A Story of Chelatase Evolution

Abstract

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 (<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 (14Thauer R. Microbiology. 1998; 144: 2377-2406Crossref PubMed Google Scholar). 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 (15Raux E. Schubert H.L. Warren M.J. Cell. Mol. Life Sci. 2000; 57: 1880-1893Crossref PubMed Scopus (166) Google Scholar), it has been suggested that the CobN homologue may in fact represent the nickel chelatase associated with coenzyme F430 (3Walker C.J. Willows R.D. Biochem. J. 1997; 327: 321-333Crossref PubMed Scopus (183) Google Scholar). 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. Cloning Procedures—All strains and plasmids used in this study are shown in Table II. Methanosarcina barkeri (strain Fusaro) and Methanobacter thermoautotrophicum genomic DNA were supplied by Prof. Thauer and Dr. Reiner Hedderich (Max Plank Institute for Terrestrial Microbiology, Marburg, Germany). Amplifications were carried out in a 50-μl reaction volume containing 1× PCR buffer (Qiagen), 0.2 mm each of the four deoxynucleotides, 0.02 μg of the appropriate genomic DNA, 0.4 μm of each primer, and one unit of HotStar Taq polymerase (Qiagen). Conditions for the amplifications were as follows: one cycle at 95 °C for 15 min followed by 35 cycles at 95 °C for 20 s, 50 °C for 20 s, and 72 °C for 45 s, ending with one cycle of 10 min at 72 °C. PCR products of the expected size were separated by agarose gel electrophoresis, extracted, and cloned into pGEMTeasy (Promega). Subsequently, the M. barkeri cbiX was cloned into pET14b (Novagen) behind a His-tag encoding sequence under the control of a T7 promoter. The M. thermoautotrophicum cbiX has a “natural” His-tag encoding sequence and was thus cloned into pET3a (Novagen), still under the control of a T7 promoter. These pET constructs were used for expression of protein for affinity purification by metal chelate chromatography.Table IIStrains and plasmids used in this studyStrainsPropertiesReference or sourceBL21(DE3)pLysSF- ompT hsdSB(rB- mB-) gal dcm (DE3) pLysSNovagenER171302Δa(pAR8086)[15Raux E. Schubert H.L. Warren M.J. Cell. Mol. Life Sci. 2000; 57: 1880-1893Crossref PubMed Scopus (166) Google Scholar]PlasmidspET3aProtein expression under the control of T7 promoterNovagenpET14bN-terminal fusion His-tag protein expression under the control of T7 promoterNovagenpETacpET14b modified with a Tac promoter instead of T7This workpET3a- MTH/cbiXOverexpresses M. thermoautotrophicum cbiXThis workpET14b- MTH/cobAOverexpresses His-tagged M. thermoautotrophicum cobAThis workpET14b- MSB/cbiXOverexpresses His-tagged M. barkeri cbiXThis workpET14b- MSB/cobAOverexpresses His-tagged M. barkeri cobAThis workpETac- MTH/cobAOverexpresses His-tagged M. thermoautotrophicum cobAThis workpETac- MTH/cobA- MTH/cbiXOverexpresses His-tagged M. thermoautotrophicum cobA and cbiXThis workpETac- MTH/cobA- MSB/cbiXOverexpresses His-tagged M. thermoautotrophicum cobA and Histagged M. barkeri cbiXThis work Open table in a new tab 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 302Δ 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 (16Raux E. Thermes C. Heathcote P. Rambach A. Warren M.J. J. Bacteriol. 1997; 179: PubMed Google Scholar). the M. barkeri and M. thermoautotrophicum were from metal chelate affinity to the pET of the to the metal chelate the was with buffer mm containing mm and The CbiX was in buffer containing mm and were by a of the protein and gel containing the His-tagged protein were and by through a that previously been in 50 mm containing mm The M. barkeri and M. thermoautotrophicum CbiX proteins were by gel on a that previously been in 50 mm containing mm and sirohydrochlorin were as described previously (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) from an enzyme containing uroporphyrinogen III uroporphyrinogen III and for sirohydrochlorin CbiX activity was with sirohydrochlorin and between and 50 μg of CbiX in a reaction volume in 50 mm were in and were on a at °C. and and were with the protein was mm and in a used CbiXL, were at °C on of protein in a an with between at was carried out R. R. 1992; 5: PubMed Scopus Google Scholar). In an to identify the of chelatase used by the for cobalamin biosynthesis, we for the of such as CbiK (16Raux E. Thermes C. Heathcote P. Rambach A. Warren M.J. J. Bacteriol. 