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In vitro characterization of a recombinant Blh protein from an uncultured marine bacterium as a β-carotene 15,15′-dioxygenase

Yeong-Su Kim,1 Nam-Hee Kim,1 Soo-Jin Yeom,1 Seon-Won Kim,2 and Deok-Kun Oh1,* Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701,1 Division of

Applied Life Science (BK21), EB-NCRC and PMBBRC, Gyeongsang National University, Jinju 660-701, Korea 2

Running title: Characterization of bacterial β-carotene 15,15′-dioxygenase Key words: Blh protein; Characterization; Retinal; β-Carotene cleavage; Substrate specificity; Dioxygenase. Address correspondence to: Deok-Kun Oh, Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, South Korea. Tel. 82-2-454-3118; Fax. 82-2-444-6176; E-mail: deokkun@konkuk.ac.kr.

Codon-optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular weight of the native purified enzyme was approximately 64 kDa as a dimer of 32 kDa subunits. The Km, kcat, and kcat/Km for β-carotene as substrate were 37 μM, 3.6 min−1, and 97 mM−1min−1, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.

Vitamin A (retinol) is a fat-soluble vitamin and

important for human health. It affects tissue development, growth, vision, and immunology. β-Carotene is the most potent vitamin A precursor of all provitamin A carotenoids. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1,2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and then retinal is converted to vitamin A in vivo (3-5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2). β-Carotene 15,15′-monooxygenase was first

isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4,8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5,9,10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).

Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.002618The latest version is at JBC Papers in Press. Published on April 14, 2009 as Manuscript M109.002618

Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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(Brp) and bacteriorhodopsin-related-protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes; bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin (PR) gene cluster, which contains PR, crtE (geranylgeranyl diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23-28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29,30), but there is no direct proof of the enzymatic activity.

In this study, we used codon-optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.

EXPERIMENTAL PROCEDURES

Reagents−The expression vector pET-24a(+) was purchased from Novagen (San Diego, CA). The expression host, E. coli ER 2566, and all restriction enzymes were purchased from New England Biolabs (Hertfordshire, UK). Luria-Bertani (LB) medium was purchased from BD Biosciences (San Jose, CA). β-Carotene and the

other carotenoid substrates were purchased from Sigma (St. Louis, MO) and Carotenature (Lupsingen, Switzerland), respectively. The pre-stained ladder for SDS-PAGE and the gel filtration calibration kit were purchased from MBI Fermentas (Hanover, MD) and Amersham Biosciences (Uppsala, Sweden), respectively. Western blot detection system and His-tagged monoclonal antibody were purchased from Intron Biotechnology (Seongnam, Gyeonggi, Korea) and Bio-Rad Laboratories (Hercules, CA), respectively. Isotopically labeled water (H2

18O) and molecular oxygen (18O2) were purchased from Sigma and Cambridge Isotope Laboratories (Andover, MA), respectively. All other regents were purchased from Sigma.

DNA cloning and site-directed

mutagenesis−The amino acid sequence used for codon optimization was obtained from the blh gene of the uncultured marine bacterium 66A03 (GenBankTM Accession number No. AAY68319). Codons of the blh gene sequence were optimized by selection based on probabilities obtained from the codon usage table without consideration of internal mRNA secondary structures or DNA repeats (31). The entire gene was synthesized (Genofocus, Daejeon, Korea), cloned into the pET-24a(+) expression vector using the restriction enzymes EcoRI and XhoI, and transformed into E. coli ER2566 as an expression host. Mutations of the conserved four histidine residues in the Blh protein were generated by site-directed mutagenesis using the QuickChange kit and protocol (Stratagene, Beverly, MA). DNA sequencing was performed at the Macrogen facility (Seoul, Korea).

Expression and purification−The recombinant

E. coli for expression of the wild-type and mutant enzymes was cultivated in 500 ml of LB medium (1.0% tryptone, 0.5% yeast extract, and 1.0% sodium chloride) in a 2,000 ml flask containing 20 μg/ml kanamycin at 37ºC with shaking at 200 rpm. When the optical density of the culture reached 0.5 at 600 nm, isopropyl-β-D-thiogalctopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce expression of the recombinant enzyme, and the culture was incubated at 16ºC for 16 h. The cells were

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harvested from the culture broth by centrifugation at 6,000 × g for 30 min at 4ºC, washed twice with 0.85% NaCl, and then resuspended in a lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) containing 1 mg/ml lysozyme. The resuspended cells were disrupted using a sonicator. The cell debris was removed by centrifugation at 13,000 × g for 20 min at 4ºC, the supernatant was filtered through a 0.45 μm filter. The filtrate applied to His-Trap HP affinity chromatography (Amersham Biosciences) equilibrated with 50 mM sodium phosphate buffer containing 300 mM NaCl. The column was washed extensively with the same buffer, and the bound protein was eluted with a linear gradient between 10 to 200 mM imidazole at a flow rate of 1 ml/min. The purification step using chromatography was carried out in a cold room at 4ºC with a fast protein liquid chromatography (FPLC) system (Bio-Rad Laboratories, Hercules, CA). The active fractions were collected and dialyzed against 100 mM Tricin-KOH buffer (pH 8.0). After dialysis, the resulting solution was used as the purified enzyme.

Amino acid sequencing−Partial amino acid

sequences of the Blh protein were investigated. After separation via SDS-PAGE and staining of the sample, each chosen band was isolated, destained, and washed. In-gel digestion was then performed with 500 ng of sequencing-grade chymotrypsin in 100 mM Tris-HCl buffer (pH 8.0) with 10 mM CaCl2 at 37 °C for 16 h. The digested peptides were extracted with 5% formic acid in acetonitrile and cleared by centrifugation at high speed for 5 min. The supernatant was dried in a SpeedVac for mass analysis. After desalting with Zip-Tip filter (Millipore, Billerica, MA) the digested peptides were loaded onto a fused silica microcapillary C18 column (75 µm × 150 mm).

LC separation was conducted using linear-gradient elution with 0.1% formic acid in H2O (solvent A) and formic acid in acetonitrile (solvent B). The elution program was as follows: a solvent composition of 97:3 (A:B) from 0 to 5 min; 60:40 from 5 to 72 min; 10:90 from 72 to 87 min; and 97:3 from 87 to 120 min. The flow rate was a constant 200 nl/min. The separated peptides were subsequently analyzed on a model LTQ linear ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA). The electrospray voltage was set to 2.0

kV, and the threshold for switching from MS to MS/MS was 250. The normalized collision energy for MS/MS was 35% of the main RF amplitude, and the duration of activation was 30 ms. All spectra were acquired in data-dependent mode. Each MS scan was followed by MS/MS scans of the three most intense peaks from the full MS scan. The repeat count of peak for dynamic exclusion was 1, and its repeat duration was 30 s. The dynamic exclusion duration was set to 180 s, and the exclusion mass width was ±1.5 Da. The list size of dynamic exclusion was 50.

