Skip to main content
NCBI home page
FEBS Open Bio logoLink to FEBS Open Bio
. 2024 Jun 24;14(8):1291–1302. doi: 10.1002/2211-5463.13849

Nickel‐chelatase activity of SirB variants mimicking the His arrangement in the naturally occurring nickel‐chelatase CfbA

Yuuma Oyamada 1, Shoko Ogawa 1, Takashi Fujishiro 1,✉
PMCID: PMC11301274  PMID: 38923868

Abstract

Metal–tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral, homodimeric‐type class II chelatase which is able to use not only Ni2+ as a physiological metal substrate, but also Co2+ as a nonphysiological substrate with higher activity than for Ni2+. The Ni/Co‐chelatase function found in CfbA is also observed in SirB, a descendant, monomeric‐type class II chelatase. This is despite the distinct active site structure of CfbA and SirB; specifically, CfbA shows a unique four His residue arrangement, unlike other monomeric class II chelatases such as SirB. Herein, we studied the Ni‐chelatase activity of SirB variants R134H, L200H, and R134H/L200H, the latter of which mimics the His alignment of CfbA. Our results showed that the SirB R134H variant exhibited the highest Ni‐chelatase activity among the SirB enzymes, which in turn suggests that the position of His134 could be more important for the Ni‐chelatase activity than that of His200. The SirB R134H/L200H variant showed lower activity than R134H, despite the four His residues found in SirB R134H/L200H. CD spectroscopy showed secondary structure denaturation and a slight difficulty in Ni‐binding of SirB R134H/L200H, which may be related to its lower activity. Finally, a docking simulation suggested that the His134 of the SirB R134H variant could function as a base catalyst for the Ni‐chelatase reaction in a class II chelatase architecture.

Keywords: chelatase, mutagenesis, nickel, porphyrin, protein–ligand docking

CfbA is a class II chelatase that utilizes Ni2+unlike other class II chelatases, such as SirB. The structural uniqueness of CfbA is due to the four His residues at its active site. We studied SirB variants mimicking this His arrangement and showed that a His‐mediated base catalysis is key for the Ni‐chelatase activity in class II chelatases.

graphic file with name FEB4-14-1291-g001.jpg

Abbreviations

CD

circular dichroism

DTT

dithiothreitol

IPTG

isopropyl‐β‐D‐thiogalactopyranoside

LB

Luria‐Bertani

SHC

sirohydrochlorin

UPI

uroporphyrin I

Chelatases are key enzymes for the biosynthesis of metal–tetrapyrrole cofactors and can catalyze the insertion of a divalent metal ion into a substrate macrocyclic tetrapyrrole [1]. There are three distinct classes of chelatases: classes I, II, and III [2]. Class II chelatases comprise the largest group among all the classes and exhibit various structures and functions [3, 4]. Most class II chelatases are monomers and exhibit a large active site pocket with tetrapyrrole‐ and metal‐binding sites. The structures of these binding sites are suitably arranged for their substrate selectivities. For example, mammalian FECH ferrochelatase uses protoporphyrin IX (PPIX) and Fe2+ [5, 6, 7, 8, 9, 10, 11, 12, 13, 14]. SirB is also a ferrochelatase in siroheme biosynthesis; however, its tetrapyrrole substrate is sirohydrochlorin (SHC) rather than PPIX [2, 15, 16, 17, 18]. Some cobalt‐chelatases can also utilize SHC with Co2+ in cobalamin biosynthesis [3, 15, 19, 20, 21, 22].

More recently, CfbA has been identified as the naturally occurring nickel‐chelatase catalyzing Ni2+ insertion into SHC in coenzyme F430 biosynthesis [23, 24]. CfbA has several unique structural features compared with other class II chelatases [2, 3, 4, 25, 26]. Uniquely, CfbA exhibits a homodimeric architecture that can be superposed onto the overall structure of other monomeric class II chelatases. Within the active site of CfbA, two metal‐binding sites are symmetrically located. In Methanocaldococcus jannaschii CfbA (Mj CfbA) [26], one subunit provides two conserved His9 and His75 residues for one metal‐binding site, while the other subunit also has another pair of His9 and His75. Overall, four His residues are arranged within the active site cavity. In addition, the SHC‐binding area exists between two pairs of His9 and His75. Under low Ni2+ concentrations, one metal‐binding site is sufficient for catalysis, in which the other pair of His residues may function as a base catalyst for SHC deprotonation, after binding of SHC and Ni2+ to the active site (Fig. 1). The coordination geometry for Ni2+ of CfbA has been studied by X‐ray crystallography (Fig. 2). Three residues, His9, His75, and Glu42 are likely to be positioned in a facial manner when the Ni‐coordination is assumed as a 6‐coordination.

Fig. 1.

