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In Vitro Characterization of a Recombinant Blh Protein froman Uncultured Marine Bacterium as a �-Carotene15,15�-Dioxygenase□S

Received for publication, April 1, 2009, and in revised form, April 13, 2009 Published, JBC Papers in Press, April 14, 2009, DOI 10.1074/jbc.M109.002618

Yeong-Su Kim‡, Nam-Hee Kim‡, Soo-Jin Yeom‡, Seon-Won Kim§, and Deok-Kun Oh‡1

From the ‡Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701 and the §Division of Applied Life Science(BK21), Environmental Biotechnology National Core Research Center and Plant Molecular Biology and Biotechnology ResearchCenter, Gyeongsang National University, Jinju 660-701, Korea

Codon optimization was used to synthesize the blh gene fromthe unculturedmarine bacterium 66A03 for expression in Esch-erichia coli. The expressed enzyme cleaved �-carotene at itscentral double bond (15,15�) to yield twomolecules of all-trans-retinal. The molecular mass of the native purified enzyme was�64 kDa as a dimer of 32-kDa subunits. The Km, kcat, andkcat/Km values for �-carotene as substrate were 37 �M, 3.6min�1, and 97mM�1min�1, respectively. The enzyme exhibitedthe highest activity for �-carotene, followed by �-cryptoxan-thin, �-apo-4�-carotenal, �-carotene, and �-carotene in decreas-ing order, but not for �-apo-8�-carotenal, �-apo-12�-carotenal,lutein, zeaxanthin, or lycopene, suggesting that thepresenceofoneunsubstituted�-ionone ring ina substratewithamolecularweightgreater thanC35 seems tobeessential for enzymeactivity.Theoxy-gen atom of retinal originated not fromwater but frommolecularoxygen, suggesting that theenzymewasa�-carotene15,15�-dioxy-genase.AlthoughtheBlhproteinand�-carotene15,15�-monooxy-genases catalyzed the same biochemical reaction, the Blh proteinwas unrelated to themammalian�-carotene 15,15�-monooxygen-ases as assessed by their different properties, including DNA andaminoacid sequences,molecularweight, formof association, reac-tionmechanism, kinetic properties, and substrate specificity. Thisis the first report of in vitro characterization of a bacterial �-caro-tene-cleaving enzyme.

VitaminA (retinol) is a fat-soluble vitamin and important forhuman health. In vivo, the cleavage of�-carotene to retinal is animportant step of vitamin A synthesis. The cleavage can pro-ceed via two different biochemical pathways (1, 2). The majorpathway is a central cleavage catalyzed by mammalian �-caro-tene 15,15�-monooxygenases (EC 1.14.99.36). �-Carotene iscleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo(3–5). The second pathway is an eccentric cleavage that occurs atdouble bonds other than the central 15,15�-double bond of�-car-otene to produce �-apo-carotenals with different chain lengths,which are catalyzed by carotenoid oxygenases from mammals,

plants, and cyanobacteria (6). These �-apo-carotenals aredegraded to one molecule of retinal, which is subsequently con-verted to vitamin A in vivo (2).

�-Carotene 15,15�-monooxygenase was first isolated as acytosolic enzyme by identifying the product of �-carotenecleavage as retinal (7). The characterization of the enzyme andthe reaction pathway from �-carotene to retinal were alsoinvestigated (4, 8). The enzyme activity has been found inmam-malian intestinal mucosa, jejunum enterocytes, liver, lung, kid-ney, and brain (5, 9, 10). Molecular cloning, expression, andcharacterization of �-carotene 15,15�-monooxygenase havebeen reported from various species, including chickens (11),fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert�-carotene to retinal include

bacterioopsin-related protein (Brp) and bacteriorhodopsin-re-lated protein-like homolog protein (Blh) (16). Brp protein isexpressed from the bop gene cluster, which encodes the structuralprotein bacterioopsin, consisting of at least three genes as follows:bop (bacterioopsin), brp (bacteriorhodopsin-related protein), andbat (bacterioopsin activator) (17). brp genes were reported inHaloarcula marismortui (18), Halobacterium sp. NRC-1 (19),Halobacterium halobium (17), Haloquadratum walsbyi, andSalinibacter ruber (20). Blh protein is expressed from the proteor-hodopsin gene cluster, which contains proteorhodopsin, crtE(geranylgeranyl-diphosphate synthase), crtI (phytoene dehydro-genase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (iso-pentenyldiphosphate isomerase), andblhgene (21). Sourcesofblhgenes were previously reported inHalobacterium sp. NRC-1 (19),Haloarcula marismortui (18), Halobacterium salinarum (22),unculturedmarine bacterium 66A03 (16), and unculturedmarinebacterium HF10 49E08 (21). �-Carotene biosynthetic genes crtE,crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for thesynthesis of�-carotene from isopentenyl diphosphate, and the Idi,IspA, CrtE, CrtB, CrtI, andCrtY proteins have been characterizedin vitro (23–28). Blh protein has been proposed to catalyze or reg-ulate the conversion of �-carotene to retinal (29, 30), but there isno direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize

the blh gene from the uncultured marine bacterium 66A03for expression in Escherichia coli, and we performed adetailed biochemical and enzymological characterization ofthe expressed Blh protein. In addition, the properties of theenzyme were compared with those of mammalian �-caro-tene 15,15�-monooxygenases.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S4.