Database analysis‒ Database searches were

performed for all MS/MS spectra using an E. coli protein database with the addition of the amino acid sequence of the Blh protein. SEQUEST was used as the peptide searching program, which included the dynamic modifications of oxidized methionine (+16 Da) and carboxyamidomethylated cysteine (+57 Da). SEQUEST criteria for peptide selection were based on the values of Xcorr, which requires values greater than 1.8, 2.3, and 3.5 for +1, +2, and +3 charge state peptides, respectively, with a delta Cn above 0.1. The criterion for protein selection was a consensus score above 10.1.

Western blot analysis−The soluble fraction of

recombinant E. coli lysate was subjected to Western blot analysis. The fraction was separated by SDS-PAGE on a 12% gels and then transferred to a PVDF membrane using a Mini-Protean II transfer apparatus (Bio-Rad Laboratories) according to the manufacturer’s instructions. For immunodetection, the membranes were first blocked with 5% (w/v) skim milk in phosphate-buffered saline containing Tween 20 (PBST) and then incubated with His-tagged monoclonal antibody for 1 h. The membrane was visualized using a WEST-oneTM Western blot detection system. The membranes were then placed between two overhead transparency films and exposed to Kodak film.

Determination of molecular weight−The

subunit molecular weight of the Blh protein from uncultured marine bacterium 66A03 was examined by SDS-PAGE under denaturing conditions, using the proteins of a prestained

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ladder as reference proteins. All protein bands were stained with Coomassie blue for visualization.

The molecular weight of the native enzyme was determined using Sephacryl S-300 HR gel filtration chromatography (Amersham Biosciences). The purified enzyme solution was applied to the chromatography and eluted with 100 mM Tricin-KOH buffer (pH 8.0) containing 0.1 M NaCl at a flow rate of 0.3 ml/min. The column was calibrated with aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and RNase A (13.7 kDa) as reference proteins and the native enzyme was calculated by comparing with migration length of reference proteins.

Enzyme assay−β-Carotene of 200 μM was

dispersed in 2 ml toluene containing 1.2% (v/v) Tween 80 to form detergent micelles, and the solution was homogenized at 10,000 rpm for 10 s using a homogenizer (IKA, Kuala Lumpur, Malaysia). The toluene was evaporated by N2 gas at room temperature and then 2 ml water was added. The resulting solution was used as the substrate solution. The substrate and enzyme solutions were mixed with the ratio of 1 : 3 (v/v) and then the mixture was used as the reaction solution. The reaction solution contained 50 μM β-carotene, 0.3% (w/v) Tween 80, 125 mM sodium chloride, 10 μM Fe2SO4, 5 mM Tris(2-carboxylethyl)phosphine hydrochloride (TCEP), and 1.0% (w/v) 1-S-octyl-β-D-thioglucopyranoside (OTG). The enzyme reaction was performed in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene and 0.04 U/ml enzyme at 40ºC for 60 min. After incubation, the reaction was stopped by 3.7% (v/v) formaldehyde and additional incubation was done at 40ºC for 10 min (32). The recovery percentage of retinal was varied under the various conditions as temperature was affecting the recovery. One unit (U) of enzyme activity was defined as the amount of enzyme required to produce 1 μmole retinal per min at 40ºC and pH 8.0.

Effects of metal ions, pH, and temperature on

enzyme activity− The Blh protein was obtained after incubating at 20ºC with 1 mM phenanthroline as an iron chelating agent for 1 h

and following by overnight dialysis at 4ºC against 100 mM Tricine-KOH buffer (pH 8.0), and then the effects of various metal ions on its activity were investigated in the presence of 10 μM Fe2+, Fe3+, Co2+, Ca2+, Cu2+, Mn2+, or Ba2+. The effect of Fe2+ was evaluated with and without phenanthroline treatment. To examine the effect of pH on the enzyme activity, the pH was varied between 6.0 and 9.0 using 100 mM potassium phosphate buffer (pH 6.0−7.0) and 100 mM Tricine-KOH buffer (pH 7.0−9.0). The reactions were performed with 50 μM β-carotene and 0.04 U/ml enzyme at 40ºC for 60 min. To investigate the effect of temperature on the enzyme activity, temperature was varied from 25 to 50ºC. The reactions were performed in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene and 0.04 U/ml enzyme for 60 min.

The influence of temperature on enzyme stability for retinal formation was monitored in 100 mM Tricine-KOH buffer (pH 8.0) for 18 h at temperatures from 35 to 55ºC. A sample was withdrawn at each time interval and was assayed for the residual relative activity in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene and 0.04 U/ml enzyme at 40ºC for 60 min. The experimental data for thermal deactivation of enzyme were fitted to a first order curve and the half-lives of the enzyme calculated using Sigma-plot software (Version 9.0, 2004).

Determination of kinetic parameters for

various carotenoid substrates−α-Carotene, β-carotene, γ-carotene, β-cryptoxanthin, zeaxanthin, lutein, β-apo-4′-carotenal, β-apo-8′-carotenal, β-apo-12′-carotenal, and lycopene were used to determine the kinetic parameters of the enzyme. The reaction was performed in 100 mM Tricine-KOH buffer (pH 8.0) at 40ºC for 30 min using various concentrations of substrates (5−800 μM). The kinetic parameters were determined by fitting the data of the Michaelis-Menten equation.

Isotope labeling for determining the

mechanism of oxygenation−To investigate the reaction mechanism of the enzyme, isotopically labeled water (H2

18O) or molecular oxygen (18O2) was used. For labeling experiments with H2

18O, freeze-dried purified enzyme and β-carotene were suspended in the H2

18O solution. For labeling

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experiments with 18O2, the enzyme and substrate were suspended in the H2O solution and then saturated by gassing the solution with 18O2 on ice for 5 min. The enzyme reaction was performed in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene and 0.04 U/ml enzyme at 40ºC for 60 min. At the end of the incubation, an equal volume of acetonitrile was added to the reaction solution. The resulting solution was centrifuged at 10,000 × g for 10 min at 4ºC, and the supernatant was evaporated. The samples were resuspended in acetonitrile and analyzed by high resolution liquid chromatography mass spectrometry (HR-MS).

Computational analysis−Theoretical weights

and pI values for proteins were calculated using the Compute pI/Mw tool at the ExPASy Web site (http://www.expasy.ch/ch2d/pi_tool.html). Prediction of transmembrane helices in the protein was performed using the TMHMM program (www.cbs.dtu.dk/services/TMHMM-2.0) (33).

