Korean Journal of Microbiology (2019) Vol. 55, No. 2, pp. 123-130 pISSN 0440-2413 DOI https://doi.org/10.7845/kjm.2019.9042 eISSN 2383-9902 Copyright ⓒ 2019, The Microbiological Society of Korea Cobalt complex structure of the sirohydrochlorin chelatase SirB from Bacillus subtilis subsp. spizizenii Mi Sun Nam, Wan Seok Song, Sun Cheol Park, and Sung-il Yoon* Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon 24341, Republic of Korea Bacillus subtilis subsp. spizizenii의 sirohydrochlorin chelatase SirB의 코발트 복합체 구조 남미선 ･ 송완석 ･ 박순철 ･ 윤성일* 강원대학교 의생명과학대학 의생명융합학부 (Received April 22, 2019; Revised June 5, 2019; Accepted June 5, 2019) Chelatase catalyzes the insertion of divalent metal into tetra- Keywords: chelatase, cobalt, crystal structure, SirB, sirohydro- pyrrole and plays a key role in the biosynthesis of metallated chlorin tetrapyrroles, such as cobalamin, siroheme, heme, and chloro- phyll. SirB is a sirohydrochlorin (SHC) chelatase that generates cobalt-SHC or iron-SHC by inserting cobalt or iron into the Metallated tetrapyrroles, including cobalamin, coenzyme center of sirohydrochlorin tetrapyrrole. To provide structural F430, siroheme, heme, and chlorophyll, function as essential insights into the metal-binding and SHC-recognition mecha- cofactors of diverse proteins and drive numerous biological nisms of SirB, we determined the crystal structure of SirB from Bacillus subtilis subsp. spizizenii (bssSirB) in complex with processes, such as metabolism, photosynthesis, and oxygen cobalt ions. bssSirB forms a monomeric α/β structure that transport (Raux et al., 2000; Schubert et al., 2002). To generate consists of two domains, an N-terminal domain (NTD) and a functional metallated tetrapyrroles, divalent metals, such as C-terminal domain (CTD). The NTD and CTD of bssSirB adopt cobalt, iron, magnesium, and nickel, should be inserted into similar structures with a four-stranded β-sheet that is decorated the center of tetrapyrroles through the enzymatic activity of by α-helices. bssSirB presents a highly conserved cavity that is chelatase. Chelatases fall into three classes (I, II, and III) based generated between the NTD and CTD and interacts with a cobalt on ATP dependence and additional catalytic activity (Brindley ion on top of the cavity using two histidine residues of the NTD. Moreover, our comparative structural analysis suggests that et al., 2003). Class I and class II chelatases are relatively well bssSirB would accommodate an SHC molecule into the inter- characterized through structural and biophysicochemical studies. domain cavity. Based on these structural findings, we propose Class I includes ATP-dependent magnesium or cobalt chelatases that the cavity of bssSirB functions as the active site where that consist of three subunits (ChlH/I/D or CobN/S/T) (Debussche cobalt insertion into SHC occurs. et al., 1992; Walker and Willows, 1997). Class II chelatases exist as a monomer or a homocomplex and include CbiX, CbiK, and SirB, which insert cobalt or iron into SHC in an *For correspondence. E-mail: [email protected]; ATP-independent manner (Al-Karadaghi et al., 1997; Schubert Tel.: +82-33-250-8385; Fax: +82-33-259-5643 124 ∙ Nam et al. et al., 1999; Leech et al., 2002, 2003; Raux et al., 2003; Yin et by the BamHI and SalI restriction enzymes and inserted into al., 2006; Romao et al., 2011; Fujishiro et al., 2019). a pET49b vector that was modified to express recombinant In the Archaea, CbiX exists as a small enzyme that consists protein in N-terminal fusion with a hexa-histidine tag and a of 120–145 residues and is regarded as the simplest form of thrombin cleavage site (Park et al., 2017). The ligated product S CbiX (CbiX ) (Brindley et al., 2003). A structural analysis of was transformed into Escherichia coli DH5α cells, and trans- S S S Archaeoglobus fulgidus CbiX (afCbiX ) revealed that afCbiX formants were selected by kanamycin. The nucleotide sequence forms a dimer that accommodates a cobalt-SHC molecule into of the bssSirB-encoding region in the expression vector was an intersubunit cavity (Yin et al., 2006; Romao et al., 2011). verified by DNA sequencing. S In contrast to the single domain structure of CbiX , CbiK is S Protein expression and purification composed of two domains, each of which resembles CbiX in size and overall structure (Schubert et al., 1999; Romao et al., To express recombinant bssSirB protein, bssSirB expression S 2011; Bali et al., 2014; Lobo et al., 2017). Therefore, CbiX is vector was transformed into E. coli BL21 (DE3) cells, and the considered as an ancestral form of class II chelatases. transformant cells containing bssSirB expression vector was S Due to extensive structural studies on CbiX and CbiK, the selected in the presence of kanamycin. The cells were first S structural features and substrate-recognition mode of CbiX grown in LB medium at 37°C. When the optical density of the and CbiK have