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A Story of Chelatase Evolution

2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês

10.1074/jbc.m302468200

1083-351X

Amanda A. Brindley, Evelyne Raux, Helen K. Leech, Heidi Schubert, Martin J. Warren,

Enzyme Structure and Function

The cobaltochelatase required for the synthesis of vitamin B12 (cobalamin) in the archaeal kingdom has been identified as CbiX through similarity searching with the CbiX from Bacillus megaterium. However, the CbiX proteins in the archaea are much shorter than the CbiX proteins found in eubacteria, typically containing less than half the number of amino acids in their primary structure. For this reason the shorter CbiX proteins have been termed CbiXS and the longer versions CbiXL. The CbiXS proteins from Methanosarcina barkeri and Methanobacter thermoautotrophicum were overproduced in Escherichia coli as recombinant proteins and characterized. Through complementation studies of a defined chelatase-deficient strain of E. coli and by direct in vitro assays the function of CbiXS as a sirohydrochlorin cobaltochelatase has been demonstrated. On the basis of sequence alignments and conserved active site residues we suggest that CbiXS may represent a primordial chelatase, giving rise to larger chelatases such as CbiXL, SirB, CbiK, and HemH through gene duplication and subsequent variation and selection. A classification scheme for chelatases is proposed. The cobaltochelatase required for the synthesis of vitamin B12 (cobalamin) in the archaeal kingdom has been identified as CbiX through similarity searching with the CbiX from Bacillus megaterium. However, the CbiX proteins in the archaea are much shorter than the CbiX proteins found in eubacteria, typically containing less than half the number of amino acids in their primary structure. For this reason the shorter CbiX proteins have been termed CbiXS and the longer versions CbiXL. The CbiXS proteins from Methanosarcina barkeri and Methanobacter thermoautotrophicum were overproduced in Escherichia coli as recombinant proteins and characterized. Through complementation studies of a defined chelatase-deficient strain of E. coli and by direct in vitro assays the function of CbiXS as a sirohydrochlorin cobaltochelatase has been demonstrated. On the basis of sequence alignments and conserved active site residues we suggest that CbiXS may represent a primordial chelatase, giving rise to larger chelatases such as CbiXL, SirB, CbiK, and HemH through gene duplication and subsequent variation and selection. A classification scheme for chelatases is proposed. Modified tetrapyrroles, such as chlorophyll, heme, siroheme, vitamin B12, and coenzyme F430 play diverse roles in a range of essential biological processes, from photosynthesis to methanogenesis (1Battersby A.R. Nat. Prod. Rep. 2000; 17: 507-526Crossref PubMed Scopus (292) Google Scholar, 2Warren M.J. Scott A.I. Trends Biochem. Sci. 1990; 15: 486-491Abstract Full Text PDF PubMed Scopus (97) Google Scholar). Although this group of biological macrocycles are structurally related, a consequence of a branched biosynthetic pathway, they differ considerably in their chemical properties and the nature of their centrally chelated metal ion. Indeed, each branch of the tetrapyrrole biosynthetic pathway has a specific metal ion chelatase that is responsible for ensuring the correct metal ion is inserted (Fig. 1 and Table I) (3Walker C.J. Willows R.D. Biochem. J. 1997; 327: 321-333Crossref PubMed Scopus (183) Google Scholar, 4Raux E. Schubert H.L. Roper J.M. Wilson S.W. Warren M.J. Bioorgan. Chem. 1999; 27: 100-118Crossref Scopus (36) Google Scholar).Table IDifferent types of chelatases involved in modified tetrapyrrole biosynthesisView Large Image Figure ViewerDownload (PPT) Open table in a new tab The tetrapyrrole biosynthetic chelatases fall into three broad classes, which vary markedly in their size and energy requirements (Fig. 1) (5Schubert H.L. Raux E. Brindley A.A. Leech H.K. Wilson K.S. Hill C.P. Warren M.J. EMBO J. 2002; 21: 1-8Crossref PubMed Scopus (52) Google Scholar). The class I chelatases require three subunits for activity and utilize ATP. Examples include the magnesium chelatase in chlorophyll and bacteriochlorophyll synthesis (3Walker C.J. Willows R.D. Biochem. J. 1997; 327: 321-333Crossref PubMed Scopus (183) Google Scholar), 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 (6Debussche L. Couder M. Thibaut D. Cameron B. Crouzet J. Blanche F. J. Bacteriol. 1992; 174: 7445-7451Crossref PubMed Google Scholar). 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 (7Fodje M.N. Hansson A. Hansson M. Olsen J.G. Gough S. Willows R.D. Al-Karadaghi S. J. Mol. Biol. 2001; 311: 111-122Crossref PubMed Scopus (140) Google Scholar), 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 (8Al-Karadaghi S. Hansson M. Nikonov S. Jonsson B. Hederstedt L. Structure. 