Molecular modeling of the Blh protein−The Blh protein had no homologous sequences. Thus, we predicted the secondary structure of the Blh protein using the protein structure prediction server (PSIPRED) (34) and compared it to other secondary structures in the protein database bank (PDB). As a result, we selected ten secondary structure candidates from among those analyzed and averaged the candidates. Based on the selected secondary structure, a simulated backbone was constructed and side chains were added using the side chain prediction program SCWRL (35). The three-dimensional structure of the Blh protein from the uncultured marine bacterium 66A03 was generated using the Accelrys Discovery Studio Modeler (Accelrys, San Diego, CA) (36). The generated structure was checked by PROCHCK (37), and then structure minimization was conducted using chemistry at harvard molecular mechanics (CHARMM) (38). After energy minimization, molecular dynamics modeling (MD) was performed at 300K, 1 atm for 500 ps with 1 fs each step. All simulation experiments were carried out on an HP XW6200 Workstation with dual Intel Xeon 3.2GHz processors.

β-Carotene, β-cryptoxanthin, and lutein were docked in the Blh models using the Surflex X docking program (Tripos, St. Louis, MO). Each docking run consisted of 100 independent docks with 1,000 iteration cycles. A random start was used to generate the substrate position within the docking box. The substrate orientation giving the lowest interaction energy was chosen for additional rounds of docking.

Analytical methods−High pressure liquid

chromatography (HPLC) analyses of carotenoids and retinoids were performed based on a previously reported method (10). The same volume of acetonitrile was added to the reaction solution and the resultant solution was mixed and kept on ice for 5 min. After centrifugation at 10,000 × g for 10 min at 4ºC, substrates and products of the supernatant were analyzed by an HPLC system (Agilent 1200 series, Santa Clara, CA) equipped with a UV detector. The substrates β-carotene and lycopene were detected at 460 and 445 nm, respectively, and lutein and zeaxanthin were determined at 450 nm using a Zorbaxsil column (250×4.6 mm, Agilent). The column was eluted with the mixture of hexane and tert-butyl methyl ether with 97:3 (v/v) as the mobile phase with a flow rate of 1 ml/min. The products (3R)-3-hydroxy-retinal, retinal, and α-retinal were detected at 370 nm with retention times of 1.3, 1.8, and 2.2 min, respectively, and the product acycloretinal was detected at 400 nm using an YMC-PAC Pro C18 column (50 × 2.0 mm, YMC, Kyoto, Japan). The column was eluted with a 90:10 (v/v) mixture of acetonitrile and water as the mobile phase at a flow rate of 0.4 ml/min. The retinal isomers 13-cis-retinal, 9-cis-retinal, and all-trans-retinal were detected at 370 nm using an YMC-PAC Pro C18 column (250 × 4.6 mm, YMC) with retention times of 26.1, 26.3, and 27.3 min, respectively. The column was eluted with 80% acetonitrile as the mobile phase at a flow rate of 1.0 ml/min.

The HR-MS data were obtained using a JMS-SX102A spectrometer (Jeol, Tokyo, Japan) operated at an accelerating voltage of 10 kV. The electron impact (EI) ionization mass spectra were collected in the positive ion mode. The HR-MS was performed under the acquisition conditions of: ion source temperature 230ºC, ionization

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energy 70 eV, and ionization current 300 μA.

RESULTS

DNA sequence of the codon-optimized blh gene−Codons of the blh gene encoding the β-carotene-cleaving enzyme from the uncultured marine bacterium 66A03 were optimized and the entire gene was synthesized. Among the 828 bp of the blh gene, including a stop codon, 213 bp (25.8%) were changed by codon optimization (Supplementary Fig. 1). The synthesized gene was cloned into the pET-24a (+) vector and expressed in E. coli. The expressed Blh protein consisted of 275 amino acids. An alignment of the Blh protein with other Blh and Brp-like proteins including membrane spanning domains is shown in Fig. 1. The proposed metal binding residues, His21, His78, His188, and His192, based on molecular modeling were absolutely conserved across all Blh and Brp-like proteins.

Expression, molecular weight, and

identification of the Blh protein−The crude extract of soluble protein obtained from harvested cells was purified by His-Trap HP affinity chromatography. The expressed enzyme was purified, without detergent, as a single band in SDS-PAGE, with a final purification of 7.3-fold, a yield of 32%, and a specific activity of 45 nmol mg–1 min–1. The molecular weight of the purified protein as analyzed by SDS-PAGE was about 32 kDa (Fig. 2A). The protein was purified again with detergent, including 0.05% OTG, 0.5% OTG, 0.5% Tween 80, 6 M urea, 1% Triton X-100, or 1% cocoamidopropyl hydroxysultaine (CAPHS). This further purification, however, did not change the purification yield. The immunoblot was performed with a His-tagged monoclonal antibody against the His-taged Blh protein. A band obtained from the soluble fraction of the cell extract confirmed the isolation of a highly purified recombinant protein. The Blh protein was expressed as a soluble form in E.coli, and the protein was purified without detergent even though it is a member of the bacteriorhodopsin family. Its soluble form may be result from the codon optimization, which is known to increase the expression and solubility of enzymes (39).

The molecular weight of native enzyme, based

on the weights of reference proteins, was estimated using gel filtration chromatography. The native Blh protein had a weight of 64 kDa as a dimer of 32 kDa subunits (Fig. 2B). A chromatogram for the gel filtration of the Blh protein confirms its purity and molecular weight (Fig. 2C).

The Blh protein on a SDS-PAGE was cut out and subjected to trypsinization. The digested peptides were recovered and characterized on a nano LC-MS/MS to obtain amino acid sequence data. The following high confidence peptide sequences were obtained: YNIAFELIG, SRRHFSFVWKQL, and FIGLPHGALD (Supplementary Fig. 2). A comparison between these data and the Blh sequence showed that all three peptides were present in the sequence (Fig. 1).

Determination of the retinal product of the Blh

protein‒The enzymatic conversion of β-carotene into retinal was examined by varying the reaction time and concentrations of enzyme and substrate, based on a standard reaction performed in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene and 0.04 U/ml enzyme at 40ºC for 60 min (Figs. 3A, 3B, and 3C). The products formed at different reaction time and concentrations of enzyme and substrate were analyzed by an HPLC system using an YMC-ODS A column and showed the same retention time as an all-trans-retinal standard. However, other retinal isomers including 13-cis-retinal and 9-cis-retinal showed different retention times, indicating the enzymatic product of β-carotene is all-trans-retinal. With increasing reaction time from 10 to 60 min, the substrate β-carotene decreased whereas the product all-trans-retinal increased (Fig. 3D).

The enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of retinal. The formation of retinal as a reaction product increased linearly up to approximately 0.2 U/ml enzyme, 20 μM β-carotene, and 30 min reaction time, respectively. However, above these parameters, the rates of product formation were not linear; increasing the concentrations of enzyme and substrate and the reaction time subsequently developed sigmoidal curves. We performed enzyme assays in the absence of

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substrate and in the presence of heat-inactivated Blh as controls, which exhibited no activity.

Effects of metal ions, pH, and temperature on

the activity of the Blh protein‒The purified enzyme obtained after the removal of metal ions by phenanthroline exhibited 2% activity relative to that without phenanthroline treatment. Among the metal ions tested, Fe2+ had the strongest effect on the activity of the Blh protein, whereas Ba2+ had the least effective. The effects of other ions decreased in the order of Fe3+, Co2+, Ca2+, Cu2+, and Mn2+ (Fig. 4A). The addition of 10 μM Fe2+ after the removal of all metal ions induced a 54% recovery of the active form of the enzyme. The optimal concentration of Fe2+ was 10 μM, regardless of phenanthroline treatment (Figs. 4B and 4C). Thus, all subsequent experiments were performed in the presence of 10 μM Fe2+.

The enzymatic conversion of β-carotene into all-trans-retinal was examined at pH values ranging from 6.0 to 9.0. The maximum enzyme activity was observed at pH 8.0 (Fig. 5A). The effect of temperature on the enzyme activity is shown in Fig. 5B. The maximum activity was recorded at 40ºC. Above this temperature, the enzyme activity decreased significantly, exhibiting 66% of the maximum activity at 50ºC. Below 40ºC, the enzyme activity decreased with decreasing temperature, exhibiting 15% of the maximum activity at 25ºC.

The thermostability of the Blh protein was measured at five incubation temperatures. The activity of the enzyme was very stable at below 45ºC but significantly decreased at above 50ºC with increasing reaction time (Fig. 5C). The enzyme followed the first-order kinetics of thermal inactivation, and the half-lives of the enzyme at 35, 40, 45, 50, and 55ºC were 17.6, 15.0, 12.5, 6.1, and 1.5 h, respectively.

Kinetic analyses of the Blh protein for various

carotenoid substrates−The kinetic parameters of the purified recombinant Blh protein on α-carotene, β-carotene, γ-carotene, β-cryptoxanthin, zeaxanthin, lutein, β-apo-4′-carotenal, β-apo-8′-carotenal, β-apo-12′-carotenal, and lycopene were determined and are shown in Table 1. The corresponding Michaelis-Menten curves are shown in Supplementary Fig. 3. The Blh protein

exhibited the highest catalytic efficiency (kcat/Km) with β-carotene among the substrates tested. This implies that β-carotene is an authentic substrate for the enzyme. The second-most kinetically favored substrate (kcat/Km) was β-cryptoxanthin, followed by β-apo-4′-carotenal, α-carotene, and γ-carotene. β-Cryptoxanthin was cleaved into retinal and (3R)-3-hydroxy-retinal. If the 15,15′-double bond of zeaxanthin or lutein were cleaved, the products would have been α-(3R)-3-hydroxy-retinal and (3R)-3-hydroxy-retinal, or two molecules of (3R)-3-hydroxy-retinal, respectively. Their common product, (3R)-3-hydroxy-retinal, was formed by the enzymatic conversion of β-cryptoxanthin but was not formed by cleavage of zeaxanthin or lutein (Supplementary Fig. 4). Neither retinal, as the enzymatic product of β-apo-8′-carotenal, nor β-apo-12′-carotenal and acycloretinal, as the products of lycopene, were detected. These results demonstrate that the Blh protein has no activity on zeaxanthin, lutein, apo-8′-carotenal, β-apo-12′-carotenal, or lycopene.

Origin of the oxygen atom of retinal in β-

carotene cleavage by the Blh protein−The molecular weight of retinal as a product was determined after the enzyme was incubated with β-carotene in the presence of H2

18O or 18O2. For labeling experiments with H2

18O, freeze-dried enzyme and β-carotene were suspended in the H2

18O solution. The mass spectrum of retinal formed under these conditions was the same as that using H2O, and the molecular weight of retinal was determined as 284 m/z (Fig. 6A), indicating that the oxygen atom of retinal did not originate from water. However, after the enzyme reaction was performed in an 18O2 atmosphere, the formed retinal was labeled with the 18O atom and its molecular weight was 286 m/z (Fig. 6B). The fragments molecular weights of labeled retinal containing isotope 18O such as 213 and 241 m/z were 2 m/z greater than those of unlabeled retinal. Thus, the oxygen atom of retinal as a reaction product of the Blh protein originated from molecular oxygen rather than from water.

DISCUSSION

Although most recent critical debates about β-

carotene-cleaving enzymes have tended to center

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around mammalian β-carotene 15,15′-monooxygenases, we became interested in β-carotene-cleaving enzymes from other sources, especially bacteria. Hence, we used codon-optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in E. coli. The expressed enzyme converts β-carotene into all-trans-retinal. Characterization of the bacterial Blh protein as a new member of the β-carotene cleavage enzymes is a worthwhile undertaking.

The Blh protein is a putative membrane protein closely related to bacteriorhodopsin (40). Bacteriorhodopsin is a membrane protein containing seven transmembrane α-helices and a covalently bound molecule of retinal. The Blh protein is predicted by the TMHMM program to have seven transmembrane segments, whereas human β-carotene 15,15′-monooxygenase has no transmembrane segments. These results suggest that the Blh protein is a membrane protein The theoretical pI values of the Blh proteins from marine bacteria (8.89−9.56) were much higher than those of the mammalian β-carotene 15,15′-monooxygenases (5.49−6.30), indicating that the two proteins are different (Table 2). There were 48−56% hydrophobic amino acids in the bacterial Blh proteins and 33−34% in mammalian β-carotene 15,15′-monooxygenases. Thus, the Blh proteins are more hydrophobic than mammalian β-carotene 15,15′-monooxygenases.

The subunit molecular weight of the Blh protein was calculated as 32 kDa based on the 281-residue amino acid structure plus a hexa-histidine tag at the carboxyl terminus. The native protein has a total molecular weight of about 64 kDa as a dimer (Fig. 2). β-Carotene 15,15′-monooxygenases from humans, mice, chickens, and rats consisted of 547, 566, 526, and 566 amino acid residues, respectively, and their molecular weights were approximately 60−64 kDa (Table 3). The molecular weight of the native human β-carotene 15,15′-monooxygenase as a tetramer is 250 kDa (41).