Fig. 1

Open in a new tab

Proposed mechanism of nickel‐chelatase reaction by CfbA with sirohydrochlroin (SHC) and Ni2+ involved in His‐mediate base catalysis. First, SHC is bound to the active site, followed by Ni2+‐binding to a pair of His9 and His75 with the support of Glu42. Then, an acetate of SHC is bound to Ni2+ via ligand exchange with Glu42, as captured by X‐ray crystallography [26]. This ligand exchange may contribute to SHC ring distortion, which is favorable for His‐mediated base catalysis to abstract protons from SHC, although the necessity of either His9 or His75, or both His9 and His75, has not been clarified so far. Finally, Ni2+ is inserted into SHC, yielding Ni‐sirohydrochlorin. The SHC's substituents Rn (n = 1–9) are as follows: R1 = R4 = R8 = CH2COO−, R2 = R3 = R5 = R7 = CH2CH2COO−, R6 = R9 = CH3.

Fig. 2.

Fig. 2

Open in a new tab

Structures of SirB and CfbA. (A) Overall and (B) active site structures of SirB in complex with Co 2+ (PDB ID: 5ZT7) [18]. (C) View for the geometry of the Co 2+‐binding site and its chemical structure. (D) Overall and (E) active site structures of CfbA in complex with Ni 2+ (PDB ID: 6M27) [26]. (F) Geometry of the Co 2+‐binding site and its chemical structure. Residues for metal binding in the N‐terminal domain of SirB and the corresponding residues of CfbA are colored slate blue. Mutated residues of the C‐terminal domain of SirB and their corresponding residues of CfbA are colored magenta. Notably, CfbA exhibits the homodimeric architecture colored in slate blue and pink for one and the other of the monomers. The overall homodimeric architecture of CfbA resembles the SirB monomeric architecture. Cobalt and nickel ions are shown in pink and green spheres, respectively. The CfbA structure contains two Ni 2+ but only one Ni 2+ bound to the blue‐colored residue is sufficient for the nickel‐chelatase reaction. The binding of the other Ni 2+ is attributed to the high Ni 2+ concentration in X‐ray crystallographic analysis [26]. It should be noted that Co 2+‐ and Ni 2+‐coordinations in these SirB and CfbA structures, especially the presence of H2O or OH− ligands, are not completely resolved, although one H2O or OH− is visible in the case of SirB. However, by considering the facial geometry of three amino acid ligands (two His and one Glu) in SirB and CfbA as well as the preference of Co 2+‐ and Ni 2+‐coordinations in water, there could be 4, 5, or 6‐coordination.

Considering the His‐mediated base catalysis and previously determined X‐ray crystal structures of CfbA with SHC and/or Ni2+, a catalytic mechanism of nickel‐insertion to SHC by CfbA has been proposed (Fig. 1) [26]: First, the SHC molecule is bound to the active site, followed by binding of Ni2+ to one pair of His9 and His75 with a supporting ligand of Glu42 (1 of Fig. 1). Then, an acetate group of SHC is bound to Ni2+ via ligand‐exchange instead of Glu42 (2 of Fig. 1), which is captured by X‐ray crystal structure of a reaction intermediate of CfbA [26]. This ligand exchange may be able to make SHC distorted, which makes NH moieties face on the His9 and/or His75, which are different from Ni2+‐bound His9/His75. Afterwards, His‐mediated base catalysis by His9 and/or His75, which is a focus of this study, is contributing to deprotonating of SHC (2 of Fig. 1). After the deprotonation of SHC, Ni2+ can be inserted into the central pyrrole‐nitrogens of SHC (3 of Fig. 1), yielding Ni‐sirohydrochlorin as a product (4 of Fig. 1).

It is known that CfbA can utilize not only Ni2+, but also Co2+ as a metal substrate [2, 26, 27]. The activity for Co2+ of CfbA is much higher than Ni2+. The Ni/Co‐chelatase function is also known in some monomeric‐types of class II chelatases, such as SirB [15]. However, the active site structures of CfbA and SirB are rather distinct (Fig. 2), e.g. the numbers of conserved His residues serving as metal‐binding ligands and/or possible base catalysts to abstract protons from their tetrapyrrole substrate. Currently, it is interesting how the arrangement of the four His residues in CfbA contributes to nickel‐chelatase activity. The uniqueness of the His arrangement of CfbA can be recognized via comparison to monomeric‐type of class II chelatases, such as SirB. For example, SirB has only two His (Fig. 2), which resembles a pair of two His residues for Ni‐binding in CfbA (Figs. S1 and S2), although other parts of the residues of SirB are different from the corresponding ones of CfbA (Fig. 2). To consider the relationship between the His residue arrangement and nickel‐chelatase activity, we herein studied monomeric SirB variants mimicking CfbA's His arrangement. We prepared three SirB variants, R134H, L200H, and R134/L200H variants, and characterized them using Ni2+ and uroporphyrin I (UPI), an SHC analog used for facile nickel‐chelatase assay [18]. Circular dichroism (CD) spectroscopy was performed to gain clues to consider the possible structure–function relationship of the SirB variants, especially the R134H/L200H variant showing lower activity than expected. Furthermore, docking simulation to make models of UPI‐bound SirB WT and the variants was performed for addressing a possible role of His residues as a base catalyst, which may be related to increased Ni‐chelatase activity of the SirB R134H variant than SirB WT.