1 To whom correspondence should be addressed: Dept. of Bioscience andBiotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul143-701, South Korea. Tel.: 82-2-454-3118; Fax: 82-2-444-6176; E-mail:[email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 23, pp. 15781–15793, June 5, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JUNE 5, 2009 • VOLUME 284 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 15781

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EXPERIMENTAL PROCEDURES

Reagents—The expression vector pET-24a(�) was pur-chased from Novagen (San Diego). The expression host,E. coli ER 2566, and all restriction enzymes were purchasedfrom New England Biolabs (Hertfordshire, UK). Luria-Ber-tani (LB) medium was purchased from BD Biosciences.�-Carotene and the other carotenoid substrates were pur-chased from Sigma and Carotenature (Lupsingen, Switzer-land), respectively. The pre-stained ladder for SDS-PAGEand the gel filtration calibration kit were purchased fromMBI Fermentas (Hanover, MD) and Amersham Biosciences,respectively. Western blot detection system and His-taggedmonoclonal antibody were purchased from Intron Biotech-nology (Seongnam, Gyeonggi, Korea) and Bio-Rad, respec-tively. Isotopically labeled water (H2

18O) and molecular oxy-gen (18O2) were purchased from Sigma and CambridgeIsotope Laboratories (Andover, MA), respectively. All otherregents were purchased from Sigma.DNA Cloning and Site-directed Mutagenesis—The amino

acid sequence used for codon optimization was obtained fromthe blh gene of the uncultured marine bacterium 66A03 (Gen-BankTM accession number AAY68319). Codons of the blh genesequence were optimized by selection based on probabilitiesobtained from the codon usage table without consideration ofinternal mRNA secondary structures or DNA repeats (31). Theentire gene was synthesized (Genofocus, Daejeon, Korea),cloned into the pET-24a(�) expression vector using the restric-tion enzymes EcoRI and XhoI, and transformed into E. coliER2566 as an expression host. Mutations of the conserved fourhistidine residues in the Blh protein were generated by site-directed mutagenesis using the QuickChange kit and protocol(Stratagene, Beverly, MA). DNA sequencing was performed atthe Macrogen facility (Seoul, Korea).Expression and Purification—The recombinant E. coli for

expression of the wild-type andmutant enzymes was cultivatedin 500ml of LBmedium (1.0% tryptone, 0.5% yeast extract, and1.0% sodium chloride) in a 2,000-ml flask containing 20 �g/mlkanamycin at 37 °C with shaking at 200 rpm.When the absorb-ance of the culture reached 0.5 at 600 nm, isopropyl �-D-thio-galactopyranoside was added to a final concentration of 0.1mM

to induce expression of the recombinant enzyme, and the cul-ture was incubated at 16 °C for 16 h. The cells were harvestedfrom the culture brothby centrifugation at 6,000� g for 30minat4 °C, washed twice with 0.85% NaCl, and then resuspended in alysis buffer (50mMNaH2PO4, 300mMNaCl, pH 8.0) containing 1mg/ml lysozyme. The resuspended cells were disrupted using asonicator. The cell debris was removed by centrifugation at13,000 � g for 20 min at 4 °C, and the supernatant was filteredthrougha0.45-�mfilter.The filtrate applied toHis-TrapHPaffin-ity chromatography (AmershamBiosciences) equilibratedwith 50mM sodium phosphate buffer containing 300 mM NaCl. The col-umnwas washed extensively with the same buffer, and the boundprotein was eluted with a linear gradient between 10 and 200 mM

imidazole at a flow rate of 1 ml/min. The purification step usingchromatography was carried out in a cold room at 4 °Cwith a fastprotein liquid chromatography system (Bio-Rad). The active frac-

tions were collected and dialyzed against 100 mM Tricine2-KOHbuffer (pH8.0).Afterdialysis, the resultingsolutionwasusedas thepurified enzyme.AminoAcid Sequencing—Partial amino acid sequences of the

Blh protein were investigated. After separation via SDS-PAGEand staining of the sample, each chosen band was isolated,destained, and washed. In-gel digestion was then performedwith 500 ng of sequencing-grade chymotrypsin in 100mMTris-HCl buffer (pH 8.0) with 10 mM CaCl2 at 37 °C for 16 h. Thedigested peptides were extracted with 5% formic acid in aceto-nitrile 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 microcap-illary C18 column (75 �m � 150 mm).

Liquid chromatography separation was conducted using lin-ear gradient elution with 0.1% formic acid in H2O (solvent A)and formic acid in acetonitrile (solvent B). The elution programwas as follows: a solvent composition of 97:3 (A:B) from 0 to 5min; 60:40 from 5 to 72 min; 10:90 from 72 to 87 min; and 97:3from 87 to 120 min. The flow rate was a constant 200 nl/min.The separated peptides were subsequently analyzed on amodelLTQ linear ion-trap mass spectrometer (ThermoFinnigan, SanJose, CA). The electrospray voltage was set to 2.0 kV, and thethreshold for switching fromMS to MS/MS was 250. The nor-malized collision energy for MS/MS was 35% of the main RFamplitude, and the duration of activationwas 30ms. All spectrawere acquired in data-dependent mode. EachMS scan was fol-lowed byMS/MS scans of the threemost intense peaks from thefull MS scan. The repeat count of peak for dynamic exclusionwas 1, and its repeat duration was 30 s. The dynamic exclusionduration was set to 180 s, and the exclusion mass width was�1.5 Da. The list size of dynamic exclusion was 50.Data Base Analysis—Data base searches were performed for

all MS/MS spectra using an E. coli protein data base with theaddition of the amino acid sequence of the Blh protein.SEQUEST was used as the peptide-searching program, whichincluded the dynamic modifications of oxidized methionine(�16 Da) and carboxyamidomethylated cysteine (�57 Da).SEQUEST criteria for peptide selection were based on the val-ues of XCorr, which requires values greater than 1.8, 2.3, and3.5 for�1,�2, and�3 charge state peptides, respectively, witha �Cn above 0.1. The criterion for protein selection was a con-sensus score above 10.1.Western Blot Analysis—The soluble fraction of recombinant

E. coli lysate was subjected to Western blot analysis. The frac-tion was separated by SDS-PAGE on 12% gels and then trans-ferred to a polyvinylidene difluoride membrane using a Mini-Protean II transfer apparatus (Bio-Rad) according to themanufacturer’s instructions. For immunodetection, the mem-branes were first blocked with 5% (w/v) skim milk in phos-phate-buffered saline containing Tween 20 (PBST) and thenincubated with His-tagged monoclonal antibody for 1 h. The

2 The abbreviations used are: Tricine, N-[2-hydroxy-1,1-bis(hydroxy-methyl)ethyl]glycine; HPLC, high pressure liquid chromatography; MS,mass spectrometry; MS/MS, tandem MS; HR-MS, high resolution liquidchromatography-mass spectrometry.