1997; 179: 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). searching revealed that of the B. megaterium CbiX were in the of a range of archaeal However, these proteins to be less than half the size of the B. megaterium CbiX (Fig. typically containing amino acids, which is shorter than the 306 amino acids found in the B. megaterium To between the versions of CbiX, such as that found in B. and the shorter found in the we termed and CbiXS To the function of the CbiXS proteins we the gene from two M. barkeri and M. thermoautotrophicum CbiXS proteins contain and amino acids, (Fig. The genes were from the appropriate genomic DNA the described under Although these two genes similar the M. thermoautotrophicum CbiXS also contains a histidine-rich similar from the histidine-rich region found in the B. megaterium (Fig. (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 was cloned into pET14b to allow its recombinant expression as a His-tagged protein, its the M. thermoautotrophicum encodes a protein with a histidine-rich which is likely to allow purification by metal chelate the M. thermoautotrophicum cbiX was cloned into The genes were also subcloned into a pETac downstream of the M. thermoautotrophicum cobA. This the CbiXS proteins to be for their to 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: PubMed Google Scholar). the genes were to that PCR been the M. barkeri and M. proteins were overproduced as recombinant proteins in E. coli and be seen on of The M. thermoautotrophicum CbiXS with its histidine-rich region was by metal chelate as described under the as for the M. barkeri This protein that was than as by (Fig. The M. barkeri CbiXS on an gel with a of 15 in to its of for the His-tag The M. thermoautotrophicum CbiXS with a of which is larger than its of This larger may the of the histidine-rich region within the protein, which may the protein on The that the M. thermoautotrophicum CbiXS be on a metal chelate that the histidine-rich region of the M. thermoautotrophicum CbiXS is of binding The of the CbiXS proteins was investigated This revealed that CbiXS proteins at a to a of that the proteins exist either as homodimers or CbiXS CbiXS proteins were found to be of the of cobalt into A reaction is shown in The reaction be by the of at and a in at (Fig. (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. CbiXS was found to have a specific activity of of of protein, the M. thermoautotrophicum CbiXS a much activity of The CbiXS proteins from M. barkeri and M. thermoautotrophicum were not found to chelate cobalt into that the cobalamin biosynthetic pathway The CbiXS proteins were also for their to chelate Only the M. thermoautotrophicum CbiXS was found to be to chelate nickel but with a specific activity than that for cobalt activity was with M. barkeri in the M. thermoautotrophicum CbiXS was in an with of cobalt and cobalt was found to be chelated These suggest that cobalt is the for this chelatase, and it is likely that CbiXS is a cobaltochelatase associated with cobalamin CbiXS as a in the of in the M. thermoautotrophicum cobA into pETac Subsequently, the from M. thermoautotrophicum and the M. barkeri were cloned downstream of to plasmids and To that proteins were overproduced from this the strains these plasmids were with the and 15 indicating that the and CbiXS proteins were not plasmids and were to the cysG of the E. coli 302Δ a strain PubMed Scopus Google Scholar). These demonstrate that the M. thermoautotrophicum is active as a uroporphyrinogen III and that CbiXS proteins are to function as sirohydrochlorin However, the of cobalt to the minimal growth which the E. coli cysG were this complementation that the activity is by an be with CbiXS a cobalt chelatase. A similar been the was used to this E. coli auxotrophy (16Raux E. Thermes C. Heathcote P. Rambach A. Warren M.J. J. Bacteriol. 1997; 179: PubMed Google Scholar, E. T. T. Warren M.J. Biochem. J. 1999; PubMed Google Scholar). nickel was to the minimal growth of complementation was with the E. coli cysG Thus, nickel not appear to with for into sirohydrochlorin with of the E. coli cysG strain with a of used to the E. coli strain 302Δ on minimal Open table in a new tab In vitro with the M. barkeri and M. thermoautotrophicum CbiXS proteins revealed that they were to into sirohydrochlorin to However, this was at a of less than 10% of that for the of of and cobalt were to the enzyme in an was to siroheme not of CbiXS to by structural is yet for of the SirB/CbiX family of it is not they have similarity to CbiK or HemH or they represent a new division of the class II chelatases. To the CbiXS proteins have structure similarity to CbiK, of the CbiXL, and SirB proteins were and with two CbiK The of the two CbiXS proteins are with the proteins containing a of structure but still with some (Fig. The B. megaterium SirB a with it containing about 60% structure and (Fig. the CbiK proteins from S. (16Raux E. Thermes C. Heathcote P. Rambach A. Warren M.J. J. Bacteriol. 1997; 179: PubMed Google Scholar) and J.M. Raux E. Brindley A.A. Schubert H.L. Warren M.