Materials and Methods

Materials

UPI and myoglobin were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Isopropyl‐β‐D‐thiogalactopyranoside (IPTG) was purchased from BLD Pharmatech (Shanghai, China). Dithiothreitol (DTT), imidazole, and chloramphenicol were purchased from FUJIFILM‐Wako Pure Chemical (Tokyo, Japan). All other chemicals were purchased from Nacalai Tesque (Kyoto, Japan). All DNA oligo primers (Table S1) were purchased from Eurofins Genomics Japan (Tokyo, Japan). Ferritin, aldolase, conalbumin, and ovalbumin were purchased from Cytiva (Tokyo, Japan).

Site‐directed mutagenesis

For the site‐directed mutagenesis of SirB, the pACYC‐sirA‐His‐sirB‐sirC plasmid [18], which is used for the expression of Bacillus subtilis SirB wildtype (WT) with an N‐terminal His6‐tag, was used as a template for inverse PCR with mutagenic primers. The amplified PCR products were digested with DpnI and self‐ligated with a 2× ligation convenience kit (Nippongene, Tokyo, Japan) and T4 polykinase (Nippongene). The ligation mixture was used to transform Escherichia coli DH5α cells. Transformed E. coli DH5α colonies were selected in Luria–Bertani (LB) medium supplemented with 25 μg/mL chloramphenicol and then cultivated at 37 °C for 12 h. The plasmid was extracted from cultivated E. coli DH5α cells and used for DNA sequencing of sirB to check for the mutation of interest. To construct the expression systems of SirB R134H, L200H, and R134H/L200H variants, three plasmids (pACYC‐sirA‐His‐sirB R134H‐sirC, pACYC‐sirA‐His‐sirB L200H‐sirC, and pACYC‐sirA‐His‐sirB R134H/L200H‐sirC) were used to transform E. coli C41(DE3).

Expression and purification of SirB WT and three variants

SirB WT and the variants were expressed and purified as previously described [18]. Transformed E. coli C41(DE3) cells harboring either of the expression plasmids for WT or the variants were cultivated in 6 L of LB medium supplemented with 25 μg/mL chloramphenicol at 37 °C for 4 h. When the OD600 value reached 0.4–0.6, IPTG was added to the culture at a final concentration of 1 mM. The culture was incubated at 20 °C for 20 h to express the SirB variants. Subsequently, E. coli cells were harvested via centrifugation at 9000  g for 20 min at 4 °C. The harvested E. coli cells were frozen in liquid nitrogen and stored at −80 °C.

SirB WT and the variants were purified either on ice or at 4 °C. E. coli cells expressing either SirB WT or the variants were disrupted via sonication. The sonicated cells were then centrifuged at 20 000  g for 40 min at 4 °C. The supernatant was then loaded onto a HisTrap FF crude column (Cytiva) equilibrated with buffer A (50 mM Tris–HCl, pH 7.8, 500 mM KCl, and 1 mM DTT). After washing the column with buffer A, SirB was eluted with buffer B (50 mM Tris–HCl, pH 7.8, 500 mM KCl, 1 mM DTT, and 250 mM imidazole). The pooled SirB fractions were concentrated using Amicon Ultra‐15 (Merck‐Millipore, Burlington, MA, USA). The concentrated SirB was loaded onto a HiPrep 16/60 Sephacryl S‐200 HR column equilibrated with buffer C (50 mM Tris–HCl, pH 7.8, 150 mM NaCl, and 1 mM DTT). SirB fractions were pooled and concentrated before further use. The purity of SirB was evaluated via SDS‐PAGE (Fig. S3).

Gel filtration analysis for determining the molecular weight of SirB

Oligomeric states of SirB WT and three variants in solution were performed by gel filtration chromatography using a Superdex™ S200 Increase 10/300 GL column installed onto Akta Go (Cytiva) at 4 °C with a flow rate of 0.3 mL/min. The column was equilibrated with buffer D (50 mM Tris–HCl buffer pH 7.8, 150 mM NaCl, 1 mM tris(2‐carboxyethyl)phosphine) For the analysis of either of SirB WT or variant samples, temperature, 100 μL of SirB was injected. Absorption at 280 nm was monitored to gain chromatograms and values of elution volumes of SirB samples. Calibration for molecular mass calculation was created by gel filtration analysis of molecular weight protein markers: ferritin (Mw = 440 000), aldolase (Mw = 158 000), conalbumin (Mw = 75 000), ovalbumin (Mw = 43 000), and myoglobin (Mw = 17 000). Using the calibration, the molecular weight of SirB WT and three variants were calculated (Fig. S4).