Characterization of Bacterial �-Carotene 15,15�-Dioxygenase

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membrane was visualized using a WEST-oneTM Western blotdetection system. The membranes were then placed betweentwo overhead transparency films and exposed to Kodak film.Determination ofMolecularWeight—The subunit molecular

weight of the Blh protein from uncultured marine bacterium66A03 was examined by SDS-PAGE under denaturing condi-tions, using the proteins of a prestained ladder as referenceproteins. All protein bands were stained with Coomassie Bluefor visualization.The molecular weight of the native enzyme was determined

using Sephacryl S-300 HR gel filtration chromatography(Amersham Biosciences). The purified enzyme solution wasapplied to the chromatography and eluted with 100 mM

Tricine-KOH buffer (pH 8.0) containing 0.1 M NaCl at a flowrate of 0.3 ml/min. The column was calibrated with aldolase(158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonicanhydrase (29 kDa), and RNase A (13.7 kDa) as reference pro-teins, and the native enzyme was calculated by comparing withmigration length of reference proteins.Enzyme Assay—�-Carotene of 200 �M was dispersed in 2 ml

of toluene containing 1.2% (v/v) Tween 80 to form detergentmicelles, and the solution was homogenized at 10,000 rpm for10 s using a homogenizer (IKA, Kuala Lumpur, Malaysia). Thetoluene was evaporated by N2 gas at room temperature, andthen 2 ml water was added. The resulting solution was used asthe substrate solution. The substrate and enzyme solutionswere mixed with the ratio of 1:3 (v/v) and then the mixture wasused as the reaction solution. The reaction solution contained50 �M �-carotene, 0.3% (w/v) Tween 80, 125 mM sodium chlo-ride, 10 �M Fe2SO4, 5 mM Tris(2-carboxyethyl)phosphinehydrochloride, and 1.0% (w/v) 1-S-octyl-�-D-thioglucopyrano-side. The enzyme reaction was performed in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 �M �-carotene and 0.04unit/ml enzyme at 40 °C for 60 min. After incubation, the reac-tion was stopped by 3.7% (v/v) formaldehyde, and additionalincubation was done at 40 °C for 10 min (32). The recoverypercentage of retinal was varied under the various conditions astemperature was affecting the recovery. One unit of enzymeactivity was defined as the amount of enzyme required to pro-duce 1 �mole of retinal per min at 40 °C and pH 8.0.Effects of Metal Ions, pH, and Temperature on Enzyme

Activity—The Blh protein was obtained after incubating at20 °C with 1 mM phenanthroline as an iron-chelating agent for1 h and following by overnight dialysis at 4 °C against 100 mM

Tricine-KOH buffer (pH 8.0), and then the effects of variousmetal ions on its activity were investigated in the presence of 10�MFe2�, Fe3�, Co2�, Ca2�, Cu2�, Mn2�, or Ba2�. The effect ofFe2� was evaluated with and without phenanthroline treat-ment. To examine the effect of pH on the enzyme activity, thepH was varied between 6.0 and 9.0 using 100 mM potassiumphosphate buffer (pH 6.0–7.0) and 100 mM Tricine-KOHbuffer (pH 7.0–9.0). The reactions were performed with 50 �M

�-carotene and 0.04 unit/ml enzyme at 40 °C for 60 min. Toinvestigate the effect of temperature on the enzyme activity,temperature was varied from 25 to 50 °C. The reactions wereperformed in 100 mM Tricine-KOH buffer (pH 8.0) containing50 �M �-carotene and 0.04 unit/ml enzyme for 60 min.

The influence of temperature on enzyme stability for retinalformationwasmonitored in 100mMTricine-KOHbuffer (pH8.0)for18hat temperatures from35to55 °C.Asamplewaswithdrawnat each time interval andwas assayed for the residual relative activ-ity in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 �M

�-carotene and 0.04 unit/ml enzyme at 40 °C for 60 min. Theexperimental data for thermal deactivation of enzyme were fittedto a first-order curve, and the half-lives of the enzymewere calcu-lated using Sigma-plot software (version 9.0, 2004).Determination of Kinetic Parameters for Various Carotenoid

Substrates—�-Carotene,�-carotene,�-carotene,�-cryptoxan-thin, zeaxanthin, lutein, �-apo-4�-carotenal, �-apo-8�-carote-nal, �-apo-12�-carotenal, and lycopene were used to determinethe kinetic parameters of the enzyme. The reaction was per-formed in 100 mM Tricine-KOH buffer (pH 8.0) at 40 °C for 30min using various concentrations of substrates (5–800 �M).The kinetic parameters were determined by fitting the data ofthe Michaelis-Menten equation.Isotope Labeling for Determining the Mechanism of Oxygena-

tion—To investigate the reaction mechanism of the enzyme, iso-topically labeled water (H2

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

18O, freeze-dried purifiedenzyme and �-carotene were suspended in the H2

18O solution.For labeling experiments with 18O2, the enzyme and substratewere suspended in the H2O solution and then saturated by gas-sing the solution with 18O2 on ice for 5 min. The enzyme reac-tion was performed in 100 mM Tricine-KOH buffer (pH 8.0)containing 50 �M �-carotene and 0.04 unit/ml enzyme at 40 °Cfor 60 min. At the end of the incubation, an equal volume ofacetonitrile was added to the reaction solution. The resultingsolution was centrifuged at 10,000 � g for 10 min at 4 °C, andthe supernatant was evaporated. The samples were resus-pended in acetonitrile and analyzed by high resolution liquidchromatography-mass spectrometry (HR-MS).Computational Analysis—Theoretical weights and pI values

for proteins were calculated using the Compute pI/Mw tool atthe ExPASy web site. Prediction of transmembrane helices inthe protein was performed using the TMHMM program (33).Molecular Modeling of the Blh Protein—The Blh protein had

no homologous sequences. Thus, we predicted the secondarystructure of the Blh protein using the protein structure predic-tion server (PSIPRED) (34) and compared it with other second-ary structures in the Protein Data Bank. As a result, we selected10 secondary structure candidates from among those analyzedand averaged the candidates. Based on the selected secondarystructure, a simulated backbone was constructed, and sidechains were added using the side chain prediction programSCWRL (35). The three-dimensional structure of the Blh pro-tein from the uncultured marine bacterium 66A03 was gener-ated using the Accelrys Discovery Studio Modeler (Accelrys,San Diego) (36). The generated structure was checked byPROCHCK (37), and then structure minimization was con-ducted using Chemistry at Harvard Molecular Mechanics (38).After energy minimization, molecular dynamics modeling wasperformed at 300 K, 1 atm for 500 ps with 1 fs each step. Allsimulation experiments were carried out on an HP XW6200Workstation with dual Intel Xeon 3.2-GHz processors.




