Biochemistry 2008, 47, 5851–5857 5851 Two Distinct Roles for Two Functional Cobaltochelatases (CbiK) in DesulfoVibrio Vulgaris Hildenborough† Susana A. L. Lobo,‡ Amanda A. Brindley,§ Ce´lia V. Roma˜o,‡ Helen K. Leech,§ Martin J. Warren,§ and Lı´gia M. Saraiva*,‡ Instituto de Tecnologia Quı´mica e Biolo´gica, UniVersidade NoVa de Lisboa, AVenida da Republica (EAN), 2780-157 Oeiras, Portugal, and Protein Science Group, Department of Biosciences, UniVersity of Kent, Canterbury, Kent CT2 7NJ, United Kingdom ReceiVed February 28, 2008; ReVised Manuscript ReceiVed March 28, 2008 ABSTRACT: The sulfate-reducing bacterium DesulfoVibrio Vulgaris Hildenborough possesses a large number of porphyrin-containing proteins whose biosynthesis is poorly characterized. In this work, we have studied two putative CbiK cobaltochelatases present in the genome of D. Vulgaris. The assays revealed that both enzymes insert cobalt and iron into sirohydrochlorin, with specific activities with iron lower than that measured with cobalt. Nevertheless, the two D. Vulgaris chelatases complement an E. coli cysG mutant strain showing that, in ViVo, they are able to load iron into sirohydrochlorin. The results showed that the functional cobaltochelatases have distinct roles with one, CbiKC, likely to be the enzyme associated with cytoplasmic cobalamin biosynthesis, while the other, CbiKP, is periplasmic located and possibly associated with an iron transport system. Finally, the ability of D. Vulgaris to produce vitamin B12 was also demonstrated in this work. Modified tetrapyrroles such as hemes, siroheme, and constituted by only around 110-145 amino acids, the short S cobalamin (vitamin B12) are characterized by a large mo- form (CbiX ). The N-terminal and C-terminal domains of lecular ring structure with a centrally chelated metal ion. This the long form of CbiXL share a high degree of amino acid family of compounds share a common pathway until the sequence similarity. CbiKs are formed by ∼300 amino acid formation of the first macrocyclic intermediate, uroporphy- residues and share a low level of similarity with CbiXL. rinogen III, at which point the pathway branches. Each Analysis of circular dichroism (CD) spectra suggested that branch contains a unique chelatase that performs the insertion the cobaltochelatases CbiK, CbiXL, and CbiXS may have a of a specific metal ion into the modified tetrapyrrole similar overall topology (5). The X-ray structure determi- ring (1, 2). In heme biosynthesis, for example, ferrous iron nation of the CbiK from Salmonella enterica revealed also is inserted into the protoheme precursor protoporphyrin IX that the enzyme is highly similar to the protoporphyrin IX by the protoporphyrin IX ferrochelatase (3). In the case of ferrochelatase from Bacillus subtilis (8, 9). More recent s vitamin B12 biosynthesis, cobalt is inserted into the macro- structural work on CbiX has confirmed that this protein cycle by a cobaltochelatase, which is either specific to the shares the same basic protein architecture as that found in aerobic (oxygen-dependent) or anaerobic pathway for co- CbiK (10). balamin biosynthesis (2). Along the aerobic pathway, pre- Sulfate-reducing bacteria (SRB1), belonging to the genus corrin-2 undergoes several enzymatic modifications that DesulfoVibrio, are considered to be early organisms on the result in the formation of hydrogenobyrinic acid a,c-diamide evolutionary scale and contain a very large number of where cobalt is inserted by the class I ATP-dependent proteins with modified porphyrins, some of them unusual. cobaltochelatase CobN-S-T (4, 5). Along the anaerobic Examples of this include the rubredoxin oxygen oxidoreduc- pathway, cobalt insertion occurs at an early stage, at the level tase from D. gigas that contains iron uroporphyrin I (11) of sirohydrochlorin (6), by the class II ATP-independent and bacterioferritin of D. desulfuricans ATCC 27774, which cobaltochelatases CbiX or CbiK (1, 5, 7). The CbiX protein has an iron-coproporphyrin III cofactor (12). Proteins was first identified as a 320 amino acid enzyme, the so-called containing cobalt-porphyrin were also reported for D. gigas long form (CbiXL), whereas the archaeal orthologue is and D. desulfuricans (Norway) (13, 14), and the nature of the cobalt-porphyrin macrocycle was identified as CoIII- syrohydrochlorin (15). Studies in D. Vulgaris suggested that † This work was financed by the FCT project PTDC/BIA-PRO/ 67107/2006. S.A.L.L. and C.V.R. are recipients of grants SFRH/BD/ in these organisms an alternative pathway for the biosynthesis 19813/2004 and SFRH/BPD/21562/2005, respectively. Financial sup- of heme may be operative since coproporphyrinogen III is port from the Biotechnology and Biological Sciences Research Council (BBSRC) is also acknowledged. * Corresponding author. Lı´gia M. Saraiva, Av. da Republica (EAN), 1 Abreviations: SRB, sulfate-reducing bacteria; LB, Luria broth 2780-157 Oeiras, Portugal. Phone: +351214469328. Fax: +351- medium IPTG, isopropyl--D-thiogalactopyranoside; ALA, 5-aminole- 214411277. E-mail: [email protected]. vulinic acid; EPR, electron paramagnetic resonance; NMR, nuclear ‡ + Universidade Nova de Lisboa. magnetic resonance; SAM, S-adenosyl-L-methionine; NAD , nicoti- § University of Kent. namide adenine dinucleotide; MM, minimal medium. 