| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 280, Issue 2, 1086-1094, January 14, 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
¶
From the
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom, the ¶Center for Biological NMR, Department of Chemistry, Texas A & M University, College Station, Texas 77843, and the ||Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom
Received for publication, October 19, 2004 , and in revised form, November 1, 2004.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-lactone derivative of precorrin-3 (6).
|
-lactone attached to ring A and a hydroxy group at C-20 (6, 8). Further oxygen labeling experiments proved conclusively that the latter was derived from molecular oxygen and that CobG was therefore a monooxygenase (6). Despite its discovery more than a decade ago, comparatively little is known about the mechanism of CobG.
However, in Rhodobacter capsulatus and a number of 
-proteobacteria, genome sequencing projects have revealed the presence of cobalamin pathways that appear aerobic in character, but are missing an orthologue of cobG. In this project we sought to try and overproduce the enzymes found in the main R. capsulatus cobalamin operon (9) in Escherichia coli to genetically engineer a strain with a capacity to synthesize hydrogenobyrinic acid (HBA) in vivo and thereby identify the protein required for initiating the ring contraction process.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Engineering E. coli for the Production of HBA
For the production of HBA, E. coli strain BL21(DE3) was transformed with the two compatible plasmids pKK223–3:RccobHIKLEM-JAForf663 and pBAD33:PdecobAI. Plasmid pKK223–3:RccobHIKLEMJAForf663 was constructed by inserting the appropriate amplified R. capsulatus genes into pKK223-3 (ColE1 ori, lacPO, AmpR) using the "Link and Lock" method, which allows the consecutive cloning of genes with the reuse of the same restriction enzyme sites (see Fig. 2). The first gene to be cloned is amplified with primers containing an EcoRI at the 5' and SpeI and BamHI sites at the 3' end. The remaining genes are amplified with primers containing an XbaI at the 5' end and SpeI and BamHI sites on the 3' end. After cloning the first gene into the plasmid via the EcoRI and BamHI sites, the remaining genes, cut with XbaI and BamHI restriction enzymes, were cloned consecutively into the plasmid after it had been restricted with SpeI and BamHI. The SpeI- and XbaI-restricted fragments form compatible cohesive ends, which after ligation do not reform a restriction site. Thus by fusing the SpeI and XbaI sites, these sites can be reused in subsequent cloning steps (so long as the sites do not occur naturally within the gene).
|
Cell Growth and Isolation of HBA
E. coli strains were grown with aeration at 37 °C in LB medium containing 0.2% glucose plus the appropriate antibiotics (50 mg/liter ampicillin, 30 mg/liter chloramphenicol, 50 mg/liter kanamycin) to an A600 = 1 and the cells collected by centrifugation. The combined cell pellets from 4 liters of Luria-Bertani (LB) broth were resuspended in 1 liter of minimal medium consisting of M9 salts containing the same antibiotics plus the following additives: 0.2% glycerol, 0.2% L-(+)-arabinose, 0.01% yeast extract, 2.0 mM MgCl2, 0.1 mM CaCl2, 20 mg of 5-aminolevulinic acid, and 0.2 mM isopropyl 
-D-thiogalactopyranoside. The cells in minimal medium were incubated with aeration at 37 °C for 24–48 h. The products found in both the culture medium and in the heated (80 °C for 15 min) cell lysate were isolated by binding them to DEAE-Sephadex. HBA was esterified and purified by TLC as previously described (13). Alternatively, HBA was purified in its free acid form and quantified by high performance liquid chromatography on a BDS Hypersil C-18 column (4.6 x 250 mm; Thermo Electron Corporation) run on a Agilent 1100 series LC/MSD Trap equipped with a diode array detector and eluted at a flow rate of 1 ml min–1 with a gradient of acetonitrile in 1 M ammonium acetate.
