From the
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom, and the ¶Department of Biochemistry, University of Utah, Salt Lake City, Utah 84132
Received for publication, March 11, 2003 , and in revised form, April 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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The tetrapyrrole biosynthetic chelatases fall into three broad classes, which vary markedly in their size and energy requirements (Fig. 1) (5). The class I chelatases require three subunits for activity and utilize ATP. Examples include the magnesium chelatase in chlorophyll and bacteriochlorophyll synthesis (3), which is constituted by ChlH, I, and D, and the cobaltochelatase found in bacteria that make cobalamin along the cobaltlate pathway and requires CobN, S, and T for chelatase activity (6). There is obvious sequence similarity between ChlH and CobN, which are thought to be the main tetrapyrrole binding subunits, but much less similarity between ChlI and D and CobS and T. A crystal structure for ChlI has recently been determined, revealing that it belongs to the chaperone-like (AAA) family of ATPases (7), with a novel arrangement of domains. However, it is not yet clear how the I and D subunits interact with the H subunit to promote catalysis or why ATP is required.
The class II chelatases tend to exist as either monomers or homodimers and do not require ATP for activity (Fig. 1). This group includes the protoporphyrin ferrochelatases (HemH) of heme synthesis (8) and the cobaltochelatase associated with the cobalt-early path for cobalamin biosynthesis, CbiK (9). The structures of HemH and CbiK have been determined by x-ray crystallography and have revealed a high level of structural similarity, indicating that the proteins have probably arisen by divergent evolution from a common ancestor (9). This structural similarity is only reflected in an approximate 10% sequence identity.
The third class of chelatase includes CysG and Met8p, which are multifunctional proteins associated with siroheme biosynthesis (Fig. 1) (5, 10). As with the class II chelatases, these proteins are homodimers and do not require ATP for activity. However, they are not structurally similar to HemH or CbiK, and it is likely that they have arisen by the acquisition of a chelatase function within a dehydrogenase catalytic framework (5).
More recently we have identified other chelatases from Bacillus megaterium that we believe belong to the class II chelatases, which are associated with cobalamin (CbiX) and siroheme (SirB) biosynthesis (Fig. 1) (11, 12). CbiX, which contains 306 amino acids, and SirB, which contains 266 amino acids, share about 60% similarity with each other in terms of primary structure. The reason why CbiX is larger than SirB is that it contains a histidine-rich region at its C terminus (12), a region that may be important for metal delivery and/or storage and which may also contain an Fe-S center (13). Although CbiX and SirB are homologous, that is they are derived from a common ancestor, they only appear to share low sequence similarity with CbiK or HemH (<15%). Thus, the SirB/CbiX family may represent a new division of the class II chelatases.
In methanogens the major tetrapyrrole derivatives are coenzyme F430 and cobalamin (14). Only some methanogens appear to have heme, there are few reports of siroheme, and they are unable to make bacteriochlorophyll. Indeed, within their genome it is only possible to identify the genes for the biosynthesis of uroporphyrinogen III and its transformation into cobalamin, because the biosynthetic genes for coenzyme F430 are unknown. Among these it is possible to identify a number of chelatase genes, one that encodes a protein with similarity to CbiX and several others with a high level of identity to the cobalt-late cobaltochelatase, CobN. However, because methanogens do not operate the cobalt-late cobalamin biosynthetic pathway (15), it has been suggested that the CobN homologue may in fact represent the nickel chelatase associated with coenzyme F430 (3). In this study, we have cloned the cbiX gene from two methanogens, overproduced CbiX as a recombinant protein, and characterized its inherent enzymatic activity to demonstrate that in methanogens CbiX represents a compact cobalt chelatase. We also highlight how this small CbiX may represent an ancestral chelatase and show that the more complex structures seen in CbiK and HemH may have arisen from this enzyme design.
