Identification and Characterization of a Novel Vitamin B12 (Cobalamin) Biosynthetic Enzyme (CobZ) from Rhodobacter capsulatus, Containing Flavin, Heme, and Fe-S Cofactors

One of the most intriguing steps during cobalamin (vitamin B12) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework. The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ. However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663. To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B12 pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA. Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction. HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG. Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B12 itself is a modified tetrapyrrole. A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed. One of the most intriguing steps during cobalamin (vitamin B12) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework. The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ. However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663. To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B12 pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA. Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction. HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG. Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B12 itself is a modified tetrapyrrole. A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed. Vitamin B12 (cobalamin) is a modified tetrapyrrole and belongs to the same class of compounds as heme, chlorophyll, siroheme, and coenzyme F430 (1Warren M.J. Scott A.I. Trends Biochem. Sci. 1990; 15: 486-491Abstract Full Text PDF PubMed Scopus (96) Google Scholar). Modified tetrapyrroles are synthesized via a branched biosynthetic pathway, with 5-aminolevulinic acid the first common intermediate and uroporphyrinogen III the first branchpoint step in the pathway (1Warren M.J. Scott A.I. Trends Biochem. Sci. 1990; 15: 486-491Abstract Full Text PDF PubMed Scopus (96) Google Scholar) (Fig. 1). The structural complexity of vitamin B12 means that its biosynthesis remains one of most enigmatic and exigent metabolic pathways in nature, requiring around 30 enzymes for the complete de novo construction of the coenzyme form (2Warren M.J. Raux E. Schubert H.L. Escalante-Semerena J.C. Nat. Prod. Rep. 2002; 19: 390-412Crossref PubMed Scopus (323) Google Scholar). For cobalamin biosynthesis two distinct yet similar routes exist, known as the oxygen-dependent (aerobic) and oxygen-independent (anaerobic) pathways (3Blanche F. Thibaut D. Debussche L. Hertle R. Zipfel F. Müller G. Angew. Chem. Int. Ed. Engl. 1993; 32: 1651-1653Crossref Scopus (46) Google Scholar). These pathways diverge at precorrin-2 and merge again at adenosylcobyric acid. The major differences between these pathways include the timing of cobalt insertion, the requirement for molecular oxygen, and the nature of the extruded carbon fragment, which is lost during the ring contraction process (4Wang J. Stolowich N.J. Santander P.J. Park J.H. Scott A.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14320-14322Crossref PubMed Scopus (26) Google Scholar). These biochemical differences are also reflected at the genetic level, where the presence of certain genes offers a diagnosis of the type of pathway in operation. Thus pathways that operate the aerobic route to B12 always contain the genes cobN, -S, and -T, which encode the subunits of a cobaltochelatase that can insert cobalt into a ring-contracted tetrapyrrole-derived macrocycle (5Debussche L. Couder M. Thibaut D. Cameron B. Crouzet J. Blanche F. J. Bacteriol. 1992; 174: 7445-7451Crossref PubMed Google Scholar), and cobG, which encodes a monooxygenase required for synthesizing the hydroxylated γ-lactone derivative of precorrin-3 (6Scott A.I. Roessner C.A. Stolowich N.J. Spencer J.B. Min C. Ozaki S.I. FEBS Lett. 1993; 331: 105-108Crossref PubMed Scopus (46) Google Scholar). CobG 1The 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.1The 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. (precorrin-3B synthase) was identified initially from sequencing of the cobalamin biosynthetic operons in Pseudomonas denitrificans (7Debussche L. Thibaut D. Cameron B. Crouzet J. Blanche F. J. Bacteriol. 1993; 175: 7430-7440Crossref PubMed Google Scholar). It encodes a protein with a molecular mass of 46 kDa and displays some sequence similarity to sulfite reductase, including a motif for a Fe-S center (7Debussche L. Thibaut D. Cameron B. Crouzet J. Blanche F. J. Bacteriol. 