Protein Purification
E. coli cells transformed with the plasmid expressing the R. capsulatus Orf663 were grown overnight on LB medium, and were harvested and concentrated by centrifugation for 15 min at 5000 x g in a refrigerated Sorvall RC-5 centrifuge. The pellet was washed with 10 volumes of a 100 mM Tris-HCl buffer, pH 7.5, 2 M NaCl and then concentrated by a second centrifugation run at 5000 x g for 15 min in a 100 mM Tris-HCl, pH 7.5, buffer, supplemented with a mixture of protease inhibitors (Sigma).
The cell pellet was broken by four passages through a French press operating at 12,000 psi, under an argon atmosphere, to obtain a homogenous cell lysate. The unbroken material was removed by centrifugation for 10 min at 5,000 x g. The pellet was kept. The supernatant was subjected to a further centrifugation at 88,000 x g for 1 h in a Sorvall Combi-plus ultracentrifuge. The supernatant was yellow in color and contained the soluble flavin-binding fragment of the Orf663. The flavin-binding fragment of the protein was further purified and concentrated by affinity chromatography on a His-bind column.
The pellet, dark-red in color, contained the full-length form of Orf663. This was salt-washed in 100 mM Tris-HCl, pH 7.5, 2 M NaCl buffer, and then centrifuged at 88,000 x g for 1 h. The supernatant was discarded and the pellet was resuspended and homogenized in a 100 mM Hepes-NaOH buffer, pH 7.5. The membrane fraction was then solubilized by the mild detergent n-dodecyl -D-maltoside for 30 min with continuous gentle stirring. The final detergent concentration was 1% (w/v) and the protein concentration was adjusted to 4 mg/ml after measuring the absorption at 280 nm.
The material that had not been solubilized by the detergent treatment was removed by centrifugation for 1 h at 88,000 x g. The full-length form of Orf663 was then purified from the cleared supernatant by affinity chromatography on a His-bind column. The protein was eluted in a buffer containing 200 mM imidazole, 0.003% n-dodecyl -D-maltoside. The protein was concentrated and the excess imidazole removed by dialysis. The samples for spectroscopic analysis were prepared after concentration.
Spectroscopic Measurements
Electron Paramagnetic Resonance—The continuous wave EPR spectra were recorded on a Bruker Alexis E580 spectrometer operating at X-band (9.7 MHz, 300 millitesla) equipped with an Oxford Helium flow cryostat. The precise experimental conditions are given in the figure legends. The temperature is controlled by an Oxford ITC unit. The samples for EPR measurements were either frozen in liquid nitrogen immediately after the preparation (oxidized), or incubated for 30 min in the presence of the reductant sodium dithionite (11.5 mM) under anaerobic conditions, and stored in liquid nitrogen.
UV-Visible Spectroscopy—The optical spectra were either recorded on a Hitachi (Model U-3310) spectrophotometer equipped with an attachment for the measurement of highly scattering solutions. The samples were kept under a gentle argon stream to maintain the anaerobic conditions.
Redox Potentiometry
Redox titrations were performed in a Belle Technology glove box under a nitrogen atmosphere, essentially as described previously (14). All solutions were degassed under vacuum with argon. Oxygen levels were maintained at less than 2 ppm. The protein solution (
5 µM) in 8 ml of 100 mM potassium phosphate, pH 7.0, containing 10% (v/v) glycerol, was titrated electrochemically according to the method of Dutton (15) using sodium dithionite as reductant and potassium ferricyanide as oxidant. Absorption versus potential data at appropriate wavelengths (reflecting maximal changes in spectral properties between oxidized and reduced enzyme) were fitted to the Nernst equation to derive the apparent midpoint reduction potential (E) for the 1 electron reduction of the bound heme iron.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
To determine whether Orf663 is involved in corrin synthesis, 10 R. capsulatus genes thought to be required for HBA synthesis from uroporphyrinogen III were introduced into pKK233.3 using a newly developed multicloning procedure that we have termed Link and Lock (Fig. 2) because genes can be linked together and locked in position with the use of a minimal number of restriction enzymes. The enzymes required to convert 5-aminolevulinic acid to uroporphyrinogen III are provided by the host, and the large plasmid was transformed into an E. coli strain harboring another plasmid bearing the cobA and cobI genes necessary to convert uroporphyrinogen III to precorrin-3A to provide sufficient amounts of this intermediate and to facilitate high flux through the pathway (10). The resulting strain was found to produce comparatively large quantities of HBA, the first intermediate on the pathway to cobalamin that is stable and can be easily isolated (17). HBA was isolated both as a free acid and in its esterified form and its structure confirmed on the basis of its UV-visible, mass, and NMR spectra (Fig. 3).