Activity assay

For the nickel‐chelatase activity assay, the reaction mixture was prepared as follows: 5 μM SirB WT or either of the variants, 50 μM UPI, and 200 μM NiCl2 in a reaction buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl) in a total volume of 10 μL of the reaction mixture, and their reaction was performed in the dark. The UV–visible spectra of the reaction mixtures were measured on the Implen UV–visible C40 spectrophotometer equipped with a submicroliter cell with a 1 mm cell path (Implen, Munich, Germany). The UV–visible spectrum of each reaction mixture was scanned from 400 to 700 nm. Furthermore, the changes in the absorbance at 552 nm, attributed to the product nickel–uroporphyrin I (Ni–UPI), were plotted over time. The absorbance at 552 nm after a 24‐h reaction was used to calculate the relative nickel‐chelatase activity. The ratio of the change in the absorbance at 552 nm (ΔA 552) of an SirB variant/WT was calculated. Thus, the relative activity for WT was 1.0. Notably, for the SirB R134H variant, curve‐fitting using a pseudo‐first‐order kinetic equation was possible, whereas it was not possible for the other datasets for WT, L200H, and R134H/L200H. The pseudo‐first‐order rate constant (k) for the SirB R134H variant with UPI and Ni2+ was calculated via nonlinear root mean square curve‐fitting to the plots with Igor Pro 8.0 (WaveMetrics, Lake Oswego, OR, USA). The reactions for each plot were repeated at least three times (n = 3) and standard deviations (SD) were represented as error bars at each point. UV–visible spectrum of Ni2+ solution (10 mM NiCl2 in 50 mM Tris–HCl buffer, pH 7.8) was recorded on an Implen C40 spectrophotometer with a 1 cm‐path quartz cuvette to check the peaks derived from Ni2+ alone.

Docking simulation

Docking of UPI to SirB WT and the variants was performed using AutoDock Vina [28] on UCSF Chimera (http://www.cgl.ucsf.edu/chimera). The model structures of the SirB R134H and L200H variants were prepared by substitution of the amino acids of interest (i.e. Arg134 and Leu200) with His in the X‐ray crystal structure of SirB WT (PDB ID: 5ZT7) [18] in open‐source PyMOL (Schrödinger, New York, NY, USA). When preparing the SirB models, cobalt ions and water molecules were removed from the SirB WT and variant models. The UPI ligand model was prepared using PRODRG [29] via the energy‐minimizing process for optimizing its conformation. Each of the SirB WT or variant and UPI models was set as a rigid receptor and ligand for Autodock Vina, respectively. The residues Arg134, or His134, and Leu200, or His200, were set as flexible residues. The UPI model was also set to have flexible conformations for its substituents, i.e. acetates and propionates. The docking parameters were similar to those used in a previous study [18]. The initial coordinate for UPI was set to (x, y, z) = (39.703, −10.618, 12.017). The size of the docking grid box was 72.0 Å × 44.0 Å × 58.0 Å. The other docking parameters were used as the default. After the docking simulation, the highest‐ranked structure based on the computed binding energy was selected as a favorable docking model. All protein figures are represented using PyMOL.

CD spectroscopy

CD spectroscopy on SirB WT and variants were performed using the J‐1500 CD spectrophotometer (JASCO, Tokyo, Japan) with a quartz cuvette with a 1‐cm cell path. In all the measurements, the temperature was maintained at 20 °C. The number of residues in SirB was 273, which was used to calculate the mean residue molar ellipticity, [θ] = (recorded degrees/cell path × protein molar concentration × residue number).

For measurement of CD spectra derived from secondary structure regions of SirB WT and variants, SirB WT and the three SirB variants in 50 mM Na‐phosphate buffer (pH 7.8) were prepared, to result in 3 mL of sample volume for each case. The protein concentrations of SirB WT and the R134H, L200H, and R134H/L200H variants were 1.28, 0.93, 1.34, and 1.97 μM, respectively.

For measurement of the near UV–visible range of the CD spectra of SirB WT and variants with NiCl2, a quartz cell with a volume of 1 mL was used. NiCl2 was titrated to prepare mixtures of 10, 20, 30, 40, or 60 μM of NiCl2 and SirB WT or variants. For the near UV–visible range of CD spectroscopy, protein concentrations of SirB WT and the R134H, L200H, and R134H/L200H variants were 12, 17, 19, and 18 μM, respectively.

Results

SirB variants mimic the His residues in the CfbA active site

To design SirB variants mimicking CfbA, we first compared the active sites of Bacillus subtilis SirB (Bs SirB) and Mj CfbA [18, 26] (Fig. 2). The metal‐binding site of Bs SirB contains His10, Glu43, and His76, which are at the equivalent positions to His9, Glu42, and His75 of Mj CfbA. However, Mj CfbA has a second set of His9, Glu42, and His75. Instead, Arg134 and Leu200 of Bs SirB are equivalently located at the second set of His9 and His75 of Mj CfbA. Furthermore, the Glu of CfbA (e.g. Glu42 of Mj CfbA) was not conserved, as observed in the amino acid sequence alignment of CfbA enzymes [26]. For example, Archaeoglobus fulgidus CfbA (Af CfbA), which is also called Af CbiXS [3, 27], has Ala instead of the Glu. In other words, the pair of two His residues are strictly conserved among CfbA enzymes, whereas the Glu is not. Therefore, we focused on His arrangement rather than the Glu, with Arg134 and Leu200 as the target sites for mutagenesis. This resulted in the construction of three Bs SirB variants: R134H, L200H, and R134H/L200H. Each of these Bs SirB variants was purified with good purity (Fig. S3) and eluted as a monomer (Fig. S4) in the gel filtration analysis, as in the case of Bs SirB WT.

