Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae

In Vibrio cholerae, the genes encoding hemC, hemD, hemE, hemN, and hemY ... this study, we serendipitously found that PBGD from V. cholerae could bind...
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Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1‑Hydroxymethylbilane Takeshi Uchida,*,†,‡ Takumi Funamizu,‡ Minghao Chen,§ Yoshikazu Tanaka,∥,⊥,# and Koichiro Ishimori†,‡ †

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628, Japan § Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan ∥ Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan ⊥ PRESTO, Japan Science and Technology Agency, Sapporo 060-0810, Japan ‡

S Supporting Information *

ABSTRACT: Porphobilinogen deaminase (PBGD) is an enzyme that catalyzes the formation of hydroxymethylbilane, a tetrapyrrole intermediate, during heme biosynthesis through the stepwise polymerization of four molecules of porphobilinogen. PBGD from Vibrio cholerae was expressed in Escherichia coli and characterized in this study. Unexpectedly, spectroscopic measurements revealed that PBGD bound one equivalent of heme with a dissociation constant of 0.33 ± 0.01 μM. The absorption and resonance Raman spectra suggested that heme is a mixture of the 5-coordinate and 6-coordinate hemes. Mutational studies indicated that the 5-coordinate heme possessed Cys105 as a heme axial ligand, and His227 was coordinated to form the 6-coordinate heme. Upon heme binding, the deamination activity decreased by approximately 15%. The crystal structure of PBGD revealed that His227 was located near Cys105, but the side chain of His227 did not point toward Cys105. The addition of the cyanide ion to heme−PBGD abolished the effect of heme binding on the enzymatic activity. Therefore, coordination of His227 to heme appeared to induce reorientation of the domains containing Cys105, leading to a decrease in the enzymatic activity. This is the first report indicating that the PBGD activity is controlled by heme, the final product of heme biosynthesis. This finding improves our understanding of the mechanism by which heme biosynthesis is regulated.

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INTRODUCTION Heme is a versatile molecule involved in many biological processes, including electron transfer, oxygen transport, and chemical catalysis.1,2 The biosynthesis of heme involves multiple steps that are conserved in most living organisms (Supplementary Figure 1).3−6 The first step is the formation of a building block of tetrapyrrole biosynthesis, 5-aminolevulinic acid (ALA), which is synthesized in two different ways. In mammals, fungi, and α-proteobacteria, glycine and succinyl coenzyme A are condensed to ALA by ALA synthase (ALAS encoded by hemA). In most bacteria, archaea, and plants, ALA is synthesized from tRNA-bound glutamate via the so-called C5 pathway.7,8 Two molecules of ALA are asymmetrically condensed to the pyrrole porphobilinogen (PBG) by porphobilinogen synthase (PBGS encoded by hemB). Four molecules of porphobilinogen are oligomerized to the linear tetrapyrrole intermediate, 1-hydroxymethylbilane, by porphobilinogen deaminase (PBGD encoded by hemC). 1-Hydroxymethylbilane is cyclized to uroporphyrinogen III by uroporphyrinogen III synthase (UROS encoded by hemD). Although 1-hydroxymethylbilane is cyclized spontaneously to the isomer (uroporphyrinogen I), UROS mediates the © XXXX American Chemical Society

inversion of the ring D of 1-hydroxymethylbilane by cyclization between ring A and ring D. Uroporphyrinogen III is a common precursor of heme and other tetrapyrroles, such as chlorophyll and vitamin B12. Uroporphyrinogen III decarboxylase (UROD encoded by hemE) decarboxylates four acetate side chains of uroporphyrinogen III to form coproporphyrinogen III. Further oxidative decarboxylation by coproporphyrinogen oxidase (CPO encoded by hemF or hemN) turns coproporphyrinogen III into protoporphyrinogen IX. The six-electron oxidation of protoporphyrinogen IX results in the formation of protoporphyrin IX via protoporphyrin IX oxidase (PPO encoded by hemG or hemY). The final step of heme synthesis involves the insertion of iron into protoporphyrin IX (PPIX), which is catalyzed by ferrochelatase encoded by hemH. Heme, the final product of this pathway, is also an effector molecule of heme biosynthesis. Heme itself is reported to function as a regulator of ALAS. Binding of heme to ALAS inhibits its migration from the cytosol to the mitochondria, Received: October 28, 2017 Accepted: January 23, 2018 Published: January 23, 2018 A

DOI: 10.1021/acschembio.7b00934 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology where ALAS is active.9−11 The catalytic activity of ALAS is also regulated by heme binding to ALAS.12 Recently, PPIX was found to inhibit ALAS more effectively than heme.13 In Bradyrhizobium japonicum, the expression of hemB (PBGS) (Supplementary Figure 1) is repressed by the iron response regulator (Irr) protein.14 Irr binds an iron response element to inhibit the translation of mRNA encoding hemB. Heme binds to the heme regulatory motif in Irr, which promotes degradation of Irr itself. Thus, the amount of hemB mRNA present increases upon heme binding to Irr.15 Except for these cases, no other enzymes included in the heme biosynthesis pathways have been known to be regulated by heme or PPIX. In Vibrio cholerae, the genes encoding hemC, hemD, hemE, hemN, and hemY, which are involved in heme biosynthesis, are located in the gene cluster VC0120−VC0116.16 The hemC gene encodes PBGD (EC 2.5.1.61), which catalyzes the polymerization of porphobilinogen to form 1-hydroxymethylbilane. The active site of PBGD is composed of a dipyrromethane cofactor that is assembled from two molecules of porphobilinogen and is covalently attached to the enzyme through a thioether bond to a conserved cysteine residue (Cys254 in Escherichia coli PBGD).17 This cofactor works as a primer to catalyze the polymerization of four molecules of porphobilinogen to form the linear tetrapyrrole hydroxymethylbilane in a stepwise head-to-tail manner.17 During the reaction, four porphobilinogen molecules react sequentially to give a hexapyrrole complex from which the tetrapyrrole product is released. In this study, we serendipitously found that PBGD from V. cholerae could bind one equivalent of heme with a dissociation constant of 0.33 μM. Furthermore, binding of heme to PBGD decreased the oligomerization activity of porphobilinogen by approximately 15%. These results suggested that an excess of heme, which is toxic to cells, bound to PBGD to repress heme synthesis itself. This report is the first observation of negative feedback in the heme biosynthesis process, with the exception of ALAS and PBGS regulatory activities.

Figure 1. Purification of PBGD. (A) SDS-PAGE gel of the purified PBGD (lane 1) and a molecular mass marker (lane M). (B) Determination of the molecular mass of PBGD by size exclusion chromatography. Analytical gel filtration was performed using a Superdex 200pg column equilibrated with 50 mM Tris-HCl, 150 mM NaCl (pH 8.0) with a flow rate of 1 mL/min. The plot shows the log of the molecular mass standards versus elution volume, including the following protein standards: ribonuclease A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), and blue dextran (2000 kDa). Kav of PBGD was 0.518, which corresponds to logMW of 4.62.

against the heme concentration (Figure 2B, inset), suggesting that PBGD bound one equivalent of heme. On the basis of pyridine hemochrome of the sample, the stoichiometry of heme binding to PBGD was also determined to be 1:1. The apparent dissociation constant (Kd,heme) calculated by fitting the absorbance difference to eq 1 was 0.33 ± 0.01 μM. Effect of Heme Binding to PBGD on the Activity. The effect of heme binding to PBGD on the PBGD enzymatic activity was next examined. The porphobilinogen deaminase activity was analyzed using absorption spectrophotometry. The substrate, porphobilinogen, was mixed with PBGD, and the product was oxidized by benzoquinone to form uroporphyrinogen I (Supplementary Figure 2). The amount of product was determined directly from the uroporphyrinogen I absorption. The obtained activity of PBDG from V. cholerae was found to be 2.04 ± 0.04 μmol h−1 mg−1 (Table 1). Heme binding to PBGD reduced the activity to 1.77 ± 0.03 μmol h−1 mg−1, about 87% of the activity of PBDG alone, suggesting that heme binding repressed the PBGD activity. Michaelis−Menten plot of the specific activity versus substrate concentrations was shown in Figure 3. The maximum velocity (Vmax) of PBGD in the presence of heme was 1.8 ± 0.1 μmol h−1 mg−1, which was smaller than that in the absence of heme (2.3 ± 0.2 μmol h−1 mg−1). This result was consistent with the observation of the above-mentioned 15% decrease in the PBGD activity upon heme binding (Table 1). The Michaelis constant (Km) was also decreased by approximately 15% (75 ± 20 μM for PBGD, 66 ± 9 μM for heme−PBGD) upon heme binding. UV−visible Absorption Spectra of Heme-Reconstituted PBGD. The heme−PBGD complex was prepared by adding a 1.1-fold molar excess of heme to the purified protein and removing excess heme using a gel filtration column. The absorption spectra of the heme-bound PBGD are shown in Figure 2C. The Soret band of the ferric heme consisted of two bands at 371 and 417 nm, similar to those reported for heme-

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RESULTS Cloning, Expression, Purification, and Heme Binding of PBGD. The E. coli strain BL21(DE3) was used to overexpress PBGD from V. cholerae. The purified protein migrated as a single band with a molecular mass of ∼38 kDa on an SDS-PAGE gel (Figure 1A), in good agreement with the calculated molecular mass of PBGD (34.6 kDa). The protein used in this study was approximately 95% pure. An analysis using size-exclusion chromatography indicated that the protein was present as a monomer (∼40 kDa) (Figure 1B). The purified protein was slightly pinkish in color, suggesting that the protein was purified along with a pigment, but the absorption spectra of the as-purified PBGD revealed that the protein was devoid of heme (Figure 2A). Addition of one equivalent of hemin to the purified protein produced solutions that provided a broad band centered at 378 nm (Figure 2A). This band position differed from that of free hemin (387 nm, Figure 2A), indicating that heme specifically bound to PBGD. The heme-binding ability of PBGD was confirmed by examining the stoichiometry and binding constant of PBGD for heme. A solution of PBGD at a fixed concentration was titrated with heme. Difference spectra generated by subtracting the spectra of PBGD from that of the buffer titrated with the same amount of heme are shown in Figure 2B. The absorbance difference at 421 nm was plotted B

DOI: 10.1021/acschembio.7b00934 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 2. Electronic absorbance spectrum of heme titration. (A) Absorption spectra of PBGD as purified (), heme (−·−), and heme-complex of PBGD (···). (B) Absorption difference spectra of heme−PBGD. (Inset) The difference at 421 nm following incremental addition of heme (1−30 μM) to PBGD (10 μM) in 50 mM Tris-HCl, 150 mM NaCl (pH 8.0) against a blank cell containing buffer alone. (C) Optical absorption spectra of heme−PBGD. Ferric (), ferrous (···), and ferrous−CO (−·−) complexes in 50 mM Tris-HCl, 150 mM NaCl (pH 8.0). (D) Deconvolution of the absorption spectrum. The Soret band of heme−PBGD was deconvoluted into two Gaussian components. The red line indicates the measured spectrum, and black dotted lines indicate fitted curves.

Table 1. Heme Dependence of the PBGD Activity activity (μmol h−1 mg−1) without heme (Awo) WT WT + CN C248S C105S C105H H227D C211S C140S a

2.04 2.00 a 2.08 2.32 2.05 2.10 2.46

± 0.04 ± 0.01 ± ± ± ± ±

0.19 0.01 0.05 0.08 0.05

with heme (Aw) 1.77 1.93 a 2.06 2.18 2.04 1.99 2.05

relative activity, Aw/Awo

± 0.03 ± 0.03

0.87 ± 0.03 0.96 ± 0.02

± ± ± ± ±

0.99 0.94 0.99 0.95 0.83

0.17 0.02 0.10 0.03 0.03

± ± ± ± ±

0.17 0.01 0.07 0.05 0.03

Figure 3. Catalytic activity of PBGD as a function of the substrate concentration. The activity was measured at 30 °C in 50 mM TrisHCl, 150 mM NaCl (pH 8.0) in the absence (●) and presence (▲) of heme. The plot in the presence of heme was measured by the addition of 3 equiv of heme to PBGD. The solid lines represent the fit of the experimental data to the classical Michaelis-Menten model.

Not detected.

bound Bach118 and heme-bound peroxiredoxin with Cyscoordination.19 The spectrum was deconvoluted into two Gaussian components, and the exact peak positions were determined to be 373 and 422 nm (Figure 2D). The Soret band at 373 nm coincided with that of Bach1 with Cyscoordination,18 whereas the band at 422 nm was closer to that of Bach1 with Cys/His coordination (423 nm).18 rather than that of P450 (417 nm, Cys/H2O coordination).20 Therefore, Cys and His were determined to be the putative heme ligands for PBGD. Reduction of ferric heme−PBGD with sodium dithionite caused a red shift in the Soret band to 425 nm with a small shoulder at 388 nm. Two distinct Q-bands at 553 and 559 nm were characteristic of the 6-coordinate low-spin heme.

Although a weak Soret band appeared at 388 nm, an unusual band for ferrous heme, Cys- or Tyr-coordinated ferrous heme complexes have exhibited similar spectra with a Soret band around 390 nm.21,22 Therefore, the presence of the 388 nm band was indicative of coordination of a weak anionic ligand, such as Cys or Tyr, to the ferrous heme of the heme−PBGD complex. The addition of carbon monoxide (CO) to the ferrous heme shifted the Soret maximum to 421 nm, and visible bands appeared at 540 and 569 nm, similar to those observed in CO-bound myoglobin and hemoglobin.23 These results C

DOI: 10.1021/acschembio.7b00934 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 4. (A) Resonance Raman spectra of heme−PBGD excited at 413.1 nm in Tris-HCl, 150 mM NaCl (pH 8.0). (Inset) Spectra of the ferrous heme−PBGD complexes of 12C16O, 13C18O, and the difference (12C16O − 13C18O) in the low-frequency (left) and high-frequency (right) regions. (B) Correlation plot between the frequencies of the νFe−CO and νC−O stretching modes. The two solid lines correspond to correlations in proteins with proximal imidazoles (●), proximal imidazolates (▲), thiolate-ligated hemoproteins (◆), and oxidases (□). The data point corresponding to heme−PBGD is presented as an open circle (○).

estimate of 40 kDa (Figure 1B). Except for residues 1−9 at the N-terminus, residues 49−67, and residues 310−315 at the Cterminus, all residues exhibited good electron density. The overall structure of PBGD from V. cholerae was quite similar to that of PBGD from E. coli (rmsd, 0.702 Å for 253 residues).27,28 The PBGD structure comprised three domains of comparable size. Domains 1 and 2 were similar, with a common topology containing a five-stranded β-sheet and three α-helices. By contrast, domain 3 contained a three-stranded antiparallel β-sheet and three α-helices. Cys248, an active site residue located in the deep cleft formed by domains 1 and 2 (Figure 5C), formed a covalent bond with a porphobilinogen dimer, the primer for the elongation reaction of the porphobilinogen chain. Thr49−Leu67, located above the active site, were disordered. This region was also disordered in the crystal structures of PBGD from other organisms (1PDA, 4MLQ, 4MLV, 3EC1, 3ECR), suggesting inherent flexibility in this region. Possible Heme Ligand Mutants. The amino acid sequence of PBGD from V. cholerae was compared with that of a homologue from E. coli, suggesting that Cys248 in PBGD from V. cholerae was present at the active site. Indeed, mutation of Cys248 to serine led to the complete loss of PBGD activity (Table 1). Thus, Cys248 is a possible heme ligand because binding of heme to the active site could prevent the substrate from accessing the active site, thereby decreasing the enzymatic activity; however, the heme titration experiment revealed that the C248S mutant bound to one equivalent of heme, as did the WT PBGD (Figure 6A). The absorption spectrum of the C248S mutant bound to one equivalent of heme and its deconvoluted spectra were indistinguishable from those of heme−WT (Figure 7A). Therefore, Cys248 was assigned to the active site but not the heme-binding residue. This conclusion was supported by the crystal structure of PBGD. In the crystal structure of PBGD, the substrate stretched linearly from Cys248 toward the outside of the substrate-binding pocket (Figure 5B). It appeared that no space was available in the substrate-binding site for heme insertion.

indicated that CO-bound heme−PBGD possessed His but not Cys as a heme axial ligand. Resonance Raman Spectra of the Heme-Reconstituted PBGD. The coordination structure of the heme-bound PBGD was further confirmed by measuring the resonance Raman spectra. The ferric form of heme−PBGD yielded a spinstate marker band, ν3, at 1501 cm−1 with a shoulder at 1491 cm−1 (Figure 4A, spectrum d). The spin-state markers of most hemoproteins occur at 1480−1510 cm−1.24 These frequencies indicated that ferric heme was a mixture of the 6-coordinate low-spin and 5-coordinate high-spin hemes. For the ferrous heme, the ν3 band consisted of two bands at 1469 and 1492 cm−1 (spectrum e). The 1469 cm−1 band was derived from the 5-coordinate high-spin heme, whereas the 1492 cm−1 band was derived from the 6-coordinate low-spin heme. Thus, the ferrous form of heme−PBGD was also a mixture of 5-coordinate and 6coordinate hemes. The spectrum of the CO-bound form revealed two isotope-sensitive bands at 494 and 1964 cm−1. These bands shifted to 486 and 1865 cm−1, respectively, upon substitution with 13C18O (Figure 4A, insets). Therefore, the 494 and 1964 cm−1 bands were assigned to the Fe−CO (νFe−CO) and C−O (νC−O) stretching modes, respectively. The positions of the νFe−CO and νC−O frequencies in the correlation plot provided insight into the donor strength of the trans ligand of the heme-bound CO.25,26 The correlation plot for PBGD was collinear with the values obtained from proteins possessing neutral histidine as heme ligands (Figure 4B). This observation coincided with the absorption spectrum of the CO-bound form (Figure 2C). Overall Structure of PBGD. The crystal structure of PBGD from V. cholerae was determined by molecular replacement with Phenix using the structure of PBGD from E. coli as a search probe. The crystallographic Rwork and Rfree values converged to 23.6% and 27.7%, respectively, and the resolution achieved was 2.7 Å. The collected data and refinement statistics are listed in Table 2. Figure 5 presents the determined X-ray structure of PBGD (PDB entry 5H6O). PBGD existed as a monomer in the crystal, in good agreement with the gel filtration results, which provided a molecular mass D

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ACS Chemical Biology Table 2. Data Collection and Refinement Statistics for PBGDa diffraction source wavelength (Å) temp (K) detector space group unit-cell params (Å) mosaicity (deg) resolution range (Å) total no. reflns no. unique reflns completeness (%) multiplicity average I/σ(I) Rmeasb Wilson B-factor resolution range (Å) no. reflns Rworkc (%) Rfree (%) no. atoms protein ligands solvent rms deviations bond (Å) angle (deg) Ramachandran favored (%) allowed (%) outliers (%) Clashscore

Data Collection BL5A, Photon Factory 1.0000 100 DSC Quantum 315r I4122 a = b = 94.2, c = 165.3 0.118 47.57−2.70 (2.87−2.70) 151113 10562 99.9 (99.8) 14.3 (14.7) 38.82 (6.48) 5.9 (50.2) 59.31 Refinement 35.13−2.70 10537 23.6 27.7

Figure 5. Crystal structure of PBGD. (A) The overall structure of PBGD consisted of three domains: domains 1, 2, and 3 are displayed in blue, cyan, and magenta, respectively. (B) The side view of PGBD. (C) Active site of PBGD. (D) Proposed conformational changes induced by heme binding to Cys105 and His227.

2140 30 20

heme-reconstituted C105S mutant differed from that of the WT enzyme (Figure 7D). A broad Soret band centered at 385 nm (Figure 7C) was comparable to the Soret band measured from free heme (Figure 7D). Deconvolution of the heme− C105S mutant spectrum yielded two Soret bands at 368 and 404 nm (Figure 7C). The position of the latter band was blueshifted by 18 nm comparing with that of heme−WT PBGD but was close to the Soret band measured from the 5-coordinate heme centers in catalase and horseradish peroxidase.29 The Kd,heme of the C105S mutant (0.73 ± 0.63 μM−1) was comparable to that of the WT protein. However, the equivalence point of the titration shifted to less than 1.0 (Figure 6C). Although the heme titration plot revealed that the C105S mutant still bound heme, the absorption spectrum suggested that Cys105 was involved in heme binding in the WT PBGD. The presence of the Soret band at 422 nm in the UV−visible spectrum (Figure 2C) and the νFe−CO versus νC−O resonance Raman spectra correlation plot (Figure 4A) for the CO-bound form of heme−PBGD suggested that histidine was also a possible heme ligand of heme−PBGD. Unlike Bach1, which includes at least four heme-binding sites,18 PBGD bound to only one equivalent of heme (Figure 2B, inset). These results suggested that PBGD contained one heme-binding site with two different coordination geometries, 6- and 5-coordinate hemes, but not two different heme-binding sites. One site featured a 6-coordinate heme and the other site featured a 5coordinate heme, indicating that histidine was present near Cys105. A closer look at the crystal structure of PBGD suggested that His227 was located proximal to Cys105 (Figure 5A). Thus, we replaced His227 with alanine or leucine to confirm whether His227 was the sixth ligand of heme. Neither mutant could be expressed. Instead, an aspartic acid mutant (H227D) was expressed and purified well. The heme titration plot of the H227D mutant more closely resembled that of the C105S mutant than of the WT PBGD. Although the titration

0.002 0.68 98.19 1.81 0 8.79

a

Values for the outer shell are given in parentheses. bRmeas= ∑hkl{N(hkl)/[N(hkl) − 1]}1/2∑i|Ii(hkl) − I(hkl)|/∑hkl∑iIi(hkl), where Ii(hkl) and N(hkl) are the mean intensity of a set of equivalent reflections and the multiplicity, respectively. cRwork(%) = ∑hkl|| Fobs(hkl)| − |Fcalc(hkl)||/∑hkl|Fobs(hkl)|.

The position of the Soret band in the absorption spectrum of ferric heme−PBGD suggested Cys- and His-coordination to heme−PBGD (Figure 2D). The solved crystal structure of PBGD (Figure 5A) included seven cysteines, two (Cys105 and Cys211) of which were located close to histidine. The hemebinding site was identified by mutating each of Cys105 and Cys211 with serine, and the heme-binding ability was evaluated (Figure 6). The difference spectra generated by subtracting the spectra of the solution containing the C211S mutant from the spectra of the buffer titrated with heme were indistinguishable from those of the wild-type (WT) protein (Figure 6B). The deconvoluted spectrum of the heme−C211S mutant (Figure 7B) revealed that the peak position of the major band at 372 nm coincided with that of heme−WT PBGD (Figure 2D). In addition, the Kd,heme of the C211S mutant was 0.53 ± 0.19 μM being close to that of WT PBGD. Thus, Cys211 was not a heme ligand, although the mutation led to some perturbation of the heme coordination, considering that the position of the minor band was blue-shifted to 412 nm. By contrast, the absorbance at 372 nm in the C105S mutant difference spectra was significantly reduced (Figure 6C). Furthermore, the absorption maximum in the absorption spectrum of the E

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Figure 6. Heme titration of PBGD mutants. Absorption difference spectra of heme−PBGD. (Inset) The difference at 417 or 421 nm following the incremental addition of heme (1−30 μM) to PBGD (10 μM) in 50 mM Tris-HCl, 150 mM NaCl (pH 8.0) against a blank cell containing buffer alone. (A) C248S, (B) C211S, (C) C105S, and (D) H227D.

tary Figure 5). Thus, CN coordinated to heme and His227 was concomitantly released from the 6-coordinate heme. The PBGD activity of the CN-bound heme-PBGD was 2.00 ± 0.01 μmol h−1 mg−1, almost identical to the activity of PBGD alone (Table 1), indicating that elimination of His227-coordination from heme led to a loss of the heme binding effect on the PBGD activity. Effect of Heme Binding on the Structure of PBGD. The mechanism underlying the measured decrease in the PBGD activity upon heme binding was explored by investigating the structural changes associated with heme binding. Because the heme complex of PBGD has not been crystallized, we first measured the far-UV CD spectra to estimate the secondary structural changes experienced by the protein. The CD spectrum of PBGD included a characteristic feature with a minimum at 222 nm, a negative shoulder at 209 nm, and a maximum at 193 nm, indicating that the protein was rich in αhelix structures (Figure 8A).30 The α-helical content estimated from mean residue ellipticity at 222 nm was 29−30%, comparable to that obtained from the solved crystal structure. The addition of up to 2 equiv of heme to PBGD did not alter the CD spectrum. These results indicated that heme binding to PBGD induced almost no changes in the secondary structure. Heme-induced oligomerization has been reported in some proteins.31,32 The oligomerization status of PBGD was next evaluated using a gel filtration column to confirm whether quaternary structural changes occurred upon heme binding. In the absence of heme, PBGD was eluted at 24.0 mL (Figure 8B). Complex formation with heme did not change the retention volume; therefore, binding of heme to PBGD did not cause

plot suggested that the H227D mutant bound to one equivalent of heme (Figure 6D), the affinity for heme (Kd,heme of 3.8 ± 0.5 μM) was much lower than that of the WT protein. In addition, the absorption spectrum of the mutant revealed that the absorbance at 413 nm was significantly reduced (Figure 7E). Deconvolution of the Soret band of the heme−H227D mutant into two Gaussian components provided two maxima at 375 and 416 nm (Figure 7E). The former peak was similar to that obtained from the heme−WT, whereas the latter was blueshifted by 6 nm. The 422 nm species corresponded to a heme with Cys/His coordination, whereas the 416 nm species corresponded to a heme with Cys/H2O coordination, as observed in substrate-free P450. 20 These observations suggested that Cys105 and His227 were a pair of hemecoordinating ligands. The PBGD activity of the C105S and H227D mutants was 2.08 ± 0.19 and 2.05 ± 0.05 μmol h−1 mg−1, respectively (Table 1, Supplementary Figure 3). Furthermore, binding of heme to these mutants did not affect the activity, supporting the idea that Cys105 and His227 were the heme ligands. A mutational study suggested that the formation of the 6coordinate heme was related to the effect of heme on the PBGD activity. To confirm this hypothesis, we mutated Cys105 to histidine to increase the amount of 6-coordinate heme; however, the absorption spectrum of the heme-reconstituted C105H mutant was typical of the 5-coordinate species (Supplementary Figure 4). Next, to eliminate the proteinbound 6-coordinate heme, we added cyanide (CN) ion to heme−PBGD. The addition of CN ion to heme−PBGD caused a red shift in the Soret band to 421 nm, indicating the formation of the CN-bound 6-coordinate heme (SupplemenF

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Figure 7. Deconvoluted absorption spectra of the heme complex of PBGD. The absorption spectra of heme−PBGD in the Soret region were deconvoluted using two-Gaussian functions. (A) C248S, (B) C211S, (C) C105S, (D) hemin, and (E) H227D mutants.

accessibility of the substrate to the active site was not perturbed upon heme binding. Only the Cys105 mutant displayed a different heme titration plot (Figure 6), indicating that Cys105 was the heme-binding site, even though heme nonspecifically bound to the C105S mutant (Figure 6C). Nonspecific heme binding to Cys mutants, which prevents determination of the heme-binding site, was often encountered in some heme proteins such as Bach118 and HBP23.33 However, the C105S mutant lost its PBGD activity dependence on heme (Table 1, Supplementary Figure 3); therefore, heme most likely functioned as a regulatory molecule only when bound to Cys105. Cys105 is located approximately 20 Å from the active site (Figure 5). Therefore, heme did not directly inhibit the binding of substrates to the active site. The CD spectrum and oligomerization status were not affected by heme binding (Figure 8), indicating that the secondary and tertiary structures of PBGD remained intact upon heme binding. A prominent tertiary structural change that could

detectable quaternary structural changes, including oligomerization.

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DISCUSSION

PBGD Activity Regulatory Mechanism upon Heme Binding. In this study, we found for the first time that heme could specifically bind to PBGD through Cys105, and binding of heme to PBGD decreased the PBGD activity by approximately 15% (Figure 3, Table 1). Unfortunately, we have not succeeded in crystallization of the heme-bound PBGD, probably owing to inhomogeneity of the heme coordination structure. Thus, it is difficult to determine the mechanism of the heme-dependent regulation of the PBGD activity. One possible explanation of the reduced activity upon heme binding is the limited accessibility of the substrate to the active site. However, considering that the Km of heme−PBGD was slightly smaller than that of PBGD alone (Figure 3), G

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ACS Chemical Biology

species. This heme binding action would cause perturbation of the extension of porphobilinogen. Deconvolution of the absorption spectrum of the hemebound PBGD provided an estimate for the ratio between the 5coordinate and 6-coordinate species, 2.5:1 (Figure 2D). If the activity of the 6-coordinate species were 60−70% the activity of PBGD prepared without heme, the total activity of heme− PBGD corresponded to 90% the activity of PBGD, as observed, even if the 5-coordinate species retained its activity. Role of Heme Binding to PBGD. The apparent Kd value of heme to PBGD was 0.33 μM (Figure 2B), well within the range of values obtained from other heme-binding proteins, such as 1-Cys peroxiredoxin (Kd = 0.17 μM),19 glutathione Stransferase (Kd = 0.52 μM),35,36 and ChuX (Kd = 2.0 μM).37 The moderate affinity of PBGD for heme suggested a regulatory role of heme in the PBGD activity. The absorption spectrum of the ferric heme-bound PBGD was quite similar to the spectra obtained from of Bach1,18 Bach2,38 circadian factor period 2 (Per2),39 and 2-Cys peroxiredoxin.19 The heme-binding sites of these proteins appear to share a common motif involving Cys and Pro (CP motif). The CP motif has recently received increasing attention as an important motif in heme-binding and heme-regulated proteins.40−43 For example, binding of heme to Bach1 induces a conformational change that inhibits DNA binding;18 however, PBGD from V. cholerae possesses no CP motif. The short sequence from Leu101 to Pro110 (L101VTICEREDP110) containing Cys105 of PBGD shares no sequence homology to the previously reported heme regulatory motif;42,44 therefore, this study expands our understanding of the Cyscontaining heme-regulatory motif that utilizes heme as a signaling molecule. Biological Implications of the Heme-Dependent Regulation of PBGD Activity. In this study, we found that heme binding to PBGD decelerated the formation of 1hydroxymethylbilane by PBGD. 1-Hydroxymethylbilane was spontaneously cyclized to uroporphyrinogen I.45 Heme was not synthesized from uroporphyrinogen I because it was not converted to uroporphyrinogen III, a precursor of heme. Thus, accumulation of uroporphyrinogen I is proposed to be toxic to cells;46 therefore, it is plausible that an excess of heme represses the synthesis of 1-hydroxymethylbilane by binding to PBGD. The degree of PBGD activity inhibition upon heme binding, by as much as 15%, is modest. Binding of heme to ALAS is known to repress the catalytic activity of ALA synthesis,47,48 which keeps the heme concentration low. The degree of repression is approximately 50%.9 Considering these results, our finding that heme affects PBGD activity is significant. Although the biosynthesis of heme involves a multistep reaction that includes more than eight enzymes,3−5 it is not known which step is the rate-determining step in V. cholerae. To ascertain the effectiveness of heme binding to PBGD as a feedback mechanism of heme biosynthesis, further studies of other heme synthesis enzymes are needed. In conclusion, PBGD was found to bind to one equivalent of heme with an apparent Kd,heme of 0.33 μM. The heme-binding site consisted of Cys105 and His227. Formation of the 6coordinate heme induced a slight structural shift in domain 1 containing Cys105 and domain 3 containing His227. This change appeared to restrict access of the substrates to the active site, thereby decreasing the PBGD activity. Heme itself was found to be an effector of heme biosynthesis through PBGD via a negative feedback mechanism. Because both Cys105 and

Figure 8. Effect of heme on the secondary and tertiary structures of PBGD. (A) CD spectra of PBGD. CD spectra in the far-UV region of 3 μM PBGD containing no heme (red) and 2 equiv of heme (blue) in 50 mM sodium phosphate (pH 8.0). (B) Gel filtration elution profile. The elution of protein was monitored by measuring the absorption at 280 nm. The total protein concentration was 10 μM in 50 mM TrisHCl, 150 mM NaCl (pH 8.0).

have decreased the activity did not occur upon heme binding to PBGD. Although the 6-coordinate heme was a minor component (Figure 2D), the presence of the 6-coordinate species suggested that heme did not bind to the surface of the protein but rather bound to the interior of the protein. The estimated hemebinding site was mapped onto the crystal structure of PBGD (Figure 5A, B). Cys105 was located at the interface between domains 1 and 2, whereas His227 belonged to domain 3 (Figure 5A, B). The side chain of Cys105 pointed away from His227 (Figure 5D); therefore, His227 binding to heme to form a 6-coordinate geometry was expected to induce a concerted displacement of domain 3 toward domains 1 and 2. The addition of CN to heme−PBGD induced release of His227 from the heme and conversion of Cys/His heme coordination to CN-bound 6-coordinate heme (Figure S4). The PBGD activity of the CN-bound heme was indistinguishable from that of PBGD alone (Table 1). These results clearly supported the idea that coordination of His227 was essential for suppressing the enzymatic activity of PBGD. An MD simulation study proposes that either the active site loop or domain 2 adjusts the active site cleft to accommodate the growing pyrrole chain.34 Because the putative heme-binding site of PBGD is between domains 2 and 3 (Figure 5A), it is likely that coordination of His227 results in reorientation of the domain−domain interaction (Figure 5D), which leads to reduction of the activity upon heme binding by inhibiting the conformational change for growing pyrrole chain. Taken together, heme bound to Cys105, which induced binding of His227 to the Cys105-bound heme to form a 6-coordinate H

DOI: 10.1021/acschembio.7b00934 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology His227 are conserved in PBGD from E. coli, our finding is not specific to PBGD from V. cholerae.

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chromator (SPEX500M, Jobin Yvon) equipped with a liquid nitrogencooled CCD detector (Spec-10:400B/LN, Roper Scientific). Samples were excited at 413.1 and 441.6 nm using a krypton ion laser (BeamLok 2060, Spectra Physics) and a helium−cadmium laser (IK5651R, Kimmon Koha), respectively. The laser power at the sample point was adjusted to ∼5 mW for the ferric and ferrous forms. A lower power (0.1 mW) was used to excite the CO-bound form to prevent photodissociation. Raman shifts were calibrated using indene, CCl4, acetone, and an aqueous solution of ferrocyanide. The welldefined Raman band peak positions were measured with an accuracy of ±1 cm−1. The sample concentration used for the resonance Raman measurements was approximately 10 μM in 50 mM Tris-HCl, 150 mM NaCl (pH 8.0). Circular dichroism (CD) spectra were measured using a Jasco J1500 CD spectrometer over the spectral range 190−250 nm using a quartz cell with a path length of 1 mm at RT. The sample was diluted to a final concentration of 5 μM in 50 mM sodium phosphate, 150 mM NaCl (pH 8.0). The α-helix content ratio ( f H) was estimated from the mean residue ellipticity [θ] at 222 nm using the following equation:30