|
Having an E. coli strain with the ability to make HBA also allowed us to address the question of whether the R. capsulatus Orf663 was required for HBA synthesis. Deleting orf663 resulted in a strain that no longer accumulated HBA. Significantly, HBA synthesis could be restored either by reintroduction of orf663 or by addition of the P. denitrificans cobG gene. Thus, cobG and orf663 encode enzymes that are isofunctional. On the basis that the R. capuslatus Orf663 is a cobalamin biosynthetic enzyme and that it is quite distinct from the P. denitrificans CobG (see below), we propose that Orf663 should be called CobZ.
The protein encoded by cobZ (orf663) is therefore able to catalyze a reaction similar to that enhanced by CobG, i.e. it has the ability to generate a hydroxylactone derivative of precorrin-3A (Fig. 1) (6). Thus CobG and CobZ are alternative precorrin-3B synthases. However, CobZ (Orf663) must mediate this reaction in a manner very different from that employed by CobG, because the two proteins display no primary sequence similarity. Moreover, CobZ (866 amino acids) is considerably larger than CobG (459 amino acids).
A detailed bioinformatics analysis reveals that CobZ is composed of two distinct moieties, an N-terminal flavin-binding region that displays substantial similarity to succinate dehydrogenase/fumarate reductase and a C-terminal region that is an integral membrane protein with similarity to proteins of unknown function (originally called CitB but now called TcuB-see below (20)). At the junction between these two domains is a cysteine-rich region that has the same consensus motif for a 2[4Fe-4S] cluster as found in a number of complex redox proteins, such as heterodisulfide reductase.
In some organisms, CobZ displays similarity to two separate proteins that are found as part of an operon recently identified as being involved in the metabolism of tricarballylate (20), a compound that is catabolized by some mircroorganisms as a source of carbon and energy (21). The tricarballylate utilization operon (tcu) contains three genes, tcuA, -B, and -C, where tcuA and tcuB align with the N and C termini of CobZ (20). Thus, TcuA is predicted to contain a flavin, whereas TcuB is predicted to be an integral membrane protein with several Fe-S centers. A detailed molecular genetics study of this operon provides convincing evidence that TcuA and -B are involved in the metabolism of tricarballylate, possibly via its oxidation to cis-aconitate (20). In this respect, TcuA and TcuB have been suggested to function in the dehydrogenation of tricarballylate.
To progress from an in silico predictive model of CobZ to experimental in vitro reality, cobZ was cloned to allow overproduction of CobZ as a recombinant protein in E. coli with an N-terminal His tag. Cells overproducing CobZ appeared to accumulate two proteins, one of the expected mass of 90 kDa and another of 50 kDa. Subsequent analysis revealed that the 50-kDa protein was a proteolytic fragment resulting from the cleavage of the 90-kDa protein product. This proteolysis is consistent with CobZ consisting of two distinct moieties, where the junction between them represents a region that is susceptible to cleavage.