Nickel‐chelatase activity of SirB variants

The nickel‐chelatase activity of the three Bs SirB variants was analyzed by inserting nickel into UPI, a commercially available SHC analog (Fig. 3A) [18]. Bs SirB WT exhibited slight nickel‐chelatase activity for UPI with Ni2+. This was similar to the findings of a study on the nickel‐chelatase activity of SHC with Ni2+ [15]. Next, we studied the nickel‐chelatase activities of the SirB variants with UPI. Among them, Bs SirB R134H exhibited the highest nickel‐chelatase activity, followed by Bs SirB R134H/L200H (Fig. 3). The activities of these two SirB variants were higher than those of WT. In contrast, the nickel‐chelatase activity of the SirB L200H variant was lower than that of WT. Furthermore, no reaction for Ni–UPI formation was observed without SirB (Fig. S5A). The observed UV–visible spectra of the mixture of SirB, Ni2+ and UPI were different from of Ni2+ alone (Fig. S5B). These data also supported that the observed nickel‐chelatase reactions were catalyzed by SirB WT and the variants.

Fig. 3.

Fig. 3

Open in a new tab

(A) Scheme for UPI–nickel–chelatase reaction. (B–E) Changes of UV–visible spectra in reaction mixtures of UPI, Ni2+ with (B) SirB WT, (C) SirB R134H variant, (D) SirB L200H variant, and (E) SirB R134H/L200H variant. Black lines of UV–visible spectra indicate the spectra just after initiating the reaction (0 h). The changes of the absorption peaks are indicated by black arrows. The time‐course changes of the UV–visible spectra are colored in blue (6 h), green (12 h), light green (18 h), orange (22 h), and red (24 h). The values of the UV–visible peak absorbance at 552 nm, derived from the product Ni–UPI, were used to calculate the relative activity. The relative activity of the WT was set to 1.0. Error bars indicate the standard deviations. Reactions for each set of conditions were performed three times. (F) Comparison of the relative Ni‐chelatase activity for UPI. The relative nickel‐chelatase activity of Af CfbA (Af CbiXS) for UPI was also calculated by using its activity value, which was reported previously [30].

Thereafter, the activity of the SirB R134H variant was analyzed using the pseudo‐first‐order kinetic equation via nonlinear root mean square curve‐fitting (Fig. S6); as a result, the apparent kinetic constant (k) was 6.6 × 10−4 ± 2.6 × 10−4 min−1. The k value of the Bs SirB R134H variant in the Ni–UPI formation reaction was 4.4‐fold lower than that of Af CfbA (k = 2.9 × 10−3 min−1) (Fig. 3F) [30]. In contrast, the activities of the other SirB variants and WT could not be analyzed in the same manner as those of SirB R134H, owing to their extremely low activities.

Partial denaturation of bs SirB R134H/L200H

Next, Bs SirB WT and the three variants were characterized using CD spectroscopy (Fig. 4). The WT, R134H, and L200H variants exhibited almost identical CD spectra by comparison of their double minimum spectra derived from α‐helices in the range of 200–250 nm. In contrast, the Bs SirB R134H/L200H variant exhibited decreased α‐helix peaks. The content of the secondary structures of the Bs SirB R134H/L200H variant was ~50%–60% lower than those of Bs SirB WT and the other variants. Therefore, CD spectroscopy of the three SirB variants revealed that the structure of the R134H/L200H variant may be different from that of the other variants.

Fig. 4.

Fig. 4

Open in a new tab

CD spectra of SirB in a range of 200–250 nm. (A) WT and the (B) R134H, (C) L200H, and (D) R134H/L200H variants.

To gain further insights into the protein structure–function relationship, we performed near UV–visible range of CD spectroscopy of SirB WT and variants with Ni2+ (Fig. S7). The CD spectral peak at 279 nm increased upon the addition of Ni2+ to SirB WT or variant. This peak at 279 nm could be considered as the ligand‐to‐metal charge transfer (LMCT) in a similar manner to CooJ, a Ni‐chaperon using His residues for binding Ni2+ [31]. Thus, the CD spectral changes at 279 nm could indicate that Ni‐binding occurred in all the cases of SirB. Although the quantitative analysis of the binding constant of Ni2+ to SirB samples could fail due to poor intensity of this spectral changes at this region, it was clear that the R134H/L200H variant could show a spectral change only at a higher concentration of Ni2+ (30 μM) compared with the other SirB enzymes in the presence of 20 μM of Ni2+. This could be interpreted as the lower‐affinity of R134H/L200H variant to Ni2+. If this feature of Ni‐binding of R134H/L200H variant may have been caused by a partial denaturation around the Ni‐binding sites, including not only H134H/H200, but also His10/His76, the lowered nickel‐binding affinity could be understood. In such a case, a largely denatured signature found in CD spectroscopy on 200–240 nm of R134H/L200H variant could also be understood: The effect of mutation on R134H/L200H may have been made the region of His10/His76 as well.