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EXPERIMENTAL PROCEDURES |
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To allow the coordinated expression of cobA and cbiX, the M. barkeri and M. thermoautotrophicum cbiX genes were also subcloned into a pETac derivative, downstream of the M. barkeri cobA. The pETac plasmid is a modified pET14b in which the T7 promoter has been substituted with a Ptac promoter, allowing the expression in E. coli strain 302 a. This is an E. coli cysG mutant that is unable to produce siroheme and, therefore, unable to synthesize cysteine. The cysteine auxotrophy was investigated by growth on minimal media as described previously (16).
Protein PurificationBriefly the M. barkeri and M. thermoautotrophicum CbiXs were purified from bacterial lysates using metal chelate affinity chromatography, according to the Novagen pET manual. After application of the crude cell lysates to the metal chelate resin, the column was washed with buffer (20 mM Tris-HCl, pH 7.9) containing 100 mM imidazole and 0.5 M NaCl. The bound CbiX was eluted in buffer containing 400 mM imidazole and 0.5 M NaCl. Protein-containing fractions were detected by a combination of the Bio-Rad protein assay and SDS-polyacrylamide gel electrophoresis. Fractions containing the His-tagged protein were pooled and desalted by passage through a PD-10 column that had previously been equilibrated in 50 mM Tris-HCl, pH 8.0, containing 100 mM NaCl. The M. barkeri and M. thermoautotrophicum CbiX proteins were further purified by gel filtration on a Superdex 75 HR column (Amersham Biosciences) that had previously been equilibrated in 50 mM Tris-HCl buffer, pH 8.0, containing 100 mM NaCl.
Chelatase AssaysPrecorrin-2 and sirohydrochlorin were generated as described previously (5) from porphobilinogen using an enzyme mixture containing porphobilinogen deaminase, uroporphyrinogen III synthase, uroporphyrinogen III methyltransferase, and for sirohydrochlorin synthesis, precorrin-2 dehydrogenase. CbiX activity was measured with sirohydrochlorin (2.5 µM), Co2+ (20 µM) and between 5 and 50 µg of CbiX in a 1-ml reaction volume in 50 mM Tris-HCl, pH 8. Assays were performed in duplicate, and initial rates were recorded on a Hewlett Packard 8452A photodiode array spectrophotometer at 37 °C.
Data Bases and Computer ProgramsSequences, alignments, and comparisons were performed with the GCG software package (Genetics Computer Group, Inc., Madison, WI).
Circular Dichroism SpectraPurified protein (>95% purity) was dialyzed against 5 mM sodium phosphate, pH 7.9, and concentrated in a Vivaspin 6-ml concentrator (VivaScience). Circular dichroism (CD)1 spectra were recorded at 25 °C on 0.1 mg/ml samples of protein in a 1-mm quartz cuvette (Hellma) using an Aviv 62DS spectrometer with data acquired between 180260 nm at 0.5 nm intervals. Deconvolution was carried out using Circular Dichroism Neural Network software (17).
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RESULTS |
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The M. barkeri cbiXS was cloned into pET14b to allow its recombinant expression as a His-tagged protein, thereby facilitating its purification. Because the M. thermoautotrophicum cbiXS encodes a protein with a naturally occurring histidine-rich region, which is likely to allow purification by metal chelate chromatography, the M. thermoautotrophicum cbiX was cloned into pET3a. The genes were also subcloned into a pETac derivative (pETac-CobA), downstream of the M. thermoautotrophicum cobA. This cloning procedure allows the encoded CbiXS proteins to be tested for their ability to act as ferrochelatases in siroheme biosynthesis through the complementation of a defined E. coli cysG strain (16). All the amplified genes were fully sequenced to ensure that no PCR errors had been incorporated.