10.1021/bi800342c CCC: $40.75 2008 American Chemical Society Published on Web 05/06/2008 5852 Biochemistry, Vol. 47, No. 21, 2008 Lobo et al. formed not directly from uroporphyrinogen III but from the imidazole were applied to the column, and the recombinant vitamin B12 precursor, precorrin-2 (16). However, the bio- protein was eluted at 250 mM imidazole. After dialysis synthesis of porphyrins in DesulfoVibrio sp. remains poorly against buffer A, the protein fraction was loaded onto a understood. Q-Sepharose High-Performance column (20 mL) (GE, The genome of D. Vulgaris Hildenborough encodes two Healthcare), previously equilibrated with buffer A. Ten putative cobaltochelatases, DVU0650 and DVU1365, that volumes of a linear gradient up to 400 mM NaCl followed share a significant degree of amino acid sequence identity by four volumes of a linear gradient from 400 mM up to 1 with S. typhimurium CbiK. In order to clarify the role of M NaCl was applied, and the protein was eluted with ∼200 these proteins and their involvement in modified tetrapyrrole mM NaCl. biosynthesis in a DesulfoVibrio sp., the genes were cloned, DVU0650, ∆28DVU0650, and DVU1365 proteins were the proteins produced recombinantly and the isolated en- purified under anaerobic conditions. The anaerobic purifica- zymes characterized. tion steps were performed in a Coy model A-2463 anaerobic chamber filled with a gas mixture of 95% argon plus 5% EXPERIMENTAL PROCEDURES hydrogen. The soluble fraction from the cells expressing DVU0650, ∆28DVU0650, and DVU1365 was applied onto Cloning and Expression of Recombinant Cobaltoch- a HiTrap Chelating HP column (GE, Healthcare). The elatases. The putative cobaltochelatases encoded by the genes recombinant DVU0650 and ∆28DVU0650 proteins were DVU0650 and DVU1365 have 894 bp and 849 bp, respec- eluted with 5 volumes of buffer A containing 0.5 M NaCl tively. Amplification of the two genes was achieved in PCR and 200 mM imidazole, while the DVU1365 protein was reactions using genomic DNA of DesulfoVibrio Vulgaris eluted with 5 volumes of buffer A plus 250 mM imidazole. Hildenborough and appropriated oligonucleotides specifically The protein fractions containing DVU0650 or ∆28DVU0650 designed for each case. A second DNA fragment of the were passed through a PD10 desalting column (GE, Health- DVU0650 gene was also amplified in order to construct a care) in order to exchange the elution buffer to 50 mM Tris- truncated protein that starts at amino acid 29 (∆28DVU0650). HCl buffer at pH 8 with 100 mM NaCl, while the DVU1365 The various DNA fragments were cloned into pET-28a(+) protein fraction was dialyzed overnight against 50 mM Tris- (Novagen) giving pET-28a(+)-DVU0650, pET-28a(+)- HCl buffer at pH 8 with 8% glycerol. ∆28DVU0650, and pET-28a(+)-DVU1365 allowing the The purity of the proteins was analyzed by SDS-PAGE encoded proteins, DVU0650, ∆28DVU0650 and DVU1365, gel (17) and the protein concentration was determined by respectively, to be produced with an 6x-His-tag in the the bicinchoninic acid method (18) using protein standards N-terminal region. Sequencing of the PCR products guar- from Sigma. Heme content was assayed by the hemochro- anteed the integrity of all gene sequences. To produce mopyridine method (19), and heme extraction was performed DVU0650 and ∆28DVU0650 proteins, the recombinant plas- according to the method described by Lubben et al. (20). mids were transformed in Escherichia coli BL21Gold(DE3) The protein molecular mass was determined by gel filtration (Stratagene) and the cells were grown, at 30 °C, in in a Superdex 200 column according to the instructions of Luria-Bertani (LB) medium containing kanamycin (30 µg/ the manufacturer (GE, Healthcare), using commercially mL) until an OD600 ) 0.3. At this point, 200 µM of IPTG available standards from GE Healthcare. (isopropyl--D-thiogalactopyranoside) was added and the Comparisons of the primary amino acid sequences were medium supplemented with 50 µM 5-aminolevulinic acid done using ClustalX (21), and the prediction of sorting (ALA) and 100 µM FeSO4. The culture was then grown signals was achieved using the SOSUI signal program (22, 23). overnight, at 15 °C. The overexpression of DVU1365 was UV-visible spectra were recorded at room temperature achieved by transforming the recombinant plasmid in the in a Shimadzu UV-1700 spectrophotometer with 4.5 µMof BL21Gold(DE3) and growing the cells, at 37 °C, in LB protein, and sodium dithionite was used as a reductant. The medium containing 30 µg/mL kanamycin until an OD600 of redox titration was performed at 25 °C, under continuous 0.8, followed by the addition of 200 µM of IPTG and 100 agitation and argon atmosphere using 2.8 µM of purified µM FeSO4. The cells were then grown at 37 °C for 4 h. protein and 12 µM of mediators, diluted in 50 mM Tris- Protein Purification and Characterization.