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) of proteins containing between and with the structure of CbiK (Fig. the SirB/CbiX family of proteins contains a with that found in The structure of the CbiXS and suggest that these an D. B. P. 2002; PubMed Scopus Google Scholar). In CbiK, the conserved active site on a between the and of the This structural is for the conserved of B. megaterium and the M. barkeri for the structural with CbiK from that the CbiX with of the class II chelatase structures with a high of J. Mol. Biol. 2000; PubMed Scopus Google Scholar). We have overproduced and characterized the CbiXS proteins from M. thermoautotrophicum and M. The proteins with of and 15 with their proteins were found to as ferrochelatases in for the biosynthesis of siroheme because they a sirohydrochlorin E. coli This complementation was in the of cobalt but not proteins were found to the of cobalt into sirohydrochlorin in vitro with specific of and The in activity is probably to the growth of the two from the genes were M. from an is also found in and has an M. thermoautotrophicum at °C. The specific activity of the B. megaterium is The M. thermoautotrophicum CbiXS was found to be to chelate nickel into sirohydrochlorin but with a specific activity that was a third than that for the M. barkeri CbiXS nickel chelatase at On the basis of these we that the CbiXS proteins as cobalamin The obvious broad of these CbiXS chelatases in to chelate and, in the of the M. thermoautotrophicum about how the correct metal ion is to the chelatase in its there are specific metal ion or the chelatases interact with the metal is not The of a region on the M. thermoautotrophicum CbiXS and the that this protein to a metal chelate suggest that this histidine-rich region may play a in either metal storage or histidine-rich are found in a number of CbiXS and not that may have for metal ion storage and/or by CbiXS as the cobalt chelatase in the archaeal this suggest that the proteins with similarity to CobN and ChlH, which are also found in these are likely to be the nickel chelatases associated with coenzyme F430 However, it is to that the CobN and are also found in the genome of which is a archaeal methanogens appear to contain of these and indicating that there is still much to be about the these proteins play in CbiXS to CbiXL, SirB, and The of the CbiXS proteins that they have a with a high with that in the of CbiXL, SirB, and the proteins have a similar thus the in between CbiXS and the other larger proteins may be because of a more compact structure with in the such that they a structure. a of CbiXL, and SirB a number of conserved two and of the M. barkeri an of the M. barkeri and a number of and (Fig. the residues are found at the active site of CbiK, the two are thought to as in from the and also in binding the metal ion. Indeed, it is possible to the active site primary structure of CbiK into the (Fig. is clear from this is that the active site are found in the region of CbiXL, the active site are in the region of On the basis of the common level of in these proteins and the of catalytic we that CbiXL, SirB, and CbiK belong to the family of because HemH is structurally to CbiK, it also be in this Indeed, that CbiXS the of the CbiK and These proteins belong to the II chelatases, and we suggest that the CbiXS proteins be to and SirB to and CbiK and HemH to of II the B. megaterium was the it was that the CbiXS proteins were the acids and acids of (Fig. This that represents the fusion of two CbiXS a that may have by gene Indeed, it is possible to for the B. megaterium into two and to each other and a CbiXS is is that the region of contains the two important catalytic residues as as a number of other the region of contains conserved and amino acids (Fig. their in the protein We that from two CbiXS and that the of the protein the catalytic the these because this more protein and/or this fusion it into through the of a containing a histidine-rich a chelatase through of a of and to function as a sirohydrochlorin of this also the of CbiK and because these proteins are of two that are to one by a These may also have from a However, in this the the catalytic the these residues as it to the protein function through and/or This protein into a sirohydrochlorin by of its metal ion to into a protoporphyrin by its tetrapyrrole from to In this the evolution of the or be by the of a fusion and of the essential catalytic in either the or of the Thus, CbiXS may represent the primordial chelatase from which these other proteins We Prof. R. Thauer and Dr. Reiner Hedderich (Max Plank Institute for Terrestrial Microbiology, Marburg, for genomic We the of and the of this