Protein ExpressionBoth the M. barkeri and M. thermoautotrophicumCbiXS proteins were overproduced as soluble recombinant proteins in E. coli and could be clearly seen on SDS-PAGE gels after electrophoresis of whole cell extracts. The M. thermoautotrophicum CbiXS with its natural histidine-rich region was purified by metal chelate chromatography as described under "Experimental Procedures," using the same procedures as for the pET14b-engineered M. barkeri CbiXS. This procedure yielded protein that was greater than 90% homogeneous as judged by SDS-PAGE (Fig. 3). The M. barkeri CbiXS migrated on an SDS gel with a molecular mass of 15 kDa, in close agreement to its gene-predicted molecular mass of 14 kDa plus 2 kDa for the N-terminal His-tag extension. The M. thermoautotrophicum CbiXS migrated with a molecular mass of 17 kDa, which is somewhat larger than its gene-predicted molecular mass of 14 kDa. This larger molecular mass may reflect the positioning of the histidine-rich region within the protein, which may give the protein unusual mobility on denaturing gels. The observation that the M. thermoautotrophicum CbiXS could be purified on a metal chelate column indicates that the histidine-rich region of the M. thermoautotrophicum CbiXS is capable of binding metal.
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The native aggregation state of the methanogenic CbiXS proteins was investigated using gel-filtration chromatography. This revealed that both CbiXS proteins eluted at a point corresponding to a molecular mass of 40 kDa, suggesting that the proteins exist either as homodimers or homotrimers.
CbiXS ActivityBoth CbiXS proteins were found to be capable of catalyzing the insertion of cobalt into sirohydrochlorin, yielding cobalt-sirohydrochlorin. A typical reaction profile is shown in Fig. 4a. The reaction can be monitored by the loss of absorption at 376 nm and a relative corresponding gain in absorption at 414 nm (Fig. 4b) (5). The M. Barkeri CbiXS was found to have a specific activity of 122 nmol of product formed/min/mg of protein, whereas the M. thermoautotrophicum CbiXS had a much lower activity of 18 nmol/min/mg. The CbiXS proteins from M. barkeri and M. thermoautotrophicum were not found to chelate cobalt into precorrin-2, suggesting that the cobalamin biosynthetic pathway proceeds via cobalt-sirohydrochlorin.
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The CbiXS proteins were also tested for their ability to chelate nickel. Only the M. thermoautotrophicum CbiXS was found to be able to chelate nickel but with a specific activity substantially lower than that observed for cobalt (6 nmol/min/mg). No activity was observed with M. barkeri CbiXS. Moreover, in competition experiments when the M. thermoautotrophicum CbiXS was challenged in an incubation with equal concentrations of cobalt and nickel, cobalt was found to be chelated first. These experiments would suggest that cobalt is the preferred substrate for this chelatase, and it is likely that CbiXS is a cobaltochelatase associated with cobalamin biosynthesis.
CbiXS Can Act as a Ferrochelatase in the Biosynthesis of Siroheme in VivoCloning the M. thermoautotrophicum cobA into pETac yielded pETac-cobA. Subsequently, both the cbiXS from M. thermoautotrophicum and the M. barkeri were cloned individually downstream of cobA, to give plasmids pETac-cobA-MBcbiXS and pETac-cobA-MTcbiXS. To ensure that both proteins were being overproduced from this plasmid, the strains harboring these plasmids were probed with antibodies against the His tag. Blots clearly demonstrated immunoreactivity against 30 and 15 kDa proteins, indicating that the CobA and CbiXS proteins were being expressed (data not shown). Moreover, both plasmids (pETac-cobA-MBcbiXS and pETac-cobA-MTcbiXS) were able to complement efficiently the cysG deficiency of the E. coli 302 a strain (Table III) (18). These results demonstrate that the M. thermoautotrophicum CobA is active as a uroporphyrinogen III methyltransferase and that both CbiXS proteins are able to function as sirohydrochlorin ferrochelatases. However, the addition of exogenous cobalt to the minimal growth medium onto which the E. coli cysG transformants were plated prevented this complementation (Table III), suggesting that the ferrochelatase activity is inhibited by cobalt. Such an observation would be consistent with CbiXS being a cobalt chelatase. A similar cobalt-dependent inhibition had been observed after the Salmonella enterica cbiK was used to complement this E. coli auxotrophy (16, 19). When exogenous nickel was added to the minimal growth medium, no metal-dependent inhibition of complementation was observed with the E. coli cysG transformants (Table III). Thus, nickel would not appear to compete competitively with ferrous iron for insertion into sirohydrochlorin with CbiXS.