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�-Carotene, �-cryptoxanthin, and lutein were docked in theBlh models using the Surflex X docking program (Tripos, St.Louis, MO). Each docking run consisted of 100 independentdocks with 1,000 iteration cycles. A random start was used togenerate the substrate position within the docking box. Thesubstrate orientation giving the lowest interaction energy waschosen for additional rounds of docking.Analytical Methods—High pressure liquid chromatography

(HPLC) analyses of carotenoids and retinoids were performedbased on a previously reported method (10). The same volumeof acetonitrile was added to the reaction solution, and theresultant solution was mixed and kept on ice for 5 min. After

centrifugation at 10,000 � g for 10min at 4 °C, substrates and productsof the supernatant were analyzed byan HPLC system (Agilent 1200series, Santa Clara, CA) equippedwith a UV detector. The substrates�-carotene and lycopene weredetected at 460 and 445 nm, respec-tively, and lutein and zeaxanthinwere determined at 450 nm using aZorbaxsil column (250 � 4.6 mm,Agilent). The column was elutedwith the mixture of hexane and tert-butyl methyl ether with 97:3 (v/v) asthemobile phase with a flow rate of 1ml/min. The products (3R)-3-hy-droxy-retinal, retinal, and �-retinalwere detected at 370 nm with reten-tion times of 1.3, 1.8, and 2.2 min,respectively, and the product acyclo-retinal was detected at 400 nm usingan YMC-ODS A column (50 � 2.0mm, YMC, Kyoto, Japan). The col-umn was eluted with a 90:10 (v/v)mixture of acetonitrile and water asthe mobile phase at a flow rate of 0.4ml/min. The retinal isomers 13-cis-retinal, 9-cis-retinal, and all-trans-retinal were detected at 370 nmusingan YMC-ODS A column (250 � 4.6mm, YMC) with retention times of26.1, 26.3, and 27.3 min, respectively.The columnwas elutedwith 80%ace-tonitrile as the mobile phase at a flowrate of 1.0 ml/min.The HR-MS data were obtained

using a JMS-SX102A spectrometer(Jeol, Tokyo, Japan) operated at anaccelerating voltage of 10 kV. Theelectron impact ionizationmass spec-tra were collected in the positive ionmode. The HR-MS was performedunder the acquisition conditions asfollows: ion source temperature230 °C, ionization energy 70 eV, andionization current 300 �A.

RESULTS

DNA Sequence of the Codon-optimized blh Gene—Codons ofthe blh gene encoding the �-carotene-cleaving enzyme from theuncultured marine bacterium 66A03 were optimized, and theentire gene was synthesized. Among the 828 bp of the blh gene,including a stop codon, 213 bp (25.8%) were changed by codonoptimization (supplemental Fig. 1). The synthesized gene wascloned into the pET-24a(�) vector and expressed inE. coli. Theexpressed Blh protein consisted of 275 amino acids. An align-ment of the Blh protein with other Blh and Brp-like proteins,includingmembrane spanning domains, is shown in Fig. 1. The

FIGURE 1. Alignment of Blh and Brp-like proteins originated from typical strains. The proposed metal-binding residues (four histidine) based on molecular modeling are highlighted with a black background. Thepredicted transmembrane regions are underlined. The digested peptide sequences of the protein expressedfrom the codon-optimized blh gene that were identified from the MS data are italicized. Sequence alignmentwas performed by ClustalW program. Asterisks indicate identity. The GenBankTM accession numbers are asfollows: uncultured marine bacterium 66A03 Blh protein, AAY68319; uncultured marine bacterium HF10 49E08Blh protein, ABL97831; Halobacterium sp. NRC-1 Blh protein, NP280794; H. marismortui ATCC 43049 Brp-likeprotein, YP136627; H. walsbyi DSM 16790 Brp-like protein, YP656806.




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proposed metal-binding residues, His-21, His-78, His-188, andHis-192, based on molecular modeling were absolutely con-served across all Blh and Brp-like proteins.Expression, Molecular Weight, and Identification of the Blh

Protein—The crude extract of soluble protein obtained fromharvested cells was purified by His-Trap HP affinity chroma-tography. The expressed enzyme was purified, without deter-gent, as a single band in SDS-PAGE, with a final purification of7.3-fold, a yield of 32%, and a specific activity of 45 nmol mg�1

min�1. The molecular weight of the purified protein as ana-lyzed by SDS-PAGE was about 32 kDa (Fig. 2A). The proteinwas purified again with detergent, including 0.05% 1-S-octyl-�-D-thioglucopyranoside, 0.5% 1-S-octyl-�-D-thioglucopyrano-side, 0.5% Tween 80, 6 M urea, 1% Triton X-100, or 1% coco-amidopropyl hydroxysultaine. This further purification,however, did not change the purification yield. The immuno-blot was performed with a His-tagged monoclonal antibody

against the His-tagged Blh protein.A band obtained from the solublefraction of the cell extract con-firmed the isolation of a highly puri-fied recombinant protein. The Blhprotein was expressed as a solubleform in E. coli, and the protein waspurified without detergent eventhough it is a member of the bacte-riorhodopsin family. Its solubleform may result from the codonoptimization, which is known toincrease the expression and solubil-ity of enzymes (39).The molecular weight of native

enzyme, based on the weights ofreference proteins, was estimatedusing gel filtration chromatography.The native Blh protein had amass of64 kDa as a dimer of 32-kDa sub-units (Fig. 2B). A chromatogram forthe gel filtration of the Blh proteinconfirms its purity and molecularweight (Fig. 2C).The Blh protein on a SDS-PAGE

was cut out and subjected totrypsinization. The digested pep-tides were recovered and character-ized on a nano liquid chromatogra-phy-MS/MS to obtain amino acidsequence data. The following highconfidence peptide sequences wereobtained: YNIAFELIG, SRRHFS-FVWKQL, and FIGLPHGALD(supplemental Fig. 2). A comparisonbetween these data and the Blhsequence showed that all three pep-tides were present in the sequence(Fig. 1).Determination of the Retinal