1993; 175: 7430-7440Crossref PubMed Google Scholar). The role of CobG in cobalamin synthesis was demonstrated when it was shown that it converted precorrin-3A into a compound with an extra 16 mass units (precorrin-3B), corresponding to the inclusion of an oxygen atom (8Debussche L. Thibaut D. Danzer M. Debu F. Frechet D. Herman F. Blanche F. Vuilhorgne M. J. Chem. Soc. Chem. Commun. 1993; : 1100-1103Crossref Google Scholar). The structure of precorrin-3B was determined from multiple 13C labeling studies coupled with infrared spectroscopy, revealing that the product of the reaction catalyzed by CobG contained a γ-lactone attached to ring A and a hydroxy group at C-20 (6Scott A.I. Roessner C.A. Stolowich N.J. Spencer J.B. Min C. Ozaki S.I. FEBS Lett. 1993; 331: 105-108Crossref PubMed Scopus (46) Google Scholar, 8Debussche L. Thibaut D. Danzer M. Debu F. Frechet D. Herman F. Blanche F. Vuilhorgne M. J. Chem. Soc. Chem. Commun. 1993; : 1100-1103Crossref Google Scholar). Further oxygen labeling experiments proved conclusively that the latter was derived from molecular oxygen and that CobG was therefore a monooxygenase (6Scott A.I. Roessner C.A. Stolowich N.J. Spencer J.B. Min C. Ozaki S.I. FEBS Lett. 1993; 331: 105-108Crossref PubMed Scopus (46) Google Scholar). 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 α-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 (9Vlcek C. Paces V. Maltsev N. Paces J. Haselkorn R. Fonstein M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9384-9388Crossref PubMed Scopus (36) Google Scholar) 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. Most chemicals were purchased from Sigma. Other materials were provided by the following suppliers: restriction enzymes and modification enzymes were from Promega, Chilworth, Southampton, United Kingdom; pKK223.3, chelating-Sepharose fast flow resin and gel filtration columns were from Amersham Biosciences, Little Chalfont, Bucks, UK; pET14b was from Novagen, Madison, WI; tryptone and yeast extract were from Oxoid, Basingstoke, UK; sodium dithionite was from Roche, Poole, UK; and primers were from Invitrogen, Paisley, UK. 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). Plasmid pBAD33:cobAI was constructed by cutting a DNA fragment bearing the two genes from pUC18:cobAI (10Roessner C.A. Park J.H. Scott A.I. Bioorg. Med. Chem. 1999; 7: 2215-2219Crossref PubMed Scopus (6) Google Scholar) with KpnI and PstI and ligating it into pBAD33 (11Guzman L-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3941) Google Scholar) (p15A ori, pBAD, ChlR). An orf663 minus strain was constructed in a similar manner except that the large plasmid did not contain orf663. The orf663 minus strain was also transformed with a third compatible plasmid, pZS*24 (pSC101 ori, Plac/ara1, KanR) (12Lutz R. Bujard H. Nucleic Acids Res. 1997; 6: 1203-1210Crossref Scopus (1221) Google Scholar), bearing either orf663 or the P. denitrificans cobG gene. 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 of medium of containing the same antibiotics plus the following 0.2% 0.2% yeast of 5-aminolevulinic and The cells in medium were with aeration at 37 °C for The found in the medium and in the °C for cell were by to HBA was and by as C.A. Spencer J.B. Stolowich N.J. J. Santander P.J. C. Min C. Scott A.I. Chem. Full Text PDF PubMed Scopus Google Scholar). HBA was in its acid form and by on a on a with a and at a flow of 1 with a of in 1 E. coli cells transformed with the plasmid the R. capsulatus Orf663 were grown on LB and were and by for at in a The was with 10 of a and by a at for in a with a of The cell was by a at an to a cell The was by for 10 at The was The was to a at for 1 in a The was in and contained the fragment of the The fragment of the protein was and by on a The in contained the form of was in and at for 1 The was and the was resuspended and in a The was by the for 30 with The was and the protein was to 4 after the at The that had not been by the was by for 1 at The form of Orf663 was from the by on a The protein was in a containing The protein was and the by The for were after were on a at with an flow The are in the The is by an The for were either in after the or for 30 in the presence of the sodium dithionite and in were either on a with an for the of The were a to the were in a a as L. PubMed Scopus Google Scholar). were with were at than The protein in of containing was to the of PubMed Scopus Google Scholar) using sodium dithionite as and as at appropriate in between and were to the to the for the 1 of the heme After R. capsulatus (aerobic) and (anaerobic) we that cobalamin to similar within the cells of A600 that this can make cobalamin in the presence of of