprecipitation experiment showed that the generated antibodies successfully precipitated recombinant CPO, PPO and FC from E. coli cell-free extracts and detected them in Western blots (data not shown).
The following strategy was employed for the detection of enzyme complexes in T. elongatus whole-cell extracts including solubilized membrane-bound proteins. The protein to be tested for potential binding partners was bound to its specific antibody. This antibody–antigen complex was immobilized on Protein A Sepharose, isolated via centrifugation and washed. In the case of a co-immunoprecipitated interaction partner, the second bound protein was visualized in Western blot experiments using a second antibody directed against it. Co-immunoprecipitation experiments with anti-CPO, anti-PPO and anti-FC antibodies were performed with T. elongatus whole-cell extracts. The cells contained the natural amount of all three enzymes since none of the corresponding genes was overexpressed. Two of the complementary co-immunoprecipitation experiments resulted in the precipitation of the postulated PPO–FC complex. Significant amounts of complexed protein were detected with the anti-FC antibodies after immunoprecipitation with anti-PPO antibodies (Fig. 2B, lane 4) and with anti-PPO antibodies after the anti-FC-mediated precipitation (Fig. 2C, lane 6). The control experiments performed with whole-cell extracts with either pre-immune serum or Protein A-Sepharose ruled out non-specific cross-reactivities of the antibodies employed with PPO (Fig. 2C, lanes 2 and 3) and FC (Fig. 2B, lanes 2 and 3), respectively. The removal of the antibody heavy chains from the anti-PPO preparation was necessary to allow detection of the PPO due to almost identical molecular masses (Fig. 2C, lanes 5 and 6). The in silico model by Koch and coworkers additionally suggested that CPO could also be a partner of the enzyme complex. Thus, our co-immunoprecipitation results exclude CPO from the stable complex; however, they do not rule out transient interactions. Although anti-CPO antibodies precipitated CPO from T. elongatus whole-cell extracts (Fig. 2A, lane 4), no co-immunoprecipitated proteins were observed using anti-FC or anti-PPO antibodies (Fig. 2A, lanes 5 and 6). In summary, these results confirm the existence of a complex of PPO with FC in T. elongatus cells.
|
shows a longitudinal section of a T. elongatus cell, conventionally embedded in epoxy resin. It shows that the bacterial cytoplasm is mostly occupied by thylakoid membranes, which are most clearly visible only in such parts of the cytoplasm where the ultrathin section is perpendicular to the thylakoid membrane (arrows in Fig. 3A). First, we incubated the ultrathin sections of T. elongatus with anti-FC antibodies labelled with small 10 nm gold particles. Next, anti-PPO antibodies decorated with large 15 nm gold particles were applied. Differently sized gold particles less than 25 nm from each other were counted as protein–protein complexes since each of the two applied antibodies can maximally span a distance of approximately 12 nm due to the hinge in the Fab region. As evident from the statistical evaluation of 25 different longitudinal sections of T. elongatus and as shown in Fig. 3(B), more than 51.7±9.1 % of the detected anti-FC antibodies were significantly co-localizing with anti-PPO antibodies at room temperature (Fig. 3B, C). In total, for the 25 analysed longitudinal sections 330 out of 640 10 nm gold particle FC (FC-10 nm) molecules were found co-localized with PPO-15 nm molecules. On average, out of 25.6±5.19 FC molecules 12.64±2.32 were co-localized with PPO on a single analysed section. These observations clearly indicated in vivo PPO–FC complex formation. It has to be taken into account that, due to the embedding procedure in low-temperature resin and counter-staining of the sections with uranyl acetate alone, the thylakoid membranes are only partially visible in the ultrathin sections compared to the osmium-fixed conventional embedding method (compare Fig. 3A, B). In cases where the thylakoid membranes were partly visible in conjunction with the gold particles, the PPO–FC complexes within T. elongatus were found localized to the cytoplasmic side of the thylakoid membrane. This is in good agreement with the cytoplasmic synthesis of the proteins as well as their missing export signals. The identical experiments were performed with the anti-CPO and either anti-PPO or anti-FC as the second antibody (Fig. 3D, E). In Fig. 3(D, E), less than 5 % of FC and PPO seem to co-localize with CPO. In total numbers for the 25 cut sections, only 26 out of 658 of the CPO-15 nm molecules were co-localized with PPO-10 nm. A similar situation was found for the CPO–FC analysis: in total numbers for the 25 cut sections only 25 out of 632 of the CPO-15 nm molecules were found co-localized with FC-10 nm molecules. For both CPO–PPO and CPO–FC the number of co-localizations was within the standard deviation, and therefore likely to be random. With regard to the size of the proteins, diameter of the section, cut orientation and antigen orientation with over 50 % pairs in double-immunogold labelling, the existence of a complex between PPO and FC seems very likely.