Product of the Blh Protein—Theenzymatic conversion of �-carotene into retinal was exam-ined by varying the reaction time and concentrations ofenzyme and substrate, based on a standard reaction per-formed in 100 mM Tricine-KOH buffer (pH 8.0) containing50 �M �-carotene and 0.04 unit/ml enzyme at 40 °C for 60min (Fig. 3, A–C). The products formed at different reactiontimes and concentrations of enzyme and substrate were ana-lyzed by an HPLC system using an YMC-ODS A column andshowed the same retention time as an all-trans-retinal stand-ard. However, other retinal isomers including 13-cis-retinaland 9-cis-retinal showed different retention times, indicat-ing the enzymatic product of �-carotene is all-trans-retinal.With increasing reaction time from 10 to 60 min, the sub-strate �-carotene decreased whereas the product all-trans-retinal increased (Fig. 3D).The enzyme cleaved �-carotene at its central double bond

(15,15�) to yield two molecules of retinal. The formation of ret-

FIGURE 2. Determination of expression and molecular weight of the Blh protein. A, SDS-PAGE stained withCoomassie Blue and Western blot of Blh protein. Lane 1, prestained marker proteins (72, 55, 43, 34, 26, 17, and11 kDa); lane 2, His-Trap HP column product; lane 3, soluble fraction of the expressed Blh protein. The solublefraction was transferred to a polyvinylidene difluoride membrane and was incubated with the His tag mono-clonal antibody for 1 h. The membrane was visualized with the Western blot detection system. B, determina-tion of molecular weight of the purified native enzyme using Sephacryl S-300 HR gel filtration chromatogra-phy. The reference proteins were aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonicanhydrase (29 kDa), and RNase A (13.7 kDa). The Blh protein eluted at a position corresponding to 64 kDa. Datarepresent the means of three experiments, and error bars represent standard deviation; C, gel filtration chro-matogram of the Blh protein to confirm purity and molecular weight.




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inal as a reaction product increased linearly up to �0.2 unit/mlenzyme, 20�M �-carotene, and 30min of reaction time, respec-tively. However, above these parameters, the rates of productformation were not linear; increasing the concentrations ofenzyme and substrate and the reaction time subsequentlydeveloped sigmoidal curves. We performed enzyme assays inthe absence of substrate and in the presence of heat-inactivatedBlh 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 theremoval of metal ions by phenanthroline exhibited 2% activityrelative to that without phenanthroline treatment. Among themetal ions tested, Fe2�had the strongest effect on the activity ofthe Blh protein, whereas Ba2� had the least effective. The

effects of other ions decreased in the order of Fe3�, Co2�, Ca2�,Cu2�, andMn2� (Fig. 4A). The addition of 10�MFe2� after theremoval of all metal ions induced a 54% recovery of the activeform of the enzyme. The optimal concentration of Fe2� was 10�M, regardless of phenanthroline treatment (Fig. 4, B and C).Thus, all subsequent experiments were performed in the pres-ence of 10 �M Fe2�.The enzymatic conversion of �-carotene into all-trans-reti-

nal was examined at pH values ranging from 6.0 to 9.0. Themaximum enzyme activity was observed at pH 8.0 (Fig. 5A).The effect of temperature on the enzyme activity is shown inFig. 5B. The maximum activity was recorded at 40 °C. Abovethis temperature, the enzyme activity decreased significantly,exhibiting 66% of the maximum activity at 50 °C. Below 40 °C,

FIGURE 3. Determination of the retinal product of the Blh protein by varying the reaction time and concentrations of enzyme and substrate. Datarepresent the means of three experiments, and error bars represent standard deviation. The experiments for determination of the retinal product werecompensated with controls that were performed with the same reactions without enzyme. A, reactions by varying the reaction time were performed in 100 mM

Tricine-KOH buffer (pH 8.0) containing 50 �M �-carotene and 0.04 unit/ml enzyme at 40 °C. B, reactions by varying the concentration of the Blh protein wereperformed in 100 mM Tricine-KOH buffer (pH 8.0) containing 50 �M �-carotene at 40 °C for 60 min. C, reactions by varying the concentration of �-carotene wereperformed in 100 mM Tricine-KOH buffer (pH 8.0) containing 0.04 unit/ml enzyme at 40 °C for 60 min. D, HPLCs of the cleavage product of �-carotene by the Blhprotein and all-trans-retinal standard. The substrate �-carotene was determined at 460 nm using a Zorbaxsil column (250 � 4.6 mm), and the product retinalwas detected at 370 nm using an YMC-PAC Pro C18 column (50 � 2.0 mm).




