Cobalt complex structure of the sirohydrochlorin chelatase SirB from Bacillus subtilis subsp. spizizenii

Bacillus subtilis subsp. spizizenii의 sirohydrochlorin chelatase SirB의 코발트 복합체 구조

Nam, Mi Sun;Song, Wan Seok;Park, Sun Cheol;Yoon, Sung-il

  • Received : 2019.04.22
  • Accepted : 2019.06.05
  • Published : 2019.06.30


Chelatase catalyzes the insertion of divalent metal into tetrapyrrole and plays a key role in the biosynthesis of metallated tetrapyrroles, such as cobalamin, siroheme, heme, and chlorophyll. SirB is a sirohydrochlorin (SHC) chelatase that generates cobalt-SHC or iron-SHC by inserting cobalt or iron into the center of sirohydrochlorin tetrapyrrole. To provide structural insights into the metal-binding and SHC-recognition mechanisms of SirB, we determined the crystal structure of SirB from Bacillus subtilis subsp. spizizenii (bssSirB) in complex with cobalt ions. bssSirB forms a monomeric ${\alpha}/{\beta}$ structure that consists of two domains, an N-terminal domain (NTD) and a C-terminal domain (CTD). The NTD and CTD of bssSirB adopt similar structures with a four-stranded ${\beta}-sheet$ that is decorated by ${\alpha}-helices$. bssSirB presents a highly conserved cavity that is generated between the NTD and CTD and interacts with a cobalt ion on top of the cavity using two histidine residues of the NTD. Moreover, our comparative structural analysis suggests that bssSirB would accommodate an SHC molecule into the interdomain cavity. Based on these structural findings, we propose that the cavity of bssSirB functions as the active site where cobalt insertion into SHC occurs.


chelatase;cobalt;crystal structure;SirB;sirohydrochlorin


  1. Al-Karadaghi S, Hansson M, Nikonov S, Jonsson B, and Hederstedt L. 1997. Crystal structure of ferrochelatase: the terminal enzyme in heme biosynthesis. Structure 5, 1501-1510.
  2. Bali S, Rollauer S, Roversi P, Raux-Deery E, Lea SM, Warren MJ, and Ferguson SJ. 2014. Identification and characterization of the 'missing' terminal enzyme for siroheme biosynthesis in $\alpha$-proteobacteria. Mol. Microbiol. 92, 153-163.
  3. Brindley AA, Raux E, Leech HK, Schubert HL, and Warren MJ. 2003. A story of chelatase evolution: identification and characterization of a small 13-15-kDa "ancestral" cobaltochelatase ($CbiX^S$) in the archaea. J. Biol. Chem. 278, 22388-22395.
  4. Debussche L, Couder M, Thibaut D, Cameron B, Crouzet J, and Blanche F. 1992. Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme $B_{12}$ biosynthesis in Pseudomonas denitrificans. J. Bacteriol. 174, 7445-7451.
  5. Emsley P and Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132.
  6. Fujishiro T, Shimada Y, Nakamura R, and Ooi M. 2019. Structure of sirohydrochlorin ferrochelatase SirB: the last of the structures of the class II chelatase family. Dalton Trans. 48, 6083-6090.
  7. Leech HK, Raux E, McLean KJ, Munro AW, Robinson NJ, Borrelly GP, Malten M, Jahn D, Rigby SE, Heathcote P, et al. 2003. Characterization of the cobaltochelatase $CbiX^L$ : evidence for a 4Fe-4S center housed within an MXCXXC motif. J. Biol. Chem. 278, 41900-41907.
  8. Leech HK, Raux-Deery E, Heathcote P, and Warren MJ. 2002. Production of cobalamin and sirohaem in Bacillus megaterium: an investigation into the role of the branchpoint chelatases sirohydrochlorin ferrochelatase (SirB) and sirohydrochlorin cobalt chelatase (CbiX). Biochem. Soc. Trans. 30, 610-613.
  9. Lobo SA, Videira MA, Pacheco I, Wass MN, Warren MJ, Teixeira M, Matias PM, Romao CV, and Saraiva LM. 2017. Desulfovibrio vulgaris $CbiK^P$ cobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues. Environ. Microbiol. 19, 106-118.
  10. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, and Read RJ. 2007. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658-674.
  11. Murshudov GN, Vagin AA, and Dodson EJ. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240-255.
  12. Otwinowski Z and Minor W. 1997. Processing X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326.
  13. Park SC, Kwak YM, Song WS, Hong M, and Yoon SI. 2017. Structural basis of effector and operator recognition by the phenolic acid-responsive transcriptional regulator PadR. Nucleic Acids Res. 45, 13080-13093.
  14. Raux E, Leech HK, Beck R, Schubert HL, Santander PJ, Roessner CA, Scott AI, Martens JH, Jahn D, Thermes C, et al. 2003. Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium. Biochem. J. 370, 505-516.
  15. Raux E, Schubert HL, and Warren MJ. 2000. Biosynthesis of cobalamin (vitamin $B_{12}$): a bacterial conundrum. Cell Mol. Life Sci. 57, 1880-1893.
  16. Romao CV, Ladakis D, Lobo SA, Carrondo MA, Brindley AA, Deery E, Matias PM, Pickersgill RW, Saraiva LM, and Warren MJ. 2011. Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization. Proc. Natl. Acad. Sci. USA 108, 97-102.
  17. Schubert HL, Raux E, Matthews MA, Phillips JD, Wilson KS, Hill CP, and Warren MJ. 2002. Structural diversity in metal ion chelation and the structure of uroporphyrinogen III synthase. Biochem. Soc. Trans. 30, 595-600.
  18. Schubert HL, Raux E, Wilson KS, and Warren MJ. 1999. Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis. Biochemistry 38, 10660-10669.
  19. Walker CJ and Willows RD. 1997. Mechanism and regulation of Mg-chelatase. Biochem. J. 327(Pt 2), 321-333.
  20. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, et al. 2011. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235-242.
  21. Yin J, Xu LX, Cherney MM, Raux-Deery E, Bindley AA, Savchenko A, Walker JR, Cuff ME, Warren MJ, and James MN. 2006. Crystal structure of the vitamin $B_{12}$ biosynthetic cobaltochelatase, $CbiX^S$, from Archaeoglobus fulgidus. J. Struct. Funct. Genomics 7, 37-50.
  22. Zheng H, Chordia MD, Cooper DR, Chruszcz M, Muller P, Sheldrick GM, and Minor W. 2014. Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server. Nat. Protoc. 9, 156-170.


Supported by : National Research Foundation of Korea, Kangwon National University