Mammalian β-carotene 15,15′-monooxygenases are reported to be Fe2+-dependent protoporphyrin (non-heme iron) enzymes and are inhibited by iron chelating agents (4,42,43). Phenanthroline behaves as an iron chelating agent and strongly inhibited the conversion of β-carotene into retinal

by the Blh protein. Furthermore, among the metal ions tested, Fe2+ had the strongest effect on the activity of the Blh protein. Fe2+ is bound by four conserved histidine residues in the active sites of mammalian β-carotene monooxygenase and apocarotenoid oxygenase (43,44) and in the molecular model of the Blh protein (Fig. 7A). In the Blh protein, Fe2+ is coordinated with His21, His78, His188, and His192, which are absolutely conserved across all Blh and Brp-like proteins (Fig. 1). The four conserved histidine residues were replaced with alanine to produce alanine-substituted mutants. Enzyme activities were determined for the mutants and were compared with that of wild-type enzyme. The relative activities of the H21A, H78A, H188A, and H192A mutants were 3, 0, 0, and 5%, respectively. Thus, we can propose that the four histidine residues in the Blh protein are metal-binding residues.

The enzyme activity in the presence of 10 μM Fe2+ before dialysis was almost the same as after dialysis. These results imply that Fe2+ is tightly bound to the Blh protein.

The maximum activity of the Blh protein was observed at pH 8.0 and 40ºC (Fig. 5). The maximum activities of β-carotene 15,15′-monooxygenases from mammalian sources occurred at pH 7.5−8.5, except for the mouse enzyme which had a maximum activity at pH 9.0 (45). The optimum reaction temperature of mammalian β-carotene 15,15′-monooxygenases from rabbits (42), hogs (46), pigs (47), chickens (11), mice (48,49), and humans (13,41) was 37ºC. The thermostability of the human β-carotene 15,15′-monooxygenase was investigated, and the half-lives of the human enzyme at 35, 40, 45, and 50ºC were 4.5, 3.4, 2.4, and 1.4 h, respectively. The half-lives of the Blh protein were aproximately 4−5-fold higher at these temperatures than those of the human β-carotene 15,15′-monooxygenase.

The Blh protein from the uncultured marine bacterium converted the substrate β-carotene into two molecules of retinal and it converted β-apo-4′-carotenal into one molecule of retinal. The enzyme cleaved the central double bonds (15,15′) of α-carotene, γ-carotene, and β-cryptoxanthin to yield not only one molecule of retinal but also one molecule of each α-retinal, acycloretinal, and

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(3R)-3-hydroxy-retinal. β-Carotene has two β-ionone rings, α-carotene has β-ionone and ε-ionone rings, and γ-carotene and β-apo-carotenals have one β-ionone ring. β-Cryptoxanthin has β-ionone and hydroxyl β-ionone rings, zeaxanthin has hydroxyl ε-ionone and hydroxyl β-ionone rings, and lutein has two hydroxyl β-ionone rings. However, lycopene does not have a β-ionone ring. The Blh protein exhibited activity for β-carotene, β-cryptoxanthin, β-apo-4′-carotenal (C35), α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal (C30), β-apo-12′-carotenal (C25), lutein, zeaxanthin, or lycopene. Based on the chemical structures of the carotenoid substrates, enzyme activity was found only for substrates containing one unsubstituted β-ionone ring with a molecular weight greater than C35. The mammalian β-carotene 15,15′-monooxygenases from humans and rats had activity for β-carotene, β-cryptoxanthin, and β-apo-8′-carotenal (C30) but not lycopene (41,50). We found that human β-carotene 15,15′-monooxygenase had activity for β-apo-4′-carotenal (C35). Although the kcat/Km values of the Blh protein and the human β-carotene 15,15′-monooxygenase for β-carotene were similar, the kcat/Km value of the Blh protein for β-cryptoxanthin (28 mM−1 min−1) as a substrate was 14-fold higher than that of the human enzyme (1.9 mM−1 min−1) (41).

To interpret the substrate specificity of the Blh protein on carotenoid substrates, a simulated backbone of the enzyme structure was constructed and side chains were added based on averaged similar secondary structures in the protein database. Simulated molecular docking was performed with β-carotene, β-cryptoxanthin, and lutein substrates using the Surflex X docking program. When β-carotene was bound in the active site, the para position of one β-ionone ring of β-carotene, located in the interior of the at active site, and the para position of the other unhydroxylated β-ionone ring interacted with the oxygen atom of the carbonyl group of Thr179, at a distance of 3.01 Å (Fig. 7B). When β-cryptoxanthin was bound in the active site, the distance between the hydroxyl group of the (3R)-3-hydroxy-β-ionone ring of β-cryptoxanthin and the oxygen atom of the carbonyl group of Thr179 decreased to 2.31 Å (Fig. 7C). The shorter distance may explain the observed higher affinity

of the Blh protein for β-cryptoxanthin than for β-carotene. When lutein (or zeaxanthin) was bound in the active site, two hydroxyl groups of two (3R)-3-hydroxy β-ionone rings of lutein (or zeaxanthin) were non-effective in anchoring to the active site because the distance to the oxygen atom of Thr179 was too long (Fig. 7D). As a result, the Blh protein exhibited no activity for lutein or zeaxanthin. The Blh protein showed activity for β-apo-4′-carotenal but not for β-apo-8′-carotenal. In the molecular modeling studies, β-apo-4′-carotenal was large enough to interact with Thr179, but β-apo-8′-carotenal was too small to interact (data not shown). However, it is necessary to obtain the actual structure of the enzyme complexed with these substrates in order to provide further evidence for this conclusion. To confirm our molecular modeling and structural analyses, determination of the crystal structure of the Blh protein and mutational analysis of the active site residues must be performed.

Carotenoid oxygenases can be divided into mono- and di-oxygenases based on two different reaction mechanisms (Fig. 8). The oxygen atom of the product from the monooxygenase is provided by molecular oxygen and water via an epoxide intermediate, whereas that from the dioxygenase is provided by molecular oxygen rather than from water via a dioxetane intermediate (51,52). The oxygenation mechanism of chicken β-carotene 15,15′-oxygenase was investigated with isotopic molecular oxygen and water. The oxygen atom in the terminal aldehyde group of retinal as a product was provided by molecular oxygen and water (51), indicating that the enzyme catalyzes the oxidative cleavage using a monooxygenase rather than a dioxygenase mechanism. The other mammalian β-carotene 15,15′-oxygenases were postulated to use the same mechanism. However, some controversies on the reaction mechanism was suggested because oxygen between retinal and water was exchanged within 5% (51). In contrast, the oxygen atom of the retinal formed by the reaction of the Blh protein originated from molecular oxygen rather than from water without oxygen exchange between retinal and water, indicating that the Blh protein used a dioxygenase mechanism unlike the mammalian β-carotene 15,15′-monooxygenases. The Blh protein cleaved β-carotenoid substrates at its central double bond

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(15,15′) and showed the highest activity for β-carotene among the substrates tested. Thus, the Blh protein can be identified as a β-carotene 15,15′-dioxygenase.