Docking simulation to the SirB variant models with UPI

The structural models of UPI‐bound Bs SirB variants were computed to understand why the R134H mutation is involved in enhancing nickel‐chelatase activity (Fig. 5). In the UPI‐docked model of Bs SirB R134H, the NH groups of UPI were positioned adjacent to the metal‐binding site and His134. The position of His134 was suitable to function as a base catalyst at neutral or weakly basic pH, resulting in deprotonation of the NH groups of UPI toward enhanced activity. In the UPI‐docked model of Bs SirB WT, the ε‐NH moiety of Arg134 interacted with UPI. In general, the basicity of Arg at neutral or weakly basic pH is not good compared to His, resulting in Arg134‐mediated base catalysis for UPI deprotonation being challenging. Although Arg134 can function as a base, the distance between Arg134 and UPI is not sufficiently close for effective base catalysis. Notably, the distance between His200 and UPI was farther in the Bs SirB L200H variant. Thus, His200 could not function as a base catalyst. The position of the ε‐NH moiety of Arg134 in the Bs SirB L200H variant was similar to that of the WT; however, this moiety was strongly anchored via polar interaction with the adjacent His200 at a distance of 3.5 Å. This fixation of Arg134 negatively affects the flexible motion of Arg134, possibly resulting in low efficiency of deprotonation of UPI by Arg134 when it can function as a base catalyst.

Fig. 5.

Fig. 5

Open in a new tab

Active site structures of the docking models of SirB with UPI. (A) SirB WT, (B) SirB R134H, and (C) SirB L200H. Black dashed lines indicate the distances. Yellow stick models indicate UPI. Dashed lines indicate possible polar interactions between UPI and Arg134, His134, Leu200, or His200, which are represented as orange‐colored stick models.

Discussion

The Bs SirB R134H variant showed a moderately enhanced Ni‐chelatase activity, which is usually lower than Co‐chelatase activity in CfbA. This result indicated that a single His, in the case of His134 of Bs SirB, is sufficient for enhancing activity, which was not clarified in the previous proposed mechanism of CfbA, possibly using two His residues as base catalysts. In other words, two His would not be essential for base catalysis toward the tetrapyrrole substrate in Ni‐chelatase function on class II chelatases.

Interestingly, the Bs SirB R134H/L200H variant is proposed to be more similar to CfbA from the viewpoint of the His residues; however, this variant exhibited lower activity than the Bs SirB R134H variant owing to its partial structural denaturation, as well as slightly less Ni‐binding compared with SirB WT and the other variants. One idea to explain the lower activity is that the four His arrangements in one monomeric class II chelatase may be unfavorable for its protein stability; although CfbA has four His residues in its active site. However, it is still elusive why the four His arrangements are structurally unfavored for SirB but acceptable for CfbA, and the molecular mechanism underlying the partial denaturation of Bs SirB R134H/L200H remains unclarified. The modeling of His134 and His200 on Bs SirB may not indicate a steric crash, suggesting that the denaturation of Bs SirB R134H/L200H may be caused by other reasons, such as partial protein misfolding. Thus, it should be noted that a posed structure shown in the docking simulation could explain only a possible close distance between His134 and UPI, but not other key factors (e.g. degree of protein structural denaturation) related to the activities.

It is also important to consider why Af CfbA (or Af CbiXS) still has higher Ni‐chelatase activity than the SirB R134H variant. We actually expected the R134H/L200H variant could have the highest activity, if its structure had not been denatured. One hypothesis for the higher activity of Af CfbA is that two His can deliver protons via His75 of CfbA, when His9 in CfbA deprotonates in adjacent to NH moieties of the tetrapyrrole (Figs. 2 and 5). In other words, if SirB R134H/L200H was structurally stable, His134/His200 may have a function in a similar way to two His out of four His in Af CfbA. Thus, maintaining the class II chelatase architecture is an important factor for Ni‐chelatase activity, if the importance of proton‐delivery via two His (His9 and His75 in the case of Mj CfbA) is hypothesized.

Interestingly, no studies have revealed a naturally occurring monomeric class II chelatase having four His residues at the active site. This may be understood based on the hypothetical structural basis for the denaturation of a four His‐containing monomeric class II chelatase, as observed in the SirB R134H/L200H variant. The limitation on the number and positions of His residues is important for considering the structural diversity and evolution of class II chelatases. It raises questions about why and how monomeric class II chelatases, which are considered as descendant types, could evolve from the ancestral CfbA via changes in His residues at the active sites through mutations.