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In vitro experiments with the M. barkeri and M. thermoautotrophicum CbiXS proteins revealed that they were able to insert ferrous iron into sirohydrochlorin to yield siroheme. However, this was at a rate of less than 10% of that observed for the chelation of cobalt. Moreover, when equal concentrations of ferrous iron and cobalt were presented to the enzyme in an assay mix, cobalt-sirohydrochlorin was always formed preferentially to siroheme (data not shown).
Structural Comparison of CbiXS to Other Chelatases by CD AnalysisNo structural information is yet available for any of the SirB/CbiX family of enzymes, so it is not known whether they have any topological similarity to CbiK or HemH or whether they represent a new division of the class II chelatases. To determine whether the CbiXS proteins have any secondary structure similarity to CbiK, CD spectra of the CbiXS, CbiXL, and SirB proteins were recorded and compared with two CbiK proteins.
The CD spectra of the two methanogenic CbiXS proteins are highly similar, consistent with the proteins containing a large amount (70%) of
-helical secondary structure but still with some
-sheet (814%) (Fig. 5). The B. megaterium SirB gave a spectrum consistent with it containing about 60%
-helical secondary structure and 12%
-sheet (Fig. 5). Similarly, the CbiK proteins from S. enterica (16) and Porphyromonas gingivalis (20) gave spectra equivalent of proteins containing between 4046%
-helix and 1519%
-sheet, consistent with the known three-dimensional structure of CbiK (Fig. 5). Thus the SirB/CbiX family of proteins contains a mixed
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architecture, consistent with that found in CbiK.
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The secondary structure predictions of the CbiXS and CbiXL suggest that these enzymes adopt an alternating helix-strand-helix topology (21). In CbiK, the conserved active site histidine His-207 lies on a surface loop between the first -strand and
-helix of the C-terminal domain. This same structural topology is predicted for the conserved histidine-glycine sequences of B. megaterium CbiXL and the M. barkeri CbiXS. Additional support for the structural alignment with CbiK comes from threading data that align all the CbiX sequences with various members of the class II chelatase structures with a high degree of confidence (22).
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DISCUSSION |
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The obvious broad specificity of these CbiXS chelatases in being able to chelate Fe2+, Co2+ and, in the case of the M. thermoautotrophicum CbiXS, Ni2+ raises interesting questions about how the correct metal ion is delivered to the chelatase in its host environment. Whether there are specific metal ion chaperones or whether the chelatases interact directly with the metal transporters is not known. The presence of a histidinerich region on the M. thermoautotrophicum CbiXS and the observation that this protein binds to a metal chelate column, suggest that this histidine-rich region may play a role in either metal storage or presentation. Such histidine-rich regions are found in a number of CbiXS and CbiXL proteins, although not all, inferring that different organisms may have evolved alternative methods for metal ion storage and/or delivery.
Nonetheless, by identifying CbiXS as the cobalt chelatase in the archaeal organisms, this would suggest that the proteins with similarity to CobN and ChlH, which are also found in these genomes, are likely to be the nickel chelatases associated with coenzyme F430 synthesis. However, it is interesting to note that the CobN and ChlH-like sequences are also found in the genome of Halobacterium, which is a non-methanogenic archaeal member. Moreover, methanogens appear to contain multiple copies of these CobN- and ChlH-like proteins, indicating that there is still much to be learned about the role these proteins play in metabolism.