In the molecular model, the active site of the Blh protein exhibited coordination of Fe2+ with His21, His78, His188, and His192 (Fig. 7A). According to the cleaving mechanism of apocarotenoid oxygenase (52), the O2 molecule in the active site of the Blh protein binds to the coordination shell of Fe2+ in a side-on fashion. The side-on complex of Fe-O2 then attacks and cleaves between the C15 and C15′ atoms when bound to the Blh protein.

Most enzymes that catalyze the same reaction sequence are structurally similar. However, structurally and mechanistically unrelated enzymes that catalyze the same biochemical reactions do occur. In many cases, one of these analogous enzymes is found in the bacteria and the other in eukaryotes (53). Although the bacterial Blh protein and the mammalian β-

carotene 15,15′-monooxygenases could both convert β-carotene into retinal, the properties of the bacterial Blh protein such as DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity were different from the mammalian β-carotene 15,15′-monooxygenases. Thus, the Blh protein is a bacterial analog to the mammalian β-carotene 15,15′-monooxygenases.

This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme. This paper contributes to the understanding of bacterial β-carotene-cleaving enzymes and provides a stepping stone for further studies. Moreover, this study will be useful in evolutionary studies investigating the relationships between bacterial and mammalian enzymes and industrial applications of retinal biosynthesis.

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FIGURE LEGENDS FIGURE 1. Alignment of Blh and Brp-like proteins originated from typical strains. The proposed metal binding (four histidine) residues based on molecular modeling are highlighted with a black background. The predicted transmembrane regions were underlined. The digested peptide sequences of the protein expressed from the codon-optimized blh gene that were identified from the MS data are italicized. Sequence alignment was performed by CLUSTAL W program. Asterisks mean identity. The GeneBankTM accession numbers are as follows: Uncultured marine bacterium 66A03 Blh protein, AAY68319; Uncultured marine bacterium HF10 49E08 Blh protein, ABL97831; Halobacterium sp. NRC-1 Blh protein, NP280794; Haloarcula marismortui ATCC 43049 Brp-like protein, YP136627; Haloquadratum walsbyi DSM 16790 Brp-like protein, YP656806. FIGURE 2. Determination of expression and molecular weight of the Blh protein. A, SDS-PAGE stained with Coomassie blue and Western blot of Blh protein. Lane 1, prestained marker proteins (72, 55, 43, 34, 26, 17 and 11 kDa); lane 2, His-Trap HP column product; lane 3, soluble fraction of the expressed Blh protein. The soluble fraction was transferred to a PVDF membrane and was incubated with the His-tag monoclonal antibody for 1 h. The membrane was visualized with the Western blot detection system. B, Determination of molecular weight of the purified native enzyme using Sephacryl S-300 HR gel filtration chromatography. The reference proteins were aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and RNase A (13.7 kDa). The Blh protein eluted at a position corresponding to 64 kDa. Data represent the means of three experiments and error bars represent standard deviation; C, A gel filtration chromatogram of the Blh protein to confirm purity and molecular weight. FIGURE 3. Determination of the retinal product of the Blh protein by varying the reaction time and concentrations of enzyme and substrate. Data represent the means of three experiments and error bars represent standard deviation. The experiments for determination of the retinal product were compensated with controls that were performed with the same reactions without enzyme. A, The reactions by varying the reaction time were performed in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene and 0.04 U/ml enzyme at 40ºC; B, The reactions by varying the concentration of the Blh protein were performed in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene at 40°C for 60 min; C, The reactions by varying the concentration of β-carotene were performed in 100 mM Tricine-KOH buffer (pH 8.0) containing 0.04 U/ml enzyme at 40ºC for 60 min; D, HPLC chromatograms of the cleavage product of β-carotene by the Blh protein and all-trans-retinal standard. The substrate β-carotene was determined at 460 nm using a Zorbaxsil column (250×4.6 mm) and the product retinal was detected at 370 nm using an YMC-PAC Pro C18 column (50 × 2.0 mm). FIGURE 4. Effect of metal ions on enzyme activity. Data represent the means of three experiments and error bars represent standard deviation. The experiments for effect of metal ions were compensated with the experiments that were performed with the same reactions without enzyme. Control indicates the purified Blh protein before phenanthroline treatment. A, The Blh protein was obtained after incubating at 20ºC with 1 mM phenanthroline as an iron chelating agent for 1 h and following by overnight dialysis at 4ºC against 100 mM Tricine-KOH buffer (pH 8.0). Each metal ion of 10 μM was added to the Blh protein obtained after the treatment of phenanthroline to investigate the effect of the kind of metal ions on the conversion of β-carotene to retinal. Phenanthroline indicates the Blh protein obtained after treating 1 mM phenanthroline.; B, The Blh protein was obtained after incubating at 20ºC with 1 mM phenanthroline as an iron chelating agent for 1 h and following by overnight dialysis at 4ºC against 100 mM Tricine-KOH buffer (pH 8.0). The effect of was investigated with the Blh protein with treating 1 mM phenanthroline. The different concentrations of Fe2+ were added to the Blh protein obtained after the treatment of phenanthroline to investigate the effect of Fe2+ concentration on the conversion of β-carotene to retinal

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with the Blh protein. C, The effect of Fe2+ concentration was investigated with the purified Blh protein without phenanthroline treatment. The different concentrations of Fe2+ were added to the purified Blh protein without phenanthroline treatment. FIGURE 5. Effects of pH, temperature, and thermostability on the activity of the Blh protein. Data represent the means of three experiments and error bars represent standard deviation. The experiments for effects of pH, temperature, and thermostability were compensated with controls that were performed with the same reactions without enzyme. A, Two different buffers for pH experiment were used: 100 mM potassium phosphate buffer for pH 6.0 to 7.0 (○) and 100 mM Tricine-KOH buffer for pH 7.0 to 9.0 (●). The reactions were performed with 50 μM β-carotene and 0.04 U/ml enzyme at 40ºC for 60 min; B, The reactions were performed 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene and 0.04 U/ml enzyme for 60 min in temperature range of 25−50ºC; C, Thermstability of the Blh protein for retinal production was measured at 35 (●), 40 (□), 45 (▲), 50 (○), and 55ºC (■). A sample was withdrawn at each time interval and the residual relative activity was measured after the reaction was performed 100 mM Tricine-KOH buffer (pH 8.0) containing 50 μM β-carotene and 0.04 U/ml enzyme at 40ºC for 60 min. FIGURE 6. Incorporation of molecular oxygen into the reaction product retinal determined by HR-MS analysis. A, Mass spectrum of retinal formed by the Blh protein from β-carotene in the H2