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 1994). However, this complex most likely is localized at the cytoplasmic side of the bacterial thylakoid membrane. The thylakoid membranes of cyanobacteria are invaginations of the cytoplasmic membrane. The proton gradient of photosynthesis is formed at this membrane as in non-phototrophic bacteria. Consequently, we expect an arrangement of the haem biosynthetic enzymes on the cytoplasmic side of the membrane similar to other non-phototrophic bacteria. This is in good agreement with the missing export signal for all three cyanobacterial enzymes. Nevertheless, the model proposed here based on the crystal structures of eukaryotic enzymes (Koch et al., 2004) was only partly verified. The exact mode of interaction of the complex with the membrane, i.e. transmembrane versus membrane association, remains to be determined. However, amino acid sequence comparisons of T. elongatus FC with membrane-associated human FC, soluble Bacillus subtilis FC and other FCs revealed that the cyanobacterial FC possesses more sequence similarity to plant enzymes than to bacterial or vertebrate counterparts. Since no structure for this type of FC is known, a prediction of the membrane localization behaviour remains difficult. The PPO–FC complexes allow the phototrophic cyanobacterium to protect the highly photoreactive intermediate protogen via channelling between the two enzymes. Earlier kinetic investigations of eukaryotic PPO and FC already suggested that substrate channelling occurs between these enzymes (Ferreira et al., 1988). Similar conclusions were drawn from an investigation of various B. subtilis FC mutants (Olsson et al., 2002). Channelling of potentially toxic or labile intermediates by enzymes seems to be a widespread solution in nature. Two other enzymes of haem biosynthesis, glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase, form a stable complex to protect the labile intermediate glutamate-1-semialdehyde (Lüer et al., 2005; Nogaj & Beale, 2005).
Due to the unstable nature of pre-uroporphyrinogen III, complex formation between the producing porphobilinogen deaminase and accepting uroporphyrinogen III synthase was postulated, but has not yet been experimentally verified (Shoolingin-Jordan, 1995). Consequently, further biochemical and immunological experiments are required to solve all the protein–protein interactions required for high-fidelity haem biosynthesis.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: J. W. B. Moir
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Brenner, D. A. & Bloomer, J. R. (1980). The enzymatic defect in variegate prophyria. Studies with human cultured skin fibroblasts. N Engl J Med 302, 765–769.[Abstract]
Dailey, H. A. (2002). Terminal steps of haem biosynthesis. Biochem Soc Trans 30, 590–595.[CrossRef][Medline]
Deybach, J. C., da Silva, V., Grandchamp, B. & Nordmann, Y. (1985). The mitochondrial location of protoporphyrinogen oxidase. Eur J Biochem 149, 431–435.[Medline]
Ferreira, G. C., Andrew, T. L., Karr, S. W. & Dailey, H. A. (1988). Organization of the terminal two enzymes of the heme biosynthetic pathway. Orientation of protoporphyrinogen oxidase and evidence for a membrane complex. J Biol Chem 263, 3835–3839.
Frankenberg, N., Moser, J. & Jahn, D. (2003). Bacterial heme biosynthesis and its biotechnological application. Appl Microbiol Biotechnol 63, 115–127.[CrossRef][Medline]
Harbin, B. M. & Dailey, H. A. (1985). Orientation of ferrochelatase in bovine liver mitochondria. Biochemistry 24, 366–370.
Heinemann, I. U., Diekmann, N., Masoumi, A., Koch, M., Messerschmidt, A., Jahn, M. & Jahn, D. (2007). Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site. Biochem J 402, 575–580.[CrossRef][Medline]
Heinemann, I. U., Jahn, M. & Jahn, D. (2008). The biochemistry of heme biosynthesis. Arch Biochem Biophys 474, 238–251.[CrossRef]
Jacobs, J. M. & Jacobs, N. J. (1987). Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles. Biochem J 244, 219–224.[Medline]
Koch, M., Breithaupt, C., Kiefersauer, R., Freigang, J., Huber, R. & Messerschmidt, A. (2004). Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis. EMBO J 23, 1720–1728.[CrossRef][Medline]
Labbe, P. (1997). Purification and properties of coproporphyrinogen III oxidase from yeast. Methods Enzymol 281, 367–378.[Medline]
Layer, G., Verfurth, K., Mahlitz, E. & Jahn, D. (2002). Oxygen-independent coproporphyrinogen-III oxidase HemN from Escherichia coli. J Biol Chem 277, 34136–34142.