The N-terminal proteolytic fragment was purified and shown to house a flavin molecule (Fig. 4A). Although the yellow coloration and UV-visible spectrum of this region of CobZ are typical of a flavin cofactor, it is not feasible to identify the particular type of flavin bound by this information alone. However, FMN has a 10-fold higher fluorescence at 600 nm than FAD thus allowing the flavin nucleotide to be differentiated by fluorescence spectroscopy. Moreover, FAD can be broken down into its FMN and AMP moieties, which therefore results in a 10-fold increase in the fluorescence (22). A purified sample of the CobZ N-terminal peptide was treated with trifluoroacetic acid and, once the precipitated protein had been removed by centrifugation, half was neutralized and its fluorescence spectrum measured at 600 nm. The other half of the sample was left to hydrolyze any FAD into its FMN and AMP components over a 4-h period, neutralized, and its fluorescence also measured. A comparison of the spectral readings revealed that the fluorescence had increased 10-fold. This result demonstrates that the flavin component of CobZ is FAD and also indicates that it is unlikely to be covalently bound to the protein.
|
2 mg/ml in buffer containing 200 mM imidazole and was titrated at 4 °C against an increasing concentration of sodium dithionite under anaerobic conditions. The titration against dithionite revealed that the flavin was reduced directly to the 2 electron reduced hydroquinone form, which can be deduced from the bleaching of the flavin chromophore with no evidence of any semiquinone formation during the reduction (Fig. 4B). A neutral or blue semiquinone would have been observed by the appearance of a band at 580–620 nm, whereas the anionic red semiquinone would have been identified by strong absorption features in the region around 380–400 nm. Moreover, the flavin can be returned to its fully oxidized state by addition of oxygen (data not shown) without any noticeable accumulation of semiquinone signal. This result is consistent with the flavin being involved in a 2-electron reduction process rather than two single electron steps, although we cannot rule out the possibility that semiquinone form(s) are transiently populated at this stage. The truncated flavin module of the protein was also incubated with NADH, to see if this could act as a reductant. Many flavoproteins are reduced by NADH or NADPH as part of their catalytic cycle (e.g. cytochrome P450 reductase (23)). However, incubation of the N-terminal truncated version of CobZ with NADH did not lead to any noticeable reduction of the flavin as monitored by UV-visible spectroscopy. The N-terminal region of CobZ would therefore not appear to interact directly with NADH (or else be oxidized much faster than it is reduced by NADH), so it probably operates another method for the reduction of the flavin.
The insolubility of CobZ is likely because of the fact that the C-terminal region is an integral membrane protein, as hydropathy plots predict that CobZ has at least six hydrophobic regions, all located within the C-terminal region. This would also be consistent with observations that the N terminus is cleaved, yielding a soluble 50-kDa fragment, whereas the rest (C-terminal) remains in the membrane. To test the hypothesis that CobZ may be a membrane protein, an extraction with detergent was undertaken. The extraction of CobZ was achieved with n-dodecyl -D-maltoside and the protein was passed through a Ni2+-affinity column to improve purity, because the intact protein was overproduced as an N-terminal His-tagged fusion. About 1–2 mg of purified CobZ could be obtained per liter of bacterial culture, which had been induced for a short 3-h period, to minimize degradation.
The purified CobZ was analyzed by UV-visible spectroscopy (Fig. 4A). The color of the protein appeared more orange/red than the bright yellow of the purified 50-kDa N-terminal region. The difference in color was presumed to be because of the Fe-S clusters. However, the UV-visible spectrum of CobZ revealed a very strong absorbance around 414 nm, consistent with the presence of an oxidized heme group. Reduction of the protein with dithionite resulted in a long wavelength shift of the Soret band from 414 to 425 nm, together with a considerable increase in the intensity of the band absorption at 560 nm (Fig. 5). The EPR spectrum of the oxidized form of CobZ also shows the presence of a cytochrome in the low spin state (data not shown). The measured g values of gz = 2.94, gy = 2.25, and gx = 1.50 are consistent with a low spin heme and suggest a bis-His coordination (24). However, we cannot rule out the possibility that the low spin state may be an artifact of the purification as the protein was purified in buffer containing imidazole. Nonetheless, extensive dialysis and gel filtration of the protein did not significantly alter the spectral characteristics of the heme, suggesting that the imidazole does not remain associated as a ligand. Significantly, the data demonstrate beyond all doubt that CobZ contains a heme group and that CobZ is therefore a novel flavin/heme-containing protein. Moreover, the heme group must be contained within the C terminus of the protein. The lack of any CXXCH motifs within this region, together with the absorption characteristic, suggests that it is not a c-type cytochrome and is more likely to harbor a b-type heme.