CbiXS Is Structurally Related to CbiXL, SirB, and CbiK The CD spectra of the CbiXS proteins reveal that they have a mixed /
structure, with a high
-helix content compared with that observed in the CD spectra of CbiXL, SirB, and CbiK. Nonetheless, all the proteins have a similar
-sheet content; thus the difference in CD spectra between CbiXS and the other larger proteins may be because of a more compact structure with greater order in the loop regions, such that they adopt a helical structure. Moreover, a pileup of CbiXS, CbiXL, and SirB sequences reveals a number of highly conserved residues, including two histidines (at positions 12 and 78 of the M. barkeri CbiXS), an aspartate/histidine (at position 82 of the M. barkeri CbiXS), and a number of prolines, glycines and leucines (Fig. 2). Interestingly, the same ionizable residues are found at the active site of CbiK, where the two histidines are thought to act as general bases in abstracting protons from the substrate and also in binding the metal ion. Indeed, it is possible to force the active site primary structure of CbiK into the CbiX/SirB pileup (Fig. 2). What is clear from this alignment is that the postulated active site histidines are found in the N-terminal region of CbiXL, whereas the equivalent active site histidines are located in the C-terminal region of CbiK. On the basis of the common level of
-sheet in these proteins and the conservation of known catalytic groups, we would predict that CbiXS, CbiXL, SirB, and CbiK belong to the same evolutionary related family of enzymes. Furthermore, because HemH is structurally related to CbiK, it should also be included in this family. Indeed, threading programs all predicted that CbiXS would adopt the fold of the CbiK and HemH. These proteins would all belong to the type II chelatases, and we suggest that the CbiXS proteins be assigned to type IIa, CbiXL and SirB to type IIb, and CbiK and HemH to type IIc (Table I).
Evolution of Class II ChelatasesWhen the B. megaterium CbiXL was blasted against the data bases, it was observed that the CbiXS proteins were being aligned against both the N-terminal (amino acids 1128) and C-terminal (amino acids 128260) regions of CbiXL (Fig. 6). This suggests that CbiXL represents the fusion of two CbiXS proteins, a process that may have happened by gene duplication. Indeed, it is possible to split, for example, the B. megaterium CbiXL into two halves and to align them against each other and against a CbiXS sequence. What is observed is that the N-terminal region of CbiXL contains the two important catalytic histidine residues as well as a number of other charged groups, whereas the C-terminal region of CbiXL contains mainly conserved proline and glycine amino acids (Fig. 6), presumably reflecting their importance in maintaining the protein fold. We can therefore envisage that CbiXL evolved from two CbiXS fused together and that the N-terminal domain of the fused protein subsequently maintained the catalytic groups, whereas the C-terminal domain lost these groups because this domain contributed more toward overall protein stability and/or control. Once this prototype CbiXS-dimer fusion appeared, it evolved into CbiXL through the addition of a MXCXXC peptide containing a histidine-rich region, whereas a SirB-type chelatase evolved through selection of a variant capable of accepting Fe and being able to function as a sirohydrochlorin ferrochelatase.
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An extension of this theory can also explain the appearance of CbiK and HemH, because these proteins are composed of two /
domains that are related to one another by a pseudo 2-fold symmetry. These enzymes may also have evolved from a CbiX-dimer fusion. However, in this case, the C-terminal domain maintained the catalytic functional groups, whereas the N-terminal domain lost these residues as it evolved to help maintain the protein function through stability and/or control. This protein then evolved further into a sirohydrochlorin ferrochelatase by modification of its metal ion specificity to accept Fe2+, before finally evolving into a protoporphyrin ferrochelatase by changing its tetrapyrrole substrate specificity from sirohydro-chlorin to protoporphyrin.
In this respect, the evolution of the CbiK/HemH or CbiXL/SirB can be explained by the generation of a CbiXS-dimer fusion and then maintenance of the essential catalytic groups in either the N- or C-terminal domain of the final protein. Thus, CbiXS may represent the primordial chelatase design from which these other proteins evolved.
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FOOTNOTES |
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Both authors contributed equally to this work.
|| To whom correspondence should be addressed. Tel.: 00-44-20-7882-7718; Fax: 00-44-20-7882-7609; E-mail: m.j.warren{at}qmul.ac.uk.
1 The abbreviations used are: CD, circular dichroism; CbiXL, long CbiX; CbiXS, short CbiX.
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ACKNOWLEDGMENTS |
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REFERENCES |
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