Layer, G., Grage, K., Teschner, T., Schünemann, V., Breckau, D., Masoumi, A., Jahn, M., Heathcote, P., Trautwein, A. X. & Jahn, D. (2005). Radical S-adenosylmethionine enzyme coproporphyrinogen III oxidase HemN: functional features of the [4Fe–4S] cluster and the two bound S-adenosyl-L-methionines. J Biol Chem 280, 29038–29046.
Lermontova, I., Kruse, E., Mock, H. P. & Grimm, B. (1997). Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase. Proc Natl Acad Sci U S A 94, 8895–8900.
Lister, R., Chew, O., Rudhe, C., Lee, M. N. & Whelan, J. (2001). Arabidopsis thaliana ferrochelatase-I and -II are not imported into Arabidopsis mitochondria. FEBS Lett 506, 291–295.[CrossRef][Medline]
Lüer, C., Schauer, S., Mobius, K., Schulze, J., Schubert, W. D., Heinz, D. W., Jahn, D. & Moser, J. (2005). Complex formation between glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase in Escherichia coli during the initial reactions of porphyrin biosynthesis. J Biol Chem 280, 18568–18572.
Matringe, M., Camadro, J. M., Block, M. A., Joyard, J., Scalla, R., Labbe, P. & Douce, R. (1992). Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides. J Biol Chem 267, 4646–4651.
Matringe, M., Camadro, J. M., Joyard, J. & Douce, R. (1994). Localization of ferrochelatase activity within mature pea chloroplasts. J Biol Chem 269, 15010–15015.
Medlock, A. E. & Dailey, H. A. (1996). Human coproporphyrinogen oxidase is not a metalloprotein. J Biol Chem 271, 32507–32510.
Nogaj, L. A. & Beale, S. I. (2005). Physical and kinetic interactions between glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase of Chlamydomonas reinhardtii. J Biol Chem 280, 24301–24307.
Olsson, U., Billberg, A., Sjovall, S., Al-Karadaghi, S. & Hansson, M. (2002). In vivo and in vitro studies of Bacillus subtilis ferrochelatase mutants suggest substrate channeling in the heme biosynthesis pathway. J Bacteriol 184, 4018–4024.
Phillips, J. D., Whitby, F. G., Kushner, J. P. & Hill, C. P. (2003). Structural basis for tetrapyrrole coordination by uroporphyrinogen decarboxylase. EMBO J 22, 6225–6233.[CrossRef][Medline]
Phillips, J. D., Whitby, F. G., Warby, C. A., Labbe, P., Yang, C., Pflugrath, J. W., Ferrara, J. D., Robinson, H., Kushner, J. P. & Hill, C. P. (2004). Crystal structure of the oxygen-dependant coproporphyrinogen oxidase (Hem13p) of Saccharomyces cerevisiae. J Biol Chem 279, 38960–38968.
Porra, R. J. & Jones, O. T. (1963). Studies on ferrochelatase. 2. An investigation of the role of ferrochelatase in the biosynthesis of various haem prosthetic groups. Biochem J 87, 186–192.[Medline]
Schulze, J. O., Masoumi, A., Nickel, D., Jahn, M., Jahn, D., Schubert, W. D. & Heinz, D. W. (2006). Crystal structure of a non-discriminating glutamyl-tRNA synthetase. J Mol Biol 361, 888–897.[CrossRef][Medline]
Shoolingin-Jordan, P. M. (1995). Porphobilinogen deaminase and uroporphyrinogen III synthase: structure, molecular biology and mechanism. J Bioenerg Biomembr 27, 181–195.[CrossRef][Medline]
Suzuki, T., Masuda, T., Singh, D. P., Tan, F. C., Tsuchiya, T., Shimada, H., Ohta, H., Smith, A. G. & Takamiya, K. (2002). Two types of ferrochelatase in photosynthetic and nonphotosynthetic tissues of cucumber: their difference in phylogeny, gene expression and localization. J Biol Chem 277, 4731–4737.
Watanabe, N., Che, F. S., Iwano, M., Takayama, S., Yoshida, S. & Isogai, A. (2001). Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons. J Biol Chem 276, 20474–20481.
Wu, C. K., Dailey, H. A., Rose, J. P., Burden, A., Sellers, V. M. & Wang, B. C. (2001). The 2.0 Å structure of human ferrochelatase, the terminal enzyme of heme biosynthesis. Nat Struct Biol 8, 156–160.[CrossRef][Medline]
Zouni, A., Witt, H. T., Kern, J., Fromme, P., Krauss, N., Saenger, W. & Orth, P. (2001). Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature 409, 739–743.[CrossRef][Medline]
Received 20 March 2008;
revised 7 August 2008;
accepted 19 August 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