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the enzyme activity decreased with decreasing temperature,exhibiting 15% of the maximum activity at 25 °C.The thermostability of the Blh protein was measured at five

incubation temperatures. The activity of the enzyme was verystable at below 45 °C but significantly decreased at above 50 °Cwith increasing reaction time (Fig. 5C). The enzyme followedthe first-order kinetics of thermal inactivation, and the half-lives of the enzyme at 35, 40, 45, 50, and 55 °C 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 recombi-nant Blh protein on �-carotene, �-carotene, �-carotene,�-cryptoxanthin, zeaxanthin, lutein, �-apo-4�-carotenal,�-apo-8�-carotenal, �-apo-12�-carotenal, and lycopene were

determined and are shown in Table 1. The correspondingMichaelis-Menten curves are shown in supplemental Fig. 3.The Blh protein exhibited the highest catalytic efficiency (kcat/Km) with �-carotene among the substrates tested. This impliesthat �-carotene is an authentic substrate for the enzyme. Thesecond-most kinetically favored substrate (kcat/Km) was�-cryptoxanthin, followed by �-apo-4�-carotenal, �-carotene,and �-carotene. �-Cryptoxanthin was cleaved into retinal and(3R)-3-hydroxy-retinal. If the 15,15�-double bond of zeaxan-thin or lutein were cleaved, the products would have been�-(3R)-3-hydroxy-retinal and (3R)-3-hydroxy-retinal, or twomolecules of (3R)-3-hydroxy-retinal, respectively. Their com-mon product, (3R)-3-hydroxy-retinal, was formed by the enzy-matic conversion of �-cryptoxanthin but was not formed by

FIGURE 4. Effect of metal ions on enzyme activity. Data represent the means of three experiments, and error bars represent S.D. The experiments for the effectof metal ions were compensated with the experiments that were performed with the same reactions without enzyme. Control indicates the purified Blh proteinbefore phenanthroline treatment. A, Blh protein was obtained after incubating at 20 °C with 1 mM phenanthroline as an iron-chelating agent for 1 h andfollowed by overnight dialysis at 4 °C against 100 mM Tricine-KOH buffer (pH 8.0). Each metal ion of 10 �M was added to the Blh protein obtained after thetreatment of phenanthroline to investigate the effect of the kind of metal ions on the conversion of �-carotene to retinal. Phenanthroline indicates the Blhprotein obtained after treating 1 mM phenanthroline. B, Blh protein was obtained after incubating at 20 °C with 1 mM phenanthroline as an iron-chelating agentfor 1 h and following by overnight dialysis at 4 °C against 100 mM Tricine-KOH buffer (pH 8.0). The effect of was investigated with the Blh protein with treating1 mM phenanthroline. The different concentrations of Fe2� were added to the Blh protein obtained after the treatment of phenanthroline to investigate theeffect of Fe2� concentration on the conversion of �-carotene to retinal with the Blh protein. C, effect of Fe2� concentration was investigated with the purifiedBlh protein without phenanthroline treatment. The different concentrations of Fe2� were added to the purified Blh protein without phenanthroline treatment.




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cleavage of zeaxanthin or lutein (supplemental Fig. 4). Neitherretinal, as the enzymatic product of �-apo-8�-carotenal, nor�-apo-12�-carotenal and acycloretinal, as the products of lyco-pene, were detected. These results demonstrate that the Blhprotein has no activity on zeaxanthin, lutein, apo-8�-carotenal,�-apo-12�-carotenal, or lycopene.Origin of the Oxygen Atom of Retinal in �-Carotene Cleavage

by the Blh Protein—The molecular weight of retinal as a prod-uct was determined after the enzymewas incubatedwith�-car-otene in the presence of H2

18O or 18O2. For labeling experi-ments with H2

18O, freeze-dried enzyme and �-carotene weresuspended in the H2

18O solution. Themass spectrum of retinalformed under these conditions was the same as that usingH2O,and the molecular weight of retinal was determined as 284m/z(Fig. 6A), indicating that the oxygen atom of retinal did notoriginate from water. However, after the enzyme reaction was

performed in an 18O2 atmosphere, the formed retinal waslabeled with the 18O atom, and themolecularmass was 286m/z(Fig. 6B). The fragment molecular masses of labeled retinalcontaining isotope 18O such as 213 and 241 m/z were 2 m/zgreater than those of unlabeled retinal containing normal 18Osuch as 211 and 239m/z. Thus, the oxygen atom of retinal as areaction product of the Blh protein originated from molecularoxygen rather than from water.

DISCUSSION

Although most recent critical debates about �-carotene-cleaving enzymes have tended to center around mammalian�-carotene 15,15�-monooxygenases, we became interested in�-carotene-cleaving enzymes from other sources, especiallybacteria. Hence, we used codon optimization to synthesize theblh gene from the uncultured marine bacterium 66A03 for

FIGURE 5. Effects of pH, temperature, and thermostability on the activity of the Blh protein. Data represent the means of three experiments, and error barsrepresent S.D. The experiments for effects of pH, temperature, and thermostability were compensated with controls that were performed with the samereactions without enzyme. A, two different buffers for pH experiment were used as follows: 100 mM potassium phosphate buffer for pH 6.0 –7.0 (E) and 100 mM

Tricine-KOH buffer for pH 7.0 –9.0 (F). The reactions were performed with 50 �M �-carotene and 0.04 unit/ml enzyme at 40 °C for 60 min. B, reactions wereperformed 100 mM Tricine-KOH buffer (pH 8.0) containing 50 �M �-carotene and 0.04 unit/ml enzyme for 60 min in temperature range of 25–50 °C. C, ther-mostability of the Blh protein for retinal production was measured at 35 (F), 40 (�), 45 (Œ), 50 (E), and 55 °C (f). A sample was withdrawn at each time interval,and the residual relative activity was measured after the reaction was performed with 100 mM Tricine-KOH buffer (pH 8.0) containing 50 �M �-carotene and 0.04unit/ml enzyme at 40 °C for 60 min.




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expression in E. coli. The expressed enzyme converts �-caro-tene into all-trans-retinal. Characterization of the bacterial Blhprotein as a newmember of the�-carotene cleavage enzymes isa worthwhile undertaking.The Blh protein is a putative membrane protein closely

related to bacteriorhodopsin (40). Bacteriorhodopsin is amem-brane protein containing seven transmembrane�-helices and acovalently bound molecule of retinal. The Blh protein is pre-dicted by the TMHMMprogram to have seven transmembrane

segments, whereas human �-carotene 15,15�-monooxygenasehas no transmembrane segments. These results suggest thatthe Blh protein is a membrane protein The theoretical pIvalues of the Blh proteins from marine bacteria (8.89–9.56)were much higher than those of the mammalian �-carotene15,15�-monooxygenases (5.49–6.30), indicating that thetwo proteins are different (Table 2). There were 48–56%hydrophobic amino acids in the bacterial Blh proteins and33–34% in mammalian �-carotene 15,15�-monooxygenases.