Conflict of interest

The authors declare no conflicts of interest.

Peer review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/2211‐5463.13849.

Author contributions

TF conceived and designed the project. YO and TF performed mutagenesis, protein expression and purification, and docking simulation. TF performed CD spectroscopy. YO performed activity assay with support from SO. TF wrote the article.

Supporting information

Fig. S1. The overall structure and coordination geometry of Ni2+‐bound CfbA.

Fig. S2. The overall structure and coordination geometry of Co2+‐bound SirB.

Fig. S3. SDS‐PAGE of SirB WT, R134H, L200H and R134H/L200H variants.

Fig. S4. Gel filtration analysis for determination of molecular weight of SirB WT and variants.

Fig. S5. UV–visible spectra in the mixture of UPI and Ni2+ without SirB and only the presence of Ni2+.

Fig. S6. Plots for time‐course changes in the difference in the absorbance at 552 nm in the Ni‐UPI formation by Bs SirB R134H variant.

Fig. S7. CD spectroscopy for analyzing the binding properties of Ni2+ to SirB WT and its variants.

Table S1. List of mutagenic primers used in this study.

FEB4-14-1291-s001.pdf (6.5MB, pdf)

Acknowledgements

The authors thank Emeritus Prof. Dr. Yasuhiro Takahashi for his continuous support. We also thank the staff at the Institute for Molecular Science (Okazaki, Japan) for measuring CD spectra under proposal No. JPMXP1222MS1003 and JPMXP1224MS1045 by ARIM Japan, MEXT. This work was supported by a Grant‐in‐Aid for Scientific Research on Innovative Areas 19H04639 (to T.F.) and 23H04542 (to T.F.) from JSPS, Japan. T.F. was also financially supported by the Heavy‐element Materials Research Group of the Strategic Research Centre of Saitama University. S.O. was grateful for the financial support of research by the Sasakawa Scientific Research Grant from The Japan Science Society.

Edited by Beata Vertessy

Data accessibility

Data available within the article and/or the supplementary material.