18O solution. The main fragment of mass spectrum in the solution of H2

18O indicate unlabeled ([M] = 284 m/z). The fragments of unlabeled retinal were [M−(CH2)4−H13], [M−O−CH2−(CH)2], [M−(CH2)2], [M−O−CH3], and [M−O], their molecular weights were 211, 227, 239, 253, and 268 m/z, respectively. B, Mass spectrum of retinal formed by the Blh protein from β-carotene in 18O2 atmosphere. The main fragment of mass spectrum in an 18O2 atmosphere indicates the labeled retinal ([M]= 286). The fragments of labeled retinal were [M−(CH2)4−H13], [M−18O−CH2−(CH)2], [M−(CH2)2], [M−18O−CH3], and [M−18O], their molecular weights were 213, 227, 241, 253, and 268 m/z, respectively. FIGURE 7. Molecular model of the Blh protein. The oxygen atom at carbonyl group of Thr179 interacted with the para position in the β-ionone ring of β-carotene or with the hydroxyl group in the (3R)-3-hydroxy-β-ionone ring of β-cryptoxanthin. Active site of the Blh protein (A panel). Docking of β-carotene (B panel), β-cryptoxanthin (C panel), lutein (D panel) into the active site of the Blh protein. FIGURE 8. Two different mechanisms for β-carotene central cleavage. A, Dioxygenase mechanism. The oxygen atom of the product is provided by molecular oxygen rather than water via dioxetane intermediate (7). B, Monooxygenase mechanism. The oxygen atom of the product is provided by molecular oxygen and water via an epoxide intermediate (51).

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Table l. Kinetic parameters of the Blh protein for various carotenoids as substrates. The reaction was performed in 100 mM Tricine-KOH buffer (pH 8.0) at 40ºC for 30 min using the amounts of substrates ranging from 5 to 800 μM. Data represent the mean of three experiments and ± values represent standard deviations. ND, kinetic parameters are not detected by the analytical methods used in this study.

Substrate Km

(μM) kcat

(min−1×10−2) kcat/Km

(mM−1 min−1)

α-Carotene 169±3 6.2±0.1 3.6±0.1

β-Carotene 37±1 360±1 97±0.1

γ-Carotene 382±8 2.1±0.1 0.55±0.01

β-Cryptoxanthin 29±1 80±0.1 28±0.4

Zeaxanthin ND ND ND

Lutein ND ND ND

β-Apo-4′-carotenal 147±3 63±1 4.2±0.2

β-Apo-8′-carotenal ND ND ND

β-Apo-12′-carotenal ND ND ND

Lycopene ND ND ND

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Table 2. Theoretical pI values and hydrophobic amino acids contents of Blh and Brp-like proteins, and β-carotene 15,15′- monooxygenases.

Protein Source pI Hydrophobic amino acids (%)a

Blh protein Uncultured marine bacterium 66A03 9.56 52.4 Uncultured marine bacterium HF10 49E08 9.30 48.4 Halobacterium sp. NRC-1 8.89 55.2

Brp-like protein Haloarcula marismortui ATCC 43049 9.12 56.3 Haloquadratum walsbyi DSM 16790 9.50 52.2

β-Carotene 15,15′-

monooxygenase

Chicken 6.01 32.8 Human 6.30 33.7 Mouse 5.49 33.3 Rat 5.90 32.9

a The hydrophobic amino acids are alanine, leucine, valine, isoleucine, phenyalanine, and methionine.

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Table 3. Biochemical properties of the Blh protein and β-carotene 15,15′-monooxygenases.

Organism Optimum temp (ºC)

OptimumpH

Km (μM)

Vmax (nmol mg−1min−1)

kcat

(min−1)kcat/Km

(mM−1min−1) Molecular

weight (kDa)Native enzyme

Number of amino acid

Half-life at 45°C (h)

Reference

Uncultured marine bacterium. 40 8.0 37.0 45.0 3.60 97 32 Dimer

(64 kDa) 275 12.5 This study

Human 37 8.0 7.1 10.4 0.66 93 64 Tetramer(250 kDa) 547 2.4 (41)

Mouse 37 9.0 6.0 0.04 64 566 (45,48)

Chicken 37 8.0 60 526 (32)

Rat 37 7.5 3.3 0.03 64 566 (4)

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H21

H78

H188 H192

Figure 1 Uncultured marine bacterium 66A03 Blh protein ------------------------------------------------MG Uncultured marine bacterium HF10 49E08 Blh protein MMKWIEWNPLTFSSSTGEYLPKSYSMGLSKLTLGLLIPLGILGLLSADWP Halobacterium sp. NRC-1 Blh protein -------------------------------------------------- Haloarcula marismortui ATCC 43049 Brp-like protein ----------MGVSYRSAVEPSVRYRVALAPGWVASLAVVAPFLAGVSIP Haloquadratum walsbyi DSM 16790 Brp-like protein ---------MAVSESLSDTTRQVLGRSVFIPAWLILIGCILLAPLVESLS Uncultured marine bacterium 66A03 Blh protein LMLIDWCALALVVFIGLPHGALDAAISFSMISSAKRIARLAGILLIYLLL Uncultured marine bacterium HF10 49E08 Blh protein ISYQLLPLYLSLFLFGMPHGGADHLLIWGMIR-KSTWLFRVSTLLLYPTL Halobacterium sp. NRC-1 Blh protein ----------------MPHGAIDYLALPRAVTGTVTVRWLAVVGVLYLVL Haloarcula marismortui ATCC 43049 Brp-like protein PALQYAPLVVSAVLLGLPHGAVDHLAVARTRGERPDWRAIARVFALYGVV Haloquadratum walsbyi DSM 16790 Brp-like protein PRLRYLPFALSLLIFGLPHGAVDHLTPARTVNTPVTVRSMLSVGGLYLSI