molecular coupled to the that R. capsulatus does not contain an orthologue of cobG (9Vlcek C. Paces V. Maltsev N. Paces J. Haselkorn R. Fonstein M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9384-9388Crossref PubMed Scopus (36) Google Scholar), by an aerobic cobalamin biosynthetic pathway J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that it an to ring within the main cobalamin operon there is a gene that encode a To Orf663 is in corrin synthesis, 10 R. capsulatus genes to be required for HBA synthesis from uroporphyrinogen III were into using a that we have and (Fig. genes can be and in with the of a number of restriction The enzymes required to 5-aminolevulinic acid to uroporphyrinogen III are provided by the and the large plasmid was transformed into an E. coli strain plasmid bearing the and genes to uroporphyrinogen III to precorrin-3A to of this intermediate and to the pathway (10Roessner C.A. Park J.H. Scott A.I. Bioorg. Med. Chem. 1999; 7: 2215-2219Crossref PubMed Scopus (6) Google Scholar). The strain was found to comparatively large of HBA, the first intermediate on the pathway to cobalamin that is and can be F. Cameron B. Crouzet J. Debussche L. Thibaut D. Vuilhorgne M. Angew. Chem. Int. Ed. Engl. 1995; Scopus Google Scholar). HBA was as a acid and in its form and its structure on the of its and (Fig. The of to which E. coli belongs is to have lost the ability to make cobalamin de novo some ago, although some of this group as appear to have the ability of the operon 1996; PubMed Scopus Google Scholar). it also been shown that E. coli can make novo after transformation of the bacterium with a plasmid containing this operon E. A. F. M.J. E. A. C. J. Bacteriol. 1996; PubMed Scopus (96) Google Scholar). However, E. coli with the to synthesize HBA is more of a HBA is a compound that E. coli HBA is aerobic the required for the genetically E. coli to make cobalamin E. A. F. M.J. E. A. C. J. Bacteriol. 1996; PubMed Scopus (96) Google Scholar). an E. coli strain with the ability to make HBA also allowed to the of the R. capsulatus Orf663 was required for HBA orf663 in a strain that no HBA. HBA synthesis be restored either by of orf663 or by of the P. denitrificans cobG gene. cobG and orf663 encode enzymes that are the that the R. Orf663 is a cobalamin biosynthetic enzyme and that it is distinct from the P. denitrificans CobG (see we that Orf663 be called The protein by is therefore to a reaction similar to that by CobG, it the ability to generate a hydroxylactone derivative of precorrin-3A (Fig. (6Scott A.I. Roessner C.A. Stolowich N.J. Spencer J.B. Min C. Ozaki S.I. FEBS Lett. 1993; 331: 105-108Crossref PubMed Scopus (46) Google Scholar). Thus CobG and are precorrin-3B However, this reaction in a manner from that by CobG, the two no sequence is than CobG A that is of two distinct an that displays similarity to and a that is an integral protein with similarity to of called but now called Escalante-Semerena J.C. J. Bacteriol. 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In this and have been to in the of To from an in of to in was cloned to overproduction of as a recombinant protein in E. coli with an to two one of the mass of kDa and of 50 revealed that the protein was a fragment from the of the protein is with of two distinct where the between a that is to The fragment was and shown to a flavin (Fig. the and of this of are of a flavin it is not to identify the type of flavin by this However, a at than the flavin to be by can be into its and which therefore in a in the F. R. C. Biochem. Soc. 1999; PubMed Scopus Google Scholar). 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Chem. 1996; Scopus Google Scholar). A similar been for more than one as some and the center and to the of B. J. C. P. H. P. J. Chem. 1993; Full Text PDF PubMed Google Scholar, J.H. PubMed Scopus Google Scholar). The presence of the was in the of the the in but not of a large be in the form of the protein not The main is at = and two can be at = and = this at an of fast of The of this are with that of a center Scholar, and of Scholar). this was not in the of CobZ, it is to of the naturally in the protein, by the In this we have demonstrated a molecular that is in the biosynthesis of vitamin B12 and that it is isofunctional with CobG. been shown to contain a flavin in the form of a two Fe-S centers, and a does this cofactor-rich the synthesis of bearing in that the reaction to be similar to that catalyzed by the monooxygenase CobG (6Scott A.I. Roessner C.A. Stolowich N.J. Spencer J.B. Min C. Ozaki S.I. 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