References

  • 1. Bryant DA, Hunter CN and Warren MJ (2020) Biosynthesis of the modified tetrapyrroles‐the pigments of life. J Biol Chem 295, 6888–6925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. 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 (CbiXS) in the archaea. J Biol Chem 278, 2238–22395. [DOI] [PubMed] [Google Scholar]
  • 3. Romão 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Fujishiro T (2022) Sirohydrochlorin nickelochelatase CfbA. Encyclopaedia Inorg Bioinorg Chem ebic2815. [Google Scholar]
  • 5. Dailey HA, Dailey TA, Gerdes S, Jahn D, Jahn M, O'Brian MR and Warren MJ (2017) Prokaryotic heme biosynthesis: multiple pathways to a common essential product. Microbiol Mol Biol Rev 81, e00048‐16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Crouse BR, Sellers VM, Finnegan MG, Dailey HA and Johnson MK (1996) Site‐directed mutagenesis and spectroscopic characterization of human ferrochelatase: identification of residues coordinating the [2Fe‐2S] cluster. Biochemistry 35, 16222–16229. [DOI] [PubMed] [Google Scholar]
  • 7. Sellers VM, Wu CK, Dailey TA and Dailey HA (2001) Human ferrochelatase: characterization of substrate‐iron binding and proton‐abstracting residues. Biochemistry 40, 9821–9827. [DOI] [PubMed] [Google Scholar]
  • 8. Wu CK, Dailey HA, Rose JP, Burden A, Sellers VM and Wang BC (2001) The 2.0 Å structure of human ferrochelatase, the terminal enzyme of heme biosynthesis. Nat Struct Biol 8, 156–160. [DOI] [PubMed] [Google Scholar]
  • 9. Al‐Karadaghi S, Franco R, Hansson M, Shelnutt JA, Isaya G and Ferreira GC (2006) Chelatases: distort to select? Trends Biochem Sci 31, 135–142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Dailey HA, Wu CK, Horanyi P, Medlock AE, Najahi‐Missaoui W, Burden AE, Dailey TA and Rose J (2007) Altered orientation of active site residues in variants of human ferrochelatase. Evidence for a hydrogen bond network involved in catalysis. Biochemistry 46, 7973–7979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Medlock A, Swartz L, Dailey TA, Dailey HA and Lanzilotta WN (2007) Substrate interactions with human ferrochelatase. Proc Natl Acad Sci USA 104, 1789–1793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Medlock AE, Dailey TA, Ross TA, Dailey HA and Lanzilotta WN (2007) A pi‐helix switch selective for porphyrin deprotonation and product release in human ferrochelatase. J Mol Biol 373, 1006–1016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Medlock AE, Carter M, Dailey TA, Dailey HA and Lanzilotta WN (2009) Product release rather than chelation determines metal specificity for ferrochelatase. J Mol Biol 393, 308–319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Gupta V, Liu S, Ando H, Ishii R, Tateno S, Kaneko Y, Yugami M, Sakamoto S, Yamaguchi Y, Nureki O et al. (2013) Salicylic acid induces mitochondrial injury by inhibiting ferrochelatase heme biosynthesis activity. Mol Pharmacol 84, 824–833. [DOI] [PubMed] [Google Scholar]
  • 15. 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. [DOI] [PubMed] [Google Scholar]
  • 16. 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Raux‐Deery E, Leech HK, Nakrieko KA, McLean KJ, Munro AW, Meathcote P, Rigby SE, Smith AG and Warren MJ (2005) Identification and characterization of the terminal enzyme of siroheme biosynthesis from Arabidopsis thaliana: a plastid‐located sirohydrochlorin ferrochelatase containing a 2Fe‐2S center. J Biol Chem 280, 4713–4721. [DOI] [PubMed] [Google Scholar]
  • 18. 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. [DOI] [PubMed] [Google Scholar]
  • 19. 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 CbiXL: evidence for a 4Fe‐4S center housed within an MXCXXC motif. J Biol Chem 278, 41900–41907. [DOI] [PubMed] [Google Scholar]
  • 20. Moore SJ, Lawrence AD, Biedendieck R, Deery E, Frank S, Howard MJ, Rigby SE and Warren MJ (2013) Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12). Proc Natl Acad Sci USA 110, 14906–14911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Lobo SA, Brindley AA, Romão CV, Leech HK, Warren MJ and Saraiva LM (2008) Two distinct roles for two functional cobaltochelatases (CbiK) in Desulfovibrio vulgaris Hildenborough. Biochemistry 47, 5851–5857. [DOI] [PubMed] [Google Scholar]
  • 22. Lobo SA, Videira MA, Pacheco I, Wass MN, Warren MJ, Teixeira M, Matias PM, Romão CV and Saraiva LM (2017) Desulfovibrio vulgaris CbiKP cobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues. Environ Microbiol 19, 106–118. [DOI] [PubMed] [Google Scholar]
  • 23. Zheng KY, Ngo PD, Owens VL, Yang XP and Mansoorabadi SO (2016) The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea. Science 354, 339–342. [DOI] [PubMed] [Google Scholar]
  • 24. Moore SJ, Sowa ST, Schuchardt C, Deery E, Lawrence AD, Ramos JV, Billig S, Birkemeyer C, Chivers PT, Howard MJ et al. (2017) Elucidation of the biosynthesis of the methane catalyst coenzyme F430 . Nature 543, 78–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Pisarchik A, Petri R and Schmidt‐Dannert C (2007) Probing the structural plasticity of an archaeal primordial cobaltochelatase CbiXS . Protein Eng Des Sel 20, 257–265. [DOI] [PubMed] [Google Scholar]
  • 26. Fujishiro T and Ogawa S (2021) The nickel‐sirohydrochlorin formation mechanism of the ancestral class II chelatase CfbA in coenzyme F430 biosynthesis. Chem Sci 12, 2172–2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Schuelke‐Sanchez AE, Stone AA and Liptak MD (2020) CfbA promotes insertion of cobalt and nickel into ruffled tetrapyrroles in vitro . Dalton Trans 49, 1065–1076. [DOI] [PubMed] [Google Scholar]
  • 28. Trott O and Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31, 455–461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Schüttelkopf AW and van Aalten DM (2004) PRODRG: a tool for high‐throughput crystallography of protein‐ligand complexes. Acta Crystallogr Sect D 60, 1355–1363. [DOI] [PubMed] [Google Scholar]
  • 30. 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 B12 biosynthetic cobaltochelatase, CbiXS, from Archaeoglobus fulgidus . J Struct Funct Genom 7, 37–50. [DOI] [PubMed] [Google Scholar]
  • 31. Aflano M, Pérard J, Carpentier P, Basset C, Zambelli B, Timm J, Crouzy S, Ciurli S and Cavazza C (2019) The carbon monoxide dehydrogenase accessaory protein CooJ is a histidine‐rich multidomain dimer containing an unexpected Ni(II)‐binding site. J Biol Chem 294, 7601–7614. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. The overall structure and coordination geometry of Ni2+‐bound CfbA.

Fig. S2. The overall structure and coordination geometry of Co2+‐bound SirB.

Fig. S3. SDS‐PAGE of SirB WT, R134H, L200H and R134H/L200H variants.

Fig. S4. Gel filtration analysis for determination of molecular weight of SirB WT and variants.

Fig. S5. UV–visible spectra in the mixture of UPI and Ni2+ without SirB and only the presence of Ni2+.

Fig. S6. Plots for time‐course changes in the difference in the absorbance at 552 nm in the Ni‐UPI formation by Bs SirB R134H variant.

Fig. S7. CD spectroscopy for analyzing the binding properties of Ni2+ to SirB WT and its variants.

Table S1. List of mutagenic primers used in this study.

FEB4-14-1291-s001.pdf (6.5MB, pdf)

Data Availability Statement

Data available within the article and/or the supplementary material.

Articles from FEBS Open Bio are provided here courtesy of Wiley

RESOURCES