FIGURE 6. Incorporation of molecular oxygen into the reaction product retinal determined by HR-MS analysis. A, mass spectrum of retinal formed by theBlh protein from �-carotene in the H2

18O solution. The main fragment of mass spectrum in the solution of H218O indicates 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], and their molecular weights were 211, 227, 239, 253,and 268 m/z, respectively. B, mass spectrum of retinal formed by the Blh protein from �-carotene in 18O2 atmosphere. The main fragment of mass spectrum inan 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], and their molecular weights were 213, 227, 241, 253, and 268 m/z, respectively.

TABLE 1Kinetic parameters of the Blh protein for various carotenoids as substratesThe reactionwas performed in 100mMTricine-KOHbuffer (pH 8.0) at 40 °C for 30min using the amounts of substrates ranging from 5 to 800�M. Data represent themeanof three experiments, and � values represent S.D. ND means kinetic parameters were not detected by the analytical methods used in this study.




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Thus, the Blh proteins are more hydrophobic than mamma-lian �-carotene 15,15�-monooxygenases.

The subunitmolecular weight of the Blh proteinwas calculatedas 32 kDa based on the 281-residue amino acid structure plus ahexahistidine tag at the carboxyl terminus. The native protein hasa total molecular mass of about 64 kDa as a dimer (Fig. 2). �-Car-otene 15,15�-monooxygenases from humans, mice, chickens, andrats consisted of 547, 566, 526, and 566 amino acid residues,respectively, and their molecular masses were �60–64 kDa(Table 3). The molecular mass of the native human �-carotene15,15�-monooxygenase as a tetramer is 250 kDa (41).Mammalian �-carotene 15,15�-monooxygenases are re-

ported to be Fe2�-dependent protoporphyrin (non-hemeiron) enzymes and are inhibited by iron-chelating agents (4, 42,43). Phenanthroline behaves as an iron-chelating agent andstrongly inhibited the conversion of �-carotene into retinal bythe 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 activesites of mammalian �-carotene monooxygenase and apocaro-tenoid oxygenase (43, 44) and in themolecularmodel of the Blhprotein (Fig. 7A). In the Blh protein, Fe2� is coordinated withHis-21,His-78,His-188, andHis-192,which are absolutely con-served across all Blh and Brp-like proteins (Fig. 1). The fourconserved histidine residues were replaced with alanine to pro-duce alanine-substituted mutants. Enzyme activities weredetermined for the mutants and were compared with that ofwild-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 Blhprotein are metal-binding residues. The enzyme activity in thepresence of 10 �M Fe2� before dialysis was almost the same asafter dialysis. These results imply that Fe2� is tightly bound tothe Blh protein.Themaximum activity of the Blh protein was observed at pH

8.0 and 40 °C (Fig. 5). The maximum activities of �-carotene

15,15�-monooxygenases from mammalian sources occurred atpH 7.5–8.5, except for the mouse enzyme that had amaximumactivity at pH 9.0 (45). The optimum reaction temperature ofmammalian �-carotene 15,15�-monooxygenases from rabbits(42), hogs (46), pigs (47), chickens (11), mice (48, 49), andhumans (13, 41) was 37 °C. The thermostability of the human�-carotene 15,15�-monooxygenase was investigated, and thehalf-lives of the human enzyme at 35, 40, 45, and 50 °Cwere 4.5,3.4, 2.4, and 1.4 h, respectively. The half-lives of the Blh proteinwere approximately 4–5-fold higher at these temperaturesthan those of the human �-carotene 15,15�-monooxygenase.

The Blh protein from the uncultured marine bacterium con-verted the substrate �-carotene into two molecules of retinal,and it converted �-apo-4�-carotenal into one molecule of reti-nal. The enzyme cleaved the central double bonds (15,15�) of�-carotene, �-carotene, and �-cryptoxanthin to yield not onlyonemolecule of retinal but also onemolecule of each �-retinal,acycloretinal, and (3R)-3-hydroxy-retinal. �-Carotene has two�-ionone rings; �-carotene has �-ionone and �-iononerings, and �-carotene and �-apo-carotenals have one �-ion-one ring. �-Cryptoxanthin has �-ionone and hydroxyl �-ionone rings; zeaxanthin has hydroxyl �-ionone and hydroxyl�-ionone rings, and lutein has two hydroxyl �-ionone rings.However, lycopene does not have a�-ionone ring. The Blh pro-tein exhibited activity for �-carotene, �-cryptoxanthin, �-apo-4�-carotenal (C35), �-carotene, and �-carotene in decreasingorder, but not for �-apo-8�-carotenal (C30), �-apo-12�-carote-nal (C25), lutein, zeaxanthin, or lycopene. Based on the chemi-cal structures of the carotenoid substrates, enzyme activity wasfound only for substrates containing one unsubstituted �-io-none ring with a molecular weight greater than C35. The mam-malian �-carotene 15,15�-monooxygenases from humans andrats had activity for �-carotene, �-cryptoxanthin, and �-apo-8�-carotenal (C30) but not lycopene (41, 50). We found thathuman �-carotene 15,15�-monooxygenase had activity for�-apo-4�-carotenal (C35). Although the kcat/Km values of the

TABLE 2Theoretical pI values and hydrophobic amino acid contents of Blh and Brp-like proteins and �-carotene 15,15�-monooxygenases

Protein Source pI Hydrophobic amino acidsa

%Blh protein Uncultured marine bacterium 66A03 9.56 52.4

Uncultured marine bacterium HF10 49E08 9.30 48.4Halobacterium sp. NRC-1 8.89 55.2

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

�-Carotene 15,15�-monooxygenase Chicken 6.01 32.8Human 6.30 33.7Mouse 5.49 33.3Rat 5.90 32.9

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

TABLE 3Biochemical properties of the Blh protein and �-carotene 15,15�-monooxygenases

Organism Optimumtemperature

OptimumpH Km Vmax kcat kcat/Km

Molecularmass Native enzyme No. of

amino acidsHalf-lifeat 45 °C Ref.

°C �M nmol mg�1 min�1 min�1 mM�1 min�1 kDa hUnculturedmarinebacterium

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 41Mouse 37 9.0 6.0 0.04 64 566 45, 48Chicken 37 8.0 60 526 32Rat 37 7.5 3.3 0.03 64 566 4




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Blh protein and the human �-carotene 15,15�-monooxygenasefor�-carotenewere similar, the kcat/Km value of the Blh proteinfor �-cryptoxanthin (28 mM�1 min�1) as a substrate was14-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 enzymestructurewas constructed, and side chainswere added based onaveraged similar secondary structures in the protein data base.Simulated molecular docking was performed with �-carotene,�-cryptoxanthin, and lutein substrates using the Surflex Xdocking program. When �-carotene was bound in the activesite, the para position of one �-ionone ring of �-carotene,

located in the interior of the active site, and the para position ofthe other unhydroxylated �-ionone ring interacted with theoxygen atom of the carbonyl group of Thr-179, at a distance of3.01Å (Fig. 7B).When�-cryptoxanthinwas bound in the activesite, the distance between the hydroxyl group of the (3R)-3-hydroxy-�-ionone ring of �-cryptoxanthin and the oxygenatomof the carbonyl group of Thr-179 decreased to 2.31Å (Fig.7C). The shorter distance may explain the observed higheraffinity of the Blh protein for �-cryptoxanthin than for �-caro-tene. When lutein (or zeaxanthin) was bound in the active site,two hydroxyl groups of two (3R)-3-hydroxy �-ionone rings oflutein (or zeaxanthin) were noneffective in anchoring to theactive site because the distance to the oxygen atom of Thr-179

FIGURE 7. Molecular model of the Blh protein. The oxygen atom at carbonyl group of Thr-179 interacted with the para position in the �-ionone ring of�-carotene or with the hydroxyl group in the (3R)-3-hydroxy-�-ionone ring of �-cryptoxanthin. A, active site of the Blh protein. Docking of �-carotene (B),�-cryptoxanthin (C), and lutein (D) into the active site of the Blh protein is shown.




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was too long (Fig. 7D). As a result, the Blh protein exhibited noactivity for lutein or zeaxanthin. The Blh protein showedactivity for �-apo-4�-carotenal but not for �-apo-8�-carote-nal. In the molecular modeling studies, �-apo-4�-carotenalwas large enough to interact with Thr-179, but �-apo-8�-carotenal was too small to interact (data not shown). However,it is necessary to obtain the actual structure of the enzyme com-plexedwith these substrates to provide further evidence for thisconclusion. To confirm our molecular modeling and structuralanalyses, determination of the crystal structure of the Blh pro-tein and mutational analysis of the active site residues must beperformed.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 themonooxygenase isprovided by molecular oxygen and water via an epoxide inter-mediate, whereas that from the dioxygenase is provided bymolecular oxygen rather than fromwater via a dioxetane inter-

mediate (51, 52). The oxygenation mechanism of chicken�-carotene 15,15�-oxygenase was investigated with isotopicmolecular oxygen and water. The oxygen atom in the terminalaldehyde group of retinal as a product was provided by molec-ular oxygen and water (51), indicating that the enzyme cata-lyzes the oxidative cleavage using amonooxygenase rather thana dioxygenase mechanism. The other mammalian �-carotene15,15�-oxygenaseswere postulated to use the samemechanism.However, some controversies on the reaction mechanism wassuggested because oxygen between retinal and water wasexchanged within 5% (51). In contrast, the oxygen atom of theretinal formed by the reaction of the Blh protein originatedfrommolecular oxygen rather than fromwater without oxygenexchange between retinal and water, indicating that the Blhprotein used a dioxygenase mechanism unlike the mammalian�-carotene 15,15�-monooxygenases. The Blh protein cleaved�-carotenoid substrates at its central double bond (15,15�) andshowed the highest activity for �-carotene among the sub-strates tested. Thus, the Blh protein can be identified as a�-car-otene 15,15�-dioxygenase.In the molecular model, the active site of the Blh protein

exhibited coordination of Fe2� with His-21, His-78, His-188,and His-192 (Fig. 7A). According to the cleaving mechanism ofapocarotenoid oxygenase (52), the O2 molecule in the activesite of the Blh protein binds to the coordination shell of Fe2� ina side-on fashion. The side-on complex of Fe-O2 then attacksand cleaves between the C-15 and C-15� atoms when bound tothe Blh protein.Most enzymes that catalyze the same reaction sequence are

structurally similar. However, structurally and mechanisticallyunrelated enzymes that catalyze the same biochemical reac-tions do occur. In many cases, one of these analogous enzymesis found in the bacteria and the other in eukaryotes (53).Although the bacterial Blh protein and the mammalian�-carotene 15,15�-monooxygenases could both convert�-carotene into retinal, the properties of the bacterial Blhprotein such as DNA and amino acid sequences, molecularweight, form of association, reaction mechanism, kineticproperties, and substrate specificity were different from themammalian �-carotene 15,15�-monooxygenases. Thus, theBlh protein is a bacterial analog to the mammalian �-caro-tene 15,15�-monooxygenases.This is the first report of in vitro characterization of a bacte-

rial �-carotene-cleaving enzyme. This study contributes to theunderstanding of bacterial �-carotene-cleaving enzymes andprovides a stepping stone for further studies. Moreover, thisstudy will be useful in evolutionary studies investigating therelationships between bacterial and mammalian enzymes andindustrial applications of retinal biosynthesis.

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Yeong-Su Kim, Nam-Hee Kim, Soo-Jin Yeom, Seon-Won Kim and Deok-Kun Oh-Dioxygenase′-Carotene 15,15βMarine Bacterium as a

Characterization of a Recombinant Blh Protein from an UnculturedIn Vitro

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invitro characterizationofarecombinantblhproteinfrom ... · membrane was visualized using a...

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