METHODS

Materials. The chemicals used in this study were purchased from Wako Pure Chemical Industries, Nacalai Tesque, or Sigma-Aldrich, and used without further purification. Protein Overexpression and Purification. A full-length hemC gene construct was purchased from the PlasmID Repository (http:// plasmid.med.harvard.edu/PLASMID/Home.xhtml; Clone ID VcCD00034062) and amplified by PCR using primers 5′-CGC CAT ATG GAT AGG AAC ATC ATA ATG ACC-3′ (forward) and 5′CGG GAA TTC TCA TTC GTG GTC ACA GTA GAG-3′ (reverse). The amplified fragment was cloned into the pET-28b vector (Merck Millipore) using the NdeI and EcoRI restriction sites, and the thrombin recognition site composed of Leu-Val-Pro-Arg-Gly-Ser of the pET-28b vector was mutated to the HRV 3C protease recognition site composed of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro, as described previously.49 The correct gene sequence was confirmed by DNA sequencing (Eurofins Genomics Inc.). E. coli strains carrying plasmids for hemC were grown at 37 °C in LB medium supplemented with 50 μg mL−1 kanamycin. Expression of the His6-tagged fusion protein in E. coli was induced with 0.8 mM isopropyl-β-D-thiogalactopyranoside after reaching an optical density at 600 nm (OD600) of 0.6−1.0. The culture was further grown at 28 °C overnight. The cells were harvested by centrifugation and stored at −80 °C until use. The pellet was thawed on ice, suspended in a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Nonidet P-40, 1 mg mL−1 lysozyme, and DNase (pH 8.0)), and incubated on ice for 60 min. The sample was centrifuged at 40 000 × g for 30 min, and the resulting supernatant was loaded onto a HisTrap HP column (GE Healthcare) pre-equilibrated with 50 mM Tris-HCl buffer containing 500 mM NaCl and 20 mM imidazole (pH 8.0). The resin was extensively washed (50 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole (pH 8.0)), and the bound protein was eluted in 50 mM Tris-HCl buffer containing 500 mM NaCl and 200 mM imidazole (pH 8.0). The eluted PBGD was incubated with Turbo3C protease (Accelagen) at 4 °C for ∼16 h to remove the His6-tag. After cleavage, the reaction mixture was again applied to the HisTrap column. The column flow-through was collected and applied to a HiLoad 16/60 Superdex 200 preparatory grade gel filtration column (GE Healthcare) equilibrated with 50 mM Tris-HCl, 150 mM NaCl (pH 8.0). The protein purity was assessed by SDS-PAGE on 12.5% polyacrylamide gels. Mutagenesis was conducted utilizing a PrimeSTAR mutagenesis basal kit from Takara Bio (Otsu, Japan). DNA oligonucleotides were purchased from Eurofins Genomics. The primers employed for mutation are listed in Supplementary Table 1. The introduced mutation was verified by sequencing (Eurofins Genomics). Measurement of Heme Binding to PBGD. Heme binding was tracked by differential absorption spectroscopy. Successive aliquots of 0.5 mM hemin in 0.1 M NaOH were added to both the sample cuvette containing 10 μM PBGD and the reference cuvette containing buffer alone. Spectra were recorded 3 min after the addition of each heme aliquot. The absorbance difference at 417 or 421 nm was plotted as a function of the heme concentration, and the dissociation constant (Kd,heme) was calculated using the equation

fH (%) = (− ([θ ]222 + 2340)/30300) × 100

Size Exclusion Chromatography. Oligomerization of the protein was analyzed using an ENrich SEC 650 gel filtration column (Bio-Rad) equilibrated with 50 mM Tris-HCl, 150 mM NaCl (pH 8.0). The elution profile was monitored at 280 and 380 nm. Standards with known molecular masses (ferritin 440 000 Da; catalase 232 000 Da; aldolase 158 000 Da; bovine serum albumin 66 000 Da; ovalbumin 43 000 Da; ribonuclease A 13 700 Da; and blue dextran 2000 kDa) (Gel Filtration Calibration Kits, GE Healthcare) were applied to the column. Crystallization. Crystallization of PBGD was carried out using a sitting drop vapor diffusion method at 4 °C, in which drops of 4 μL were prepared by adding 2 μL of 10 mg mL−1 protein and 2 μL of a crystallization solution. The best diffraction-quality crystals grew from 0.2 M magnesium nitrate and 20% (w/v) PEG 3350. Crystals were soaked in a crystallization solution containing 20% ethylene glycol as a cryoprotectant and were then frozen in liquid nitrogen for cryogenic data collection. Structure Determination. X-ray diffraction data were collected at 100 K on the BL5A beamline of the Photon Factory (Tsukuba, Japan) using DSC Quantum 315r CCD detector. The wavelength of the incident X-ray was 1.0 Å. Diffraction data were processed and scaled with XDS.50 The crystal belonged to the space group P4122 with unit cell dimensions of a = b = 94.2 Å and c = 165.3 Å. The structure was determined using the molecular replacement method using the program Phaser-MR51 along with the previously reported structure of PBGD from E. coli (PDB ID code 1GTK) as a search model. A significant solution was identified with a TFZ of 23.1, and automatic model building was performed using the program AutoBuild (Terwilliger TC, Acta D, 2008). The programs Coot52,53 and phenix.refine54 were used for manual model building and automatic refinement, respectively. To monitor the refinement, a random 5% subset of structures was set aside to calculate the Rfree factor. The diffraction and refinement statistics are summarized in Table 2. The coordinates and structure factors of PBGD were deposited in the Protein Data Bank with the ID code 5H6O. PBGD Assay. PBGD activity was assayed essentially as described previously.55 Briefly, 100 nmol of porphobilinogen was added to 6.7 μM enzyme solution in 50 mM Tris-HCl, 150 mM NaCl (pH 8.0) to a final volume of 450 μL. The reaction mixture was incubated at 30 °C for 5 min, and the reaction was stopped by adding 125 μL of 5.0 M HCl. The product was cyclized by adding 50 μL of 0.1% (w/v) benzoquinone at 0 °C and incubating for 20 min. Then, 0.1 mL of the supernatant obtained after centrifugation at 15 000 rpm for 5 min was diluted to 1.0 mL with 1.0 M HCl. The quantity of product was determined from the absorbance spectrum using a molar absorption coefficient of 5.48 × 105 M−1 cm−1 at 405 nm. The enzymatic activity was expressed as micromoles of uroporphyrin I formed per hour per

ΔA = ΔA max [P] + [H] + Kd,heme −

(2)

([P] + [H] + Kd,heme)2 − 4[P][H] 2[P] (1)

where ΔA is the absorption difference between the sample and reference cells upon the addition of hemin, ΔAmax is the maximum of the absorption difference upon the addition of hemin, and [P] and [H] represent the total protein and hemin concentrations, respectively. Spectroscopy. Optical spectra of the purified protein were recorded using a V-660 spectrophotometer (Jasco) at 25 °C. Resonance Raman spectra were obtained using a single monoI

DOI: 10.1021/acschembio.7b00934 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology milligram of the enzyme. The effect of heme was monitored in the presence of 3 equiv of heme. An excess amount of heme was added to completely convert PBGD to the heme-bound form. It was confirmed that a heme solution did not show PBGD activity. Michaelis−Menten plot was drawn as previously reported.56 To prepare the heme-bound form, we added 3 equiv of heme to the PBGD solution. In the presence of 3 equiv of heme, approximately 98% of PBGD was estimated to be in a heme-bound form on the basis of the obtained Kd,heme value. To avoid the effect of alkaline, in which heme was dissolved, we added the same amount of an alkaline solution to the sample in the absence of heme.