1997; 5: 1501-1510Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and the cobaltochelatase associated with the cobalt-early path for cobalamin biosynthesis, CbiK (9Schubert H.L. Raux E. Wilson K.S. Warren M.J. Biochemistry. 1999; 38: 10660-10669Crossref PubMed Scopus (90) Google Scholar). 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 (9Schubert H.L. Raux E. Wilson K.S. Warren M.J. Biochemistry. 1999; 38: 10660-10669Crossref PubMed Scopus (90) Google Scholar). 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) (5Schubert H.L. Raux E. Brindley A.A. Leech H.K. Wilson K.S. Hill C.P. Warren M.J. EMBO J. 2002; 21: 1-8Crossref PubMed Scopus (52) Google Scholar, 10Warren M.J. Bolt E.L. Roessner C.A. Scott A.I. Spencer J.B. Woodcock S.C. Biochem. J. 1994; 302: 837-844Crossref PubMed Scopus (84) Google Scholar). 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 (5Schubert H.L. Raux E. Brindley A.A. Leech H.K. Wilson K.S. Hill C.P. Warren M.J. EMBO J. 2002; 21: 1-8Crossref PubMed Scopus (52) Google Scholar). 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) (11Raux E. Lanois A. Rambach A. Warren M.J. Thermes C. Biochem. J. 1998; 335: 167-173Crossref PubMed Scopus (32) Google Scholar, 12Raux E. Leech H.K. Beck R. Schubert H.L. Santander P.J. Roessner C.A. Scott A.I. Martens J.H. Jahn D. Thermes C. Rambach A. Warren M.J. Biochem. J. 2003; 370: 505-516Crossref PubMed Google Scholar). 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 (12Raux E. Leech H.K. Beck R. Schubert H.L. Santander P.J. Roessner C.A. Scott A.I. Martens J.H. Jahn D. Thermes C. Rambach A. Warren M.J. Biochem. J. 2003; 370: 505-516Crossref PubMed Google Scholar), a region that may be important for metal delivery and/or storage and which may also contain an Fe-S center (13Leech H.K. Raux-Deery E. Heathcote P. Warren M.J. Biochem. Soc. Trans. 2002; 30: 610-613Crossref PubMed Google Scholar). 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 ( 95% purity) was dialyzed against 5 mm sodium phosphate, pH 7.9, and concentrated in a Vivaspin 6-ml concentrator (VivaScience). Circular dichroism (CD) 1The abbreviations used are: CD, circular dichroism; CbiXL, long CbiX; CbiXS, short CbiX. 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 180–260 nm at 0.5 nm intervals. Deconvolution was carried out using Circular Dichroism Neural Network software (17Bohm G. Muhr R. Jaenicke R. Protein Eng. 1992; 5: 191-195Crossref PubMed Scopus (1022) Google Scholar). In an attempt to identify the type of chelatase used by the Archaea for cobalamin biosynthesis, we searched for the presence of known cobaltochelatases such as CbiK (16Raux E. Thermes C. Heathcote P. Rambach A. Warren M.J. J. Bacteriol. 1997; 179: 3202-3212Crossref PubMed Google Scholar) and CbiX (12Raux E. Leech H.K. Beck R. Schubert H.L. Santander P.J. Roessner C.A. Scott A.I. Martens J.H. Jahn D. Thermes C. Rambach A. Warren M.J. Biochem. J. 2003; 370: 505-516Crossref PubMed Google Scholar). Data base searching revealed that homologues of the B. megaterium CbiX were present in the genomes of a range of archaeal members. However, these proteins tended to be less than half the size of the B. megaterium CbiX (Fig. 2), typically containing 110–145 amino acids, which is substantially shorter than the 306 amino acids found in the B. megaterium CbiX. To distinguish between the long versions of CbiX, such as that found in B. megaterium, and the shorter ones found in the Archaea, we termed them CbiXL (long) and CbiXS (short). To investigate the function of the CbiXS proteins we selected the cbiXS gene from two Methanogens, M. barkeri and M. thermoautotrophicum whose CbiXS proteins contain 130 and 143 amino acids, respectively (Fig. 2). The genes were amplified from the appropriate genomic DNA using the primers described under “Experimental Procedures.” Although these two genes clearly encode similar proteins, the M. thermoautotrophicum CbiXS also contains a histidine-rich region, similar though quite distinct from the histidine-rich region found in the B. megaterium CbiXL (Fig. 2) (11Raux E. Lanois A. Rambach A. Warren M.J. Thermes C. Biochem. J. 1998; 335: 167-173Crossref PubMed Scopus (32) Google Scholar). 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 (16Raux E. Thermes C. Heathcote P. Rambach A. Warren M.J. J. Bacteriol. 1997; 179: 3202-3212Crossref PubMed Google Scholar). All the amplified genes were fully sequenced to ensure that no PCR errors had been incorporated. Protein Expression—Both 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. 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 Activity—Both 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) (5Schubert H.L. Raux E. Brindley A.A. Leech H.K. Wilson K.S. Hill C.P. Warren M.J. EMBO J. 2002; 21: 1-8Crossref PubMed Scopus (52) Google Scholar). 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. 