|
-band of the heme at 560 nm in the ferrous state (Fig. 5). An isosbestic point for the Fe3+/Fe2+ Soret transition is at 419 nm. Data at the reduced Soret maximum (425 nm) were fitted to the Nernst function to produce a midpoint reduction potential of E = –123 ± 3 mV for the Fe3+/Fe2+ transition. Data fitting at longer wavelength is more complex because of likely large overlapping contributions from the different bound cofactors. However, it is notable that similar analysis of the data at 475 nm (where significant contributions from flavin and Fe-S centers are expected) suggests a more negative potential (about –145 mV). This estimate is undoubtedly a composite value from the contributions of the heme and the other cofactors (some of which may not be bound stoichiometrically). However, it does indicate that one or both of the other cofactors have a rather more negative reduction potential than the heme iron. Work is ongoing to resolve the potentials of the other centers in CobZ. The EPR spectrum of the reduced form of CobZ produced evidence for the presence of two [4Fe-4S] centers. The complex EPR spectrum of the dithionite-reduced CobZ is presented in Fig. 6, as detected at 15 K. The features at gz and gx shows a clear band splitting with peaks at gz1 = 2.12, gz2 = 2.08, and gx1 = 1.93, gx2 = 1.91 and g = 1.89. The EPR signals are completely suppressed at 70 K, an indication of fast magnetic relaxation, typical of metal centers, and the measured value of g are in the range reported for [4Fe-4S] clusters. Given the marked temperature dependence, the observed EPR spectrum can be safely assigned to a [4Fe-4S] cluster (25, 26). The fact that the transitions assignable to the gz and gx are each split into two closely lying peaks indicates the presence of two, interacting, Fe-S clusters (27). A similar behavior has been previously observed for proteins binding more than one [4Fe-4S] cluster, such as some ferredoxins, and the iron-sulfur center FA and FB bound to the PsaC subunit of Photosystem I (28, 29).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
Finally, the presence of heme within a cobalamin biosynthetic enzyme extends a web of intrigue concerning tetrapyrrole inter-relationships within R. capsulatus. It is known that cobalamin is required for bacteriochlorophyll synthesis (33). As R. capsulatus contains a metH, cobalamin is required for methionine and, thus, S-adenosylmethionine synthesis (34), which in turn, is needed for heme synthesis (35). Now, it is apparent that heme is required for cobalamin biosynthesis. Presumably, there is tight co-ordination of modified tetrapyrrole biosynthesis within this organism, but this unique interdependence may represent an early signaling mechanism that is still apparent in higher plants where intermediates in chlorophyll synthesis are involved in plastid to nucleus communication (36).
|
FOOTNOTES |
|---|
Both authors contributed equally to this work.
** Supported by the European Community through the X-TB program.
Supported by a Royal Society Leverhulme Trust Senior Research Fellowship.
To whom correspondence should be addressed. Tel.: 020-7882-7718; Fax: 020-7882-7609; E-mail: m.j.warren{at}qmul.ac.uk.
1 The abbreviations used are: CobG, precorrin-3B synthase; CobZ, gene product of orf663 and an enzyme isofunctional protein to CobG, but containing flavin and heme group as well as Fe-S centers; HBA, hydrogenobyrinic acid.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. A. Lewis and J. C. Escalante-Semerena The FAD-Dependent Tricarballylate Dehydrogenase (TcuA) Enzyme of Salmonella enterica Converts Tricarballylate into cis-Aconitate. J. Bacteriol., August 1, 2006; 188(15): 5479 - 5486. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