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(6) Layer, G., Reichelt, J., Jahn, D., and Heinz, D. W. (2010) Structure and function of enzymes in heme biosynthesis. Protein Sci. 19, 1137−1161. (7) Beale, S. I., and Castelfranco, P. A. (1973) 14C incorporation from exogenous compounds into δ-aminolevulinic acid by greening cucumber cotyledons. Biochem. Biophys. Res. Commun. 52, 143−149. (8) Jahn, D., Verkamp, E., and Söll, D. (1992) Glutamyl-transfer RNA: a precursor of heme and chlorophyll biosynthesis. Trends Biochem. Sci. 17, 215−218. (9) Yamauchi, K., Hayashi, N., and Kikuchi, G. (1980) Translocation of δ-aminolevulinate synthase from the cytosol to the mitochondria and its regulation by hemin in the rat liver. J. Biol. Chem. 255, 1746− 1751. (10) Srivastava, G., Borthwick, I. A., Brooker, J. D., Wallace, J. C., May, B. K., and Elliott, W. H. (1983) Hemin inhibits transfer of pre-δaminolevulinate synthase into chick embryo liver mitochondria. Biochem. Biophys. Res. Commun. 117, 344−349. (11) Munakata, H., Sun, J.-Y., Yoshida, K., Nakatani, T., Honda, E., Hayakawa, S., Furuyama, K., and Hayashi, N. (2004) Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase. J. Biochem. 136, 233−238. (12) Scholnick, P. L., Hammaker, L. E., and Marver, H. S. (1972) Soluble δ-aminolevulinic acid synthetase of rat liver. II. Studies related to the mechanism of enzyme action and hemin inhibition. J. Biol. Chem. 247, 4132−4137. (13) Kitatsuji, C., Ogura, M., Uchida, T., Ishimori, K., and Aono, S. (2014) Molecular mechanism for heme-mediated inhibition of 5aminolevulinic acid synthase 1. Bull. Chem. Soc. Jpn. 87, 997−1004. (14) Hamza, I., Chauhan, S., Hassett, R., and O'Brian, M. R. O. (1998) The bacterial Irr protein is required for coordination of heme biosynthesis with iron availability. J. Biol. Chem. 273, 21669−21674. (15) Qi, Z., Hamza, I., and O’Brian, M. R. (1999) Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein. Proc. Natl. Acad. Sci. U. S. A. 96, 13056−13061. (16) Heidelberg, J. F., Eisen, J. a, Nelson, W. C., Clayton, R. a, Gwinn, M. L., Dodson, R. J., Haft, D. H., Hickey, E. K., Peterson, J. D., Umayam, L., Gill, S. R., Nelson, K. E., Read, T. D., Tettelin, H., Richardson, D., Ermolaeva, M. D., Vamathevan, J., Bass, S., Qin, H., Dragoi, I., Sellers, P., McDonald, L., Utterback, T., Fleishmann, R. D., Nierman, W. C., White, O., Salzberg, S. L., Smith, H. O., Colwell, R. R., Mekalanos, J. J., Venter, J. C., and Fraser, C. M. (2000) DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406, 477−483. (17) Jordan, P. M., Warren, M. J., Williams, H. J., Stolowich, N. J., Roessner, C. A., Grant, S. K., and Scott, A. I. (1988) Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase. FEBS Lett. 235, 189−193. (18) Hira, S., Tomita, T., Matsui, T., Igarashi, K., and Ikeda-Saito, M. (2007) Bach1, a heme-dependent transcription factor, reveals presence of multiple heme binding sites with distinct coordination structure. IUBMB Life 59, 542−551. (19) Watanabe, Y., Ishimori, K., and Uchida, T. (2017) Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding. Biochem. Biophys. Res. Commun. 483, 930− 935. (20) Dawson, J. H., Andersson, L. A., and Sono, M. (1982) Spectroscopic investigations of ferric cytochrome P-450-CAM ligand complexes. Identification of the ligand trans to cysteinate in the native enzyme. J. Biol. Chem. 257, 3606−3617. (21) Liu, Y., Moënne-Loccoz, P., Hildebrand, D. P., Wilks, A., Loehr, T. M., Mauk, A. G., and Ortiz de Montellano, P. R. (1999) Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase. Biochemistry 38, 3733−3743. (22) Sekine, Y., Tanzawa, T., Tanaka, Y., Ishimori, K., and Uchida, T. (2016) Cytoplasmic heme-binding protein (HutX) from Vibrio cholerae is an intracellular heme transport protein for the hemedegrading enzyme, HutZ. Biochemistry 55, 884−893.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00934. Heme biosynthesis process, absorption spectra of the reaction product of PBGD, Michaelis−Menten plot, heme-bound C105H mutant PBGD and CN-bound form of heme-WT PBGD, and primers used for the construction of expression vectors for mutants (PDF) Accession Codes

The structure factors and coordinates for the PBGD structure have been deposited in the Protein Data Bank under the accession code 5H6O.

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AUTHOR INFORMATION

Corresponding Author

*Takeshi Uchida. E-mail: [email protected]. ORCID

Takeshi Uchida: 0000-0001-9270-8329 Present Address #

Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by Grants-in-Aid for Scientific Research (16K05835 to T.U., 16H00748 and 24000011 to Y.T., and 15H00909 to K.I.) from the Ministry of Culture, Education, Sports, Science, and Technology (MEXT) of Japan.

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ABBREVIATIONS WT, wild-type; PBG, porphobilinogen; PBGD, PBG deaminase; ALA, 5-aminolevulinic acid REFERENCES

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DOI: 10.1021/acschembio.7b00934 ACS Chem. Biol. XXXX, XXX, XXX−XXX