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 Vivo—Cloning 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 302Δ a strain (Table III) (18Griffiths L.A. Cole J.A. Arch. Microbiol. 1987; 147: 364-369Crossref PubMed Scopus (43) Google Scholar). 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 (16Raux E. Thermes C. Heathcote P. Rambach A. Warren M.J. J. Bacteriol. 1997; 179: 3202-3212Crossref PubMed Google Scholar, 19Raux E. McVeigh T. Peters S.E. Leustek T. Warren M.J. Biochem. J. 1999; 338: 701-708Crossref PubMed Google Scholar). 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.Table IIIComplementation of the E. coli cysG strain 302Δa with a variety of chelatasesPlasmid used to transform the E. coli cysG-deficient strain 302Δ aGrowth on minimal medium supplemented withCysteineNoneCobaltNickelpETac- MTH/cobA+---pkk- Pd/cobA-Pg/cbiK++-+pETac- MTH/cobA-MTH/cbiX++-+pETac-MTH/cobA-MSB/cbiX++-+ Open table in a new tab 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 Analysis—No 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 α-helical secondary structure but still with some β-sheet (8–14%) (Fig. 5). The B. megaterium SirB gave a spectrum consistent with it containing about 60% α-helical secondary structure and 12% β-sheet (Fig. 5). Similarly, the CbiK proteins from S. enterica (16Raux E. Thermes C. Heathcote P. Rambach A. Warren M.J. J. Bacteriol. 1997; 179: 3202-3212Crossref PubMed Google Scholar) and Porphyromonas gingivalis (20Roper J.M. Raux E. Brindley A.A. Schubert H.L. Gharbia S.E. Shah H.N. Warren M.J. J. Biol. Chem. 2000; 275: 40316-40323Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) gave spectra equivalent of proteins containing between 40–46% α-helix and 15–19% β-sheet, consistent with the known three-dimensional structure of CbiK (Fig. 5). Thus the SirB/CbiX family of proteins contains a mixed α/β architecture, consistent with that found in CbiK. The secondary structure predictions of the CbiXS and CbiXL suggest that these enzymes adopt an alternating helix-strand-helix topology (21Pollastri G. Przybylski D. Rost B. Baldi P. Proteins. 2002; 47: 228-235Crossref PubMed Scopus (623) Google Scholar). In CbiK, the conserved active site histidine His-207 lies on a surface loop between the first β-strand and α-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 (22Kelley L.A. MacCallum R.M. Sternberg M.J.E. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1121) Google Scholar). We have overproduced and characterized the CbiXS proteins from M. thermoautotrophicum and M. barkeri. The proteins purified with masses of 17 and 15 kDa, roughly consistent with their gene-predicted sizes. Both proteins were found to act as ferrochelatases in vivo for the biosynthesis of siroheme because they complemented a sirohydrochlorin ferrochelatase-deficient E. coli mutant. This functional complementation ability was inhibited in the presence of exogenous cobalt but not nickel. Both proteins were found to catalyze the insertion of cobalt into sirohydrochlorin in vitro with specific activities of 18 and 122 nmol/min/mg, respectively. The difference in activity is probably related to the different growth optima of the two organisms from where the cbiXS genes were obtained. M. barkeri, although initially isolated from an Italian lake, is also found in ruminants and therefore has an optimal temperature-activity dependence around 37 °C, whereas M. thermoautotrophicum grows optimally at around 65 °C. The specific activity of the B. megaterium CbiXL is 318 nmol/min/mg. The M. thermoautotrophicum CbiXS was found to be able to chelate nickel into sirohydrochlorin but with a specific activity that was a third lower than that observed for cobalt, whereas the M. barkeri CbiXS had no measurable nickel chelatase at all. On the basis of these experiments we would conclude that the methanogenic CbiXS proteins act as cobaltochelatases during cobalamin biosynthesis. 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 α/β structure, with a high α-helix content compared with that observed in the CD spectra of CbiXL, SirB, and CbiK. Nonetheless, all the proteins have a similar β-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 β-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 Chelatases—When 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 1–128) and C-terminal (amino acids 128–260) 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. An extension of this theory can also explain the appearance of CbiK and HemH, because these proteins are composed of two α/β 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. We thank Prof. R. Thauer and Dr. Reiner Hedderich (Max Plank Institute for Terrestrial Microbiology, Marburg, Germany) for providing genomic DNA. We acknowledge the excellent technical support of Anna Sargsyan and Shiasta Ali during the course of this research.