been well characterized. However, the three- culture at 600 nm reached 0.6~0.8, IPTG was added into the dimensional structure of SirB has been elusive until recently. In culture to the final concentration of 1 mM for the induction of 2019, the crystal structure of Bacillus subtilis strain 168 SirB protein expression. The cells were further cultured overnight at (bs168SirB) was reported (Fujishiro et al., 2019). Despite the 18°C and harvested by centrifugation. biological significance of SirB in the biosynthesis of metallated bssSirB protein-containing cells were lysed by sonication in tetrapyrroles, only bs168SirB has been structurally defined to a solution containing 50 mM Tris (pH 8.0), 200 mM NaCl, 5 date. Thus, it is unclear whether the structural features of bs168SirB mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl are applied to other SirB proteins. To further characterize the fluoride. The cell lysate was cleared by centrifugation, and the interaction of SirB with metal and provide structural insights resultant supernatant was incubated with Ni-NTA resin at 4°C. into the active site of SirB, we determined the crystal structure The resin was harvested using an Econo-Column (Bio-Rad) and of SirB from B. subtilis subsp. spizizenii (bssSirB) in complex washed using a solution containing 50 mM Tris (pH 8.0), 200 with cobalt ions. Based on a comparative structural analysis of mM NaCl, 5 mM β-mercaptoethanol, and 10 mM imidazole. S SirB, CbiX , and CbiK, we provide a unique cobalt-binding bssSirB protein was eluted by the stepwise increase of imi- mechanism of SirB and propose that SirB accommodates an dazole concentration (20 mM, 50 mM, and 250 mM). bssSirB SHC molecule into an interdomain cavity. protein was mainly eluted in a solution containing 50 mM Tris (pH 8.0), 200 mM NaCl, 5 mM β-mercaptoethanol, and 250 mM imidazole. bssSirB protein was dialyzed against 20 mM Tris Materials and Methods (pH 8.0) and 5 mM β-mercaptoethanol and subjected to throm- bin digestion at 18°C for 3.5 h to remove the N-terminal Construction of protein expression vector hexa-histidine tag. The resulting tag-free bssSirB protein was bssSirB-encoding DNA fragment was amplified by PCR further purified by anion exchange chromatography using a using the genomic DNA of B. subtilis subsp. spizizenii strain Mono Q 10/100 column with a gradient of 0~500 mM NaCl in W23 with DNA primers (forward primer, 5'-TAAGGATCCG 20 mM Tris (pH 8.0) and 5 mM β-mercaptoethanol. The purified ATGAAGCAAGCAATTTTATATGTCGGTC-3'; reverse primer, bssSirB protein was concentrated to ~13 mg/ml for crystalli- 5'-GCCGATGTCGACCTAATGTGCAGCGGGAGCATAT zation. GAACC-3') containing the recognition site of either BamHI or SalI restriction enzyme. The PCR-amplified DNA was digested 미생물학회지 제55권 제2호 Crystal structure of sirohydrochlorin chelatase SirB ∙ 125 Crystallization and X-ray diffraction (www.rcsb.org). bssSirB protein was crystallized by a sitting-drop vapor- diffusion method at 18°C in a drop containing 0.5 µl of ~13 mg/ml bssSirB protein and 0.5 µl of reservoir solution. Initial Results and Discussion crystals of bssSirB were obtained in the JCSG Core Suite kit Overall structure of bssSirB (Qiagen) using a reservoir solution containing 10 mM cobalt chloride, 1.8 M ammonium sulfate, 0.1 M MES (pH 6.5). The As a first step to determine the crystal structure of bssSirB, initial bssSirB crystallization condition was optimized to 10 mM recombinant bssSirB protein was expressed in E. coli cells and cobalt chloride, 0.1 M magnesium chloride, 1.3 M ammonium purified by Ni-NTA affinity chromatography and anion exchange sulfate, and 0.1 M MES (pH 6.5). chromatography. SDS-PAGE and gel-filtration chromatography X-ray diffraction of bssSirB crystals was performed at beamline analyses showed that purified bssSirB protein is monodisperse 7A, the Pohang Accelerator Laboratory (Republic of Korea). in protein size and oligomeric state (Fig. 1). Moreover, bssSirB A single bssSirB crystal was transferred to a cryoprotectant whose molecular weight is 29.3 kDa was eluted between 17 solution containing 10 mM cobalt chloride, 0.1 M magnesium kDa and 44 kDa protein standards, indicating that bssSirB is chloride, 1.4 M ammonium sulfate, 0.1 M MES (pH 6.5), and monomeric in solution (Fig. 1C). 25% glycerol, and the crystal was mounted on a goniometer bssSirB protein was crystallized in a cobalt-containing solution, using a nylon loop under a cryo-stream at -173°C. X-ray with and a bssSirB crystal diffracted X-ray to 2.15 Å resolution. a wavelength of 1.00004 Å was applied to the mounted crystal, The crystal structure of bssSirB was determined by molecular and X-ray diffraction data were collected using an ADSC replacement and refined to an Rfree value of 24.8% (Fig. 2A and Quantum 270 detector. The full dataset was indexed, integrated, scaled, and merged using the HKL2000 program (Otwinowski (A) and Minor, 1997).