:***. * :* : Uncultured marine bacterium 66A03 Blh protein ATAFFLIWYQLPAFSLLIFLLISIIHFGMADFNASP----------SKLK Uncultured marine bacterium HF10 49E08 Blh protein SIFYLICWHFQPVASAIFFLVITIFHWGQGDRYLSVKLHRACYLERSNLL Halobacterium sp. NRC-1 Blh protein GGGYAAAWFFAPVPAAFAFVAITWLHWGQGDLYPLLDFLDVDYLD-TRPR Haloarcula marismortui ATCC 43049 Brp-like protein GGAYAVTWFLAPAAAFVLFIAVTWFHWGQGDLYALRALADADHLR-SLPQ Haloquadratum walsbyi DSM 16790 Brp-like protein GGVYLGWWFFAPTSAAIMFVFITLLHWGQGDLYAILSFLDADHLP-TRFE . : *. *. : . *: :: :*:* .* : Uncultured marine bacterium 66A03 Blh protein WPHIIAHGGVVTVWLPLIQKNEVTKLFSILTNGPTP---------ILWDI Uncultured marine bacterium HF10 49E08 Blh protein KILHITARGSIPILLPGYLGNETYRMFLEEMIRQGG---QIDYDLSWVSE Halobacterium sp. NRC-1 Blh protein RAATVLIRGGLPMLVPLLGFPERYRSVVDAFAAPFG--GSVGDLAVFDPR Haloarcula marismortui ATCC 43049 Brp-like protein RIGTVLVRGGLPMLVPLLAFPEWYRRVATDLVSLFAPDAVAAIGWAFRTD Haloquadratum walsbyi DSM 16790 Brp-like protein RALSIVTRGAMPMLIPLIAHPMSYQRVLIGFIELFS-VNTISIGFLFSSP : * :.: :* : . Uncultured marine bacterium 66A03 Blh protein LLIFFLCWSIGVCLHTYETLRSKHYN---------IAFELIGLIFLAWYA Uncultured marine bacterium HF10 49E08 Blh protein NQLFFLLVPLSLMVMQLFVSIFQLEKD-ERVAWGLDLSEACFLFLWFLFL Halobacterium sp. NRC-1 Blh protein VRLWLGVAFAAATVAVLAAGRRRTHS---PGAWRVDAAETLLLWVFFFVV Haloarcula marismortui ATCC 43049 Brp-like protein VRTALALAYGALVVATLAVGFARADA---RRPWLLDAGETLGLLAYFALV Haloquadratum walsbyi DSM 16790 Brp-like protein IRNVVTAVIIGLTIMTIAAGAWRVYTGASARPYLIDAGEIGLLWAFFIAL . . : . : * * Uncultured marine bacterium 66A03 Blh protein PPLVTFATYFCFIHSRRHFS----------FVWKQLQHMSSKKMMIGSAI Uncultured marine bacterium HF10 49E08 Blh protein PPLWALGIYFALWHSLRHGLRIIWLDPTGKQSLVAGAYLKLKARWLQISG Halobacterium sp. NRC-1 Blh protein PPVFAVGVYFCVWHSVRHVARAIAVDGSVHPSLRAGDILGPLARFGVEAA Haloarcula marismortui ATCC 43049 Brp-like protein PPVLAIGVYFCLWHSLRHVARLLLVDDDATAALQNRDPSAALARFARDAA Haloquadratum walsbyi DSM 16790 Brp-like protein PPIFAVGVYFCIWHSLRHIARLAIIDHPSRQALDSAHIRPAVWRFVRDAA **: :.. **..** ** : Uncultured marine bacterium 66A03 Blh protein ILSCTSWLIGGGIYFFLNSKMIASEAALQTVFIGLAALTVPHMILIDFIF Uncultured marine bacterium HF10 49E08 Blh protein LMTILALVGLWFLLALPLSFRGIQLDWLGKAMLGISILTLPHTVVVCLMD Halobacterium sp. NRC-1 Blh protein PMTAAALALGGVLWWAVPNPPTTLESGAALYLVLIAVLTLPHVAVVTWMD Haloarcula marismortui ATCC 43049 Brp-like protein PLTAASLALLGGLYFLVPNPPGSVPEWVALYLVFIAVVTLPHVVVVSIMD Haloquadratum walsbyi DSM 16790 Brp-like protein PLTAAALLFFASLYILVPRQPNNIESLAAFYLVGIAALTAPHVVIVSWMD :: : : :: :: :* ** :: : Uncultured marine bacterium 66A03 Blh protein RPHSSRIKIKN Uncultured marine bacterium HF10 49E08 Blh protein RFQFKRVGLIK Halobacterium sp. NRC-1 Blh protein RVQGVL----- Haloarcula marismortui ATCC 43049 Brp-like protein REQGVWA---- Haloquadratum walsbyi DSM 16790 Brp-like protein RMQAVW----- * :

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32 kDa

1 2 3

1 2 3

Figure 2

A

B

Retention time (min)

80 100 120 140 160 180 200

Log

(mol

ecul

ar w

eigh

t, kD

a)

20

40

60

80

200

400

10

10064 kDa

C

kDa 72 55 43 34

26

17

11

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Figure 3

A

Time (min)

0 20 40 60 80 100 120 140

All-

tran

s-re

tinal

( μm

o l/m

g)

0

20

40

60

80

100

B

Protein (U/ml)

0.0 0.2 0.4 0.6 0.8 1.0 1.2

All-

tran

s-re

tinal

( μm

ol/h

)

0

4

8

12

16

C

β-Carotene (μM)

0 20 40 60 80 100 120

All-

tran

s-re

tinal

( μm

ol/m

g/h)

0

10

20

30

40

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Figure 3- continued

D

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Figure 4

A

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

120

Fe2+

Fe3+

Mn2+

Co2+

Ba2+

Cu2+

Mg2+

Ca2+

PhenanthrolineControl

B

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

120

Control

3 μM Fe2+

5 μM Fe2+

10 μM Fe2+

20 μM Fe2+

1 μM Fe2+

50 μM Fe2+

0 μM Fe2+

C

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

120

140

Control

50 μM Fe2+

100 μM Fe2+

500 μM Fe2+

10 μM Fe2+

1000 μM Fe2+

5 μM Fe2+

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Figure 5

A

pH

5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

120

B

Temperature (oC)

20 25 30 35 40 45 50 55

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

120

C

Time (h)

0 4 8 12 16 20

Rel

ativ

e ac

tivity

(%)

1

10

100

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Figure 6

A

B

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Figure 7

A B

C D

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Figure 8

A B

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Yeong-Su Kim, Nam-Hee Kim, Soo-Jin Yeom, Seon-Won Kim and Deok-Kun Ohbacterium as a beta-carotene 15,15¡Ç-dioxygenase

In vitro characterization of a recombinant Blh protein from an uncultured marine

published online April 14, 2009J. Biol. Chem. 

  10.1074/jbc.M109.002618Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2009/04/14/M109.002618.DC1

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