Identification of a substrate-binding site in a peroxisomal β-oxidation enzyme by photoaffinity labeling with a novel palmitoyl derivative

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 34, pp. 26315–26325, August 20, 2010

Identification of a Substrate-binding Site in a Peroxisomal

␤-Oxidation Enzyme by Photoaffinity Labeling with a Novel

□

S

Palmitoyl Derivative^*

Received for publication, January 15, 2010, and in revised form, May 28, 2010 Published, JBC Papers in Press, June 21, 2010, DOI 10.1074/jbc.M110.104547

Yoshinori Kashiwayama^‡1, Takenori Tomohiro^§1, Kotomi Narita^‡, Miyuki Suzumura^§, Tuomo Glumoff^¶,

J. Kalervo Hiltunen^¶, Paul P. Van Veldhoven^ʈ, Yasumaru Hatanaka^§2, and Tsuneo Imanaka^‡3

From the ^‡Department of Biological Chemistry and ^§Laboratory of Biorecognition Chemistry, Graduate School of Medicine and

Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan, the ^¶Department of Biochemistry and

Biocenter Oulu, University of Oulu, P. O. Box 3000, FIN-90014, Finland, and the ^ʈDepartment of Molecular Cell Biology, LIPIT,

Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium

Peroxisomes play an essential role in a number of impor-

tant metabolic pathways including ␤-oxidation of fatty acids

and their derivatives. Therefore, peroxisomes possess various

␤-oxidation enzymes and specialized fatty acid transport sys-

tems. However, the molecular mechanisms of these proteins,

especially in terms of substrate binding, are still unknown. In

this study, to identify the substrate-binding sites of these

proteins, we synthesized a photoreactive palmitic acid ana-

logue bearing a diazirine moiety as a photophore, and per-

formed photoaffinity labeling of purified rat liver pero-

xisomes. As a result, an 80-kDa peroxisomal protein was

specifically labeled by the photoaffinity ligand, and the label-

ing efficiency competitively decreased in the presence of

palmitoyl-CoA. Mass spectrometric analysis identified the

80-kDa protein as peroxisomal multifunctional enzyme type

2 (MFE2), one of the peroxisomal ␤-oxidation enzymes.

Recombinant rat MFE2 was also labeled by the photoaffinity

ligand, and mass spectrometric analysis revealed that a frag-

ment of rat MFE2 (residues Trp²⁴⁹to Arg²⁵¹) was labeled by

the ligand. MFE2 mutants bearing these residues, MFE2(W249A)

and MFE2(R251A), exhibited decreased labeling efficiency.

Furthermore, MFE2(W249G), which corresponds to one of

the disease-causing mutations in human MFE2, also exhib-

ited a decreased efficiency. Based on the crystal structure of

rat MFE2, these residues are located on the top of a hydro-

phobic cavity leading to an active site of MFE2. These data

suggest that MFE2 anchors its substrate around the region

from Trp²⁴⁹to Arg²⁵¹and positions the substrate along the

hydrophobic cavity in the proper direction toward the cata-

lytic center.

Peroxisomes are organelles bound by a single membrane,

which are present in almost all eukaryotic cells. Peroxisomes

are involved in a variety of important metabolic processes

including the ␤-oxidation of fatty acids, synthesis of plasmalo-

gen, and bile acids (1). A specialized set of enzymes responsible

for the peroxisomal functions are compartmentalized in the

organelle, and the deficiency of peroxisomal enzymes causes

severe metabolic disease such as acyl-CoA oxidase deficiency

and ^D-bifunctional protein deficiency (2–4). Fibroblasts from

patients with these deficiencies exhibit the disturbed peroxiso-

mal ␤-oxidation of very long-chain fatty acids (VLCFAs),⁴and

VLCFAs are accumulated in the plasma and tissues of these

patients (5). Analyses of these peroxisomal diseases reflect the

importance of the peroxisomal ␤-oxidation in organisms along

with disclosing the enzymatic organization of the peroxisomal

␤-oxidation system. Peroxisomes are involved in the ␤-oxida-

tion of VLCFAs, branched chain fatty acids such as pristanic

acid, and bile acid intermediates such as dihydroxycholestanoic

acid and trihydroxycholestanoic acid, which are incompatible

with mitochondrial ␤-oxidation (6). Peroxisomal ␤-oxidation

of fatty acids proceeds via a four-step pathway as in mitochon-

dria, and multiple enzymes are involved in each step of the

pathway. The first step of peroxisomal ␤-oxidation is the de-

saturation of acyl-CoA to the corresponding 2E-enoyl-CoA.

In mammals, acyl-CoA oxidase 1 (ACOX1) oxidizes straight

chain acyl-CoAs, whereas trihydroxycholestanoyl-CoA oxidase

(ACOX2) and pristanoyl-CoA oxidase (ACOX3) oxidize both

straight and 2-methyl-branched acyl-CoAs (7, 8). The second

and third reactions are catalyzed by two multifunctional

enzymes (MFEs) by stereochemically distinct pathways. MFE1

hydrates 2E-enoyl-CoA to 3S-hydroxyacyl-CoA and subse-

quently dehydrogenates the 3S-hydroxyacyl-CoA to 3-keto-

acyl-CoAs, which is a naturally occurring pathway in the ␤-ox-

idation of straight chain fatty acids (9, 10). MFE2, the other

peroxisomal multifunctional enzyme, which also possesses

2E-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogen-

ase activities, converts the 2E-enoyl-CoA into the correspond-

ing 3-ketoacyl-CoA via a 3R-hydroxyacyl-CoA intermediate

* This work was supported in part by Grants-in-aid for the Research on Mea-

sures for Intractable Diseases from the Ministry of Health, Labour and Wel-

fare of Japan and for Scientific Research from the Ministry of Education,

Culture, Sports, Science and Technology of Japan (18590049, 18790050,

18390036, and 20390032). This work also supported in part by grants from

CLUSTER (Cooperative Link of Unique Science and Technology for Econ-

omy Revitalization) and the Fugaku Trust for Medicinal Research.

□

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The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. S1 and S2 and Table S1 .

¹Both authors contributed equally to this work.

²To whom correspondence may be addressed. Tel.: 81-76-434-7515; Fax: ⁴The abbreviations used are: VLCFA, very long-chain fatty acid; ACOX, acyl-

81-76-434-5063; E-mail: yasu@pha.u-toyama.ac.jp.

CoA oxidase; H2, 2E-enoyl-CoA hydratase; HD, 3R-hydroxyacyl-CoA dehy-

drogenase; LCFA, long-chain fatty acid; MFE1 and MFE2, multifunctional

enzymetype1andtype2;PMP70, 70-kDaperoxisomalmembraneprotein.

³To whom correspondence may be addressed. Tel.: 81-76-434-7545; Fax:

81-76-434-7545; E-mail: imanaka@pha.u-toyama.ac.jp.

AUGUST 20, 2010•VOLUME 285•NUMBER 34

JOURNAL OF BIOLOGICAL CHEMISTRY 26315

Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

(11–15). MFE2 has a broad substrate spectrum, and is able to zirin-3-yl]phenyl]-^L-alanine tert-Butyl Ester (2)—tert-Butylgly-

hydrate the enoyl-CoA esters of straight chain fatty acids, cinate benzophenone imine (0.36 g, 1.2 mmol) and O-allyl-N-

2-methyl-branched fatty acids, and bile acid intermediates, and 9-anthracenylmethylcinchonidium bromide (31) (0.06 g, 0.1

it can dehydrogenate the straight chain and branched chain mmol) were dissolved in CH₂Cl₂(7 ml) and the mixture was

3-hydroxyacyl-CoAs (14, 16–19). The final step in peroxisomal then cooled at Ϫ78 °C under nitrogen. 2-tert-Butylimino-2-

␤-oxidation is the thiolytic cleavage of 3-ketoacyl-CoAs into diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine

chain-shortened acyl-CoAs and acetyl-CoA or propionyl-CoA. (0.5 g, 1.8 mmol) in CH₂Cl₂(1 ml) and 2-[2-[2-(2-tert-

It can be catalyzed by 3-ketoacyl-CoA thiolase or sterol carrier butoxylcarbonylaminoethoxy)ethoxy]ethoxy]-4-[3-(trifluo-

protein x, which cleaves straight chain ketoacyl-CoAs or both romethyl)-3H-diazirin-3-yl]benzyl bromide (32) (0.55 g, 1

straight and 2-methyl-3-ketoacyl-CoAs, respectively (20). In mmol) in CH₂Cl₂(2 ml) were slowly added to the mixture fol-

addition to these enzymes, peroxisomes contain other impor- lowed by stirring for an additional 7 h. After removal of the

tant proteins that are directly or indirectly involved in the solvent, the residue was dissolved in ether. The solution was

␤-oxidation.

washed with water twice and brine and dried over MgSO₄.

In terms of the transport of fatty acid derivatives destined The crude product was purified by column chromatography on

for peroxisomal ␤-oxidation and their metabolites across the silica gel (hexane:EtOAc ϭ 1:1) to afford compound 2 (0.63 g,

peroxisomal membrane, peroxisomal ATP-binding cassette 85%) as a pale yellow oil; ¹H NMR (CDCl₃) ␦ 7.50–7.52 (m, 2H),

proteins are suggested to be involved in this process. To date, 7.22–7.38 (m, 6H), 7.10 (d, 1H, J ϭ 7.7 Hz), 6.62 (d, 1H, J ϭ 7.7

three ATP-binding cassette transporters have been identified Hz), 6.54 (br s, 2H), 6.48 (s, 1H), 5.02 (br s, 1H), 4.26 (dd, 1H, J ϭ

on mammalian peroxisomal membranes: the 70-kDa peroxiso- 3.4, 9.8 Hz), 3.92 (m, 1H), 3.50–3.62 (m, 9H), 3.36 (dd, 1H, J ϭ

mal membrane protein (PMP70) (21–23), adrenoleukodystro- 3.4, 13.2 Hz), 3.32 (br q, 2H, J ϭ 5.1 Hz), 3.01 (dd, 1H, J ϭ 9.8,

phy protein (24), and adrenoleukodystrophy protein related 13.2 Hz), 1.45 (s, 9H), 1.44 (s, 9H).

protein (25, 26). A defect in adrenoleukodystrophy protein is

3-[2-[2-[2-(2-tert-Butoxylcarbonylaminoethoxy)ethoxy]-

known to be responsible for the X-linked neurodegeneration ethoxy]-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-^L-

disorder, adrenoleukodystrophy. Adrenoleukodystrophy pa- alanine tert-Butyl Ester (3)—An 80% ethanol solution of

tient fibroblasts exhibit decreased ␤-oxidation of VLCFA, and NH₂OH/HCl (1 M, 5 ml) was added to a solution of compound 2

VLCFA is accumulated in plasma and tissues (27, 28). Further- (0.66 g, 0.81 mmol) in CH₂Cl₂(5 ml). The reaction mixture was

more, we have reported that PMP70 is involved in the meta- stirredat45 °Cfor1h. Afterremovalofthesolvent, theresiduewas

bolic transport of long-chain acyl-CoA across peroxisomal dissolved in CH₂Cl₂and purified by column chromatography on

membranes, based on studies with CHO cells overexpressing silica gel (CH₂Cl₂:methanol ϭ 10:1) to afford compound 3 (0.44 g,

wild- and mutant-type PMP70 (29).

92%) as a pale yellow oil; ¹H NMR (CDCl₃) ␦ 7.16 (d, 1H, J ϭ 7.7

The enzymatic organization of the peroxisomal ␤-oxidation Hz), 6.75 (d, 1H, J ϭ 7.7 Hz), 6.60 (s, 1H), 5.36 (br s, 1H), 4.18 (ddd,

system and peroxisomal fatty acid transport system has been 1H, J ϭ 3.4, 7.3, 9.8 Hz), 4.12 (ddd, 1H, J ϭ 3.4, 5.1, 9.8 Hz), 4.02 (br

made relatively clear. However, the precise molecular mecha- s, 1H), 3.89 (br s, 1H), 3.83 (ddd, 1H, J ϭ 3.4, 5.1, 11.1 Hz), 3.77–

nisms of these proteins, especially in terms of substrate recog- 3.80 (m, 1H), 3.69 (m, 1H), 3.63–3.66 (m, 2H), 3.55–3.60 (m, 1H),

nition, are poorly understood. In this study, we synthesized a 3.49–3.54 (m, 1H), 3.33 (br s, 2H), 3.27 (br s, 1H), 2.91 (dd, 1H, J ϭ

photoreactive palmitic acid analogue bearing a diazirine moiety 8.3, 13.9 Hz), 1.42 (s, 9H), 1.34 (s, 9H).

as a photophore, and performed a photochemical approach

N-Palmitoyl-3-[2-[2-[2-(2-tert-butoxylcarbonylaminoe-

with the novel photoreactive fatty acid analogue to identify fatty thoxy)ethoxy]ethoxy]-4-[3-(trifluoromethyl)-3H-diazirin-3-

acid-binding proteins and analyze the substrate binding of the yl]phenyl]-^L-alanine tert-Butyl Ester (4)—A CH₂Cl₂solution (1

peroxisomal proteins.

ml) of compound 3 (200 mg, 0.35 mmol) was added to a CH₂Cl₂

solution (3 ml) of palmitic acid (90 mg, 0.35 mmol), 1-(3-di-

methylaminopropyl)-3-ethylcarbodiimide (74 mg, 0.38 mmol),

and Et₃N (39 mg, 0.38 mmol). The reaction mixture was stirred

overnight at room temperature. After removal of the solvent,

the residue was purified by column chromatography on silica

gel (hexane:EtOAc ϭ 1:1) to afford compound 4 (223 mg, 79%)

as a pale yellow solid; ¹H NMR (CDCl₃) ␦ 7.17 (d, 1H, J ϭ 7.7

Hz), 6.73 (d, 1H, J ϭ 7.7 Hz), 6.59 (s, 1H), 6.37 (d, 2H, J ϭ 8.1

Hz), 5.12 (br s, 1H), 4.71–4.77 (m, 1H), 4.08–4.18 (m, 2H),

3.88–3.96 (m, 2H), 3.71–3.79 (m, 2H), 3.67 (t, 2H, J ϭ 4.7 Hz),

3.55 (t, 2H, J ϭ 5.1 Hz), 3.31 (q, 2H, J ϭ 5.1 Hz), 3.07 (dd, 1H, J ϭ

9.6, 13.5 Hz), 2.98 (dd, 1H, J ϭ 5.8, 13.5 Hz), 2.04–2.14 (m, 2H),

1.40–1.50 (m, 11H), 1.38 (s, 9H), 1.12–1.32 (m, 26H), 0.88 (t,

3H, J ϭ 6.8 Hz).

EXPERIMENTAL PROCEDURES

Materials

pET21a was obtained from Novagen (Madison, WI). Talon

metal affinity resin was purchased from Clontech (Palo Alto,

CA). Nycodenz was from Axis-Shield (Oslo, Norway). Strepta-

vidin-conjugated horseradish peroxidase was purchased from

GE Healthcare. SoftLink SoftRelease Avidin Resin and se-

quencing grade modified trypsin were obtained from Promega

(Madison, WI). ␣-Cyano-4-hydroxycinnamic acid was from

Sigma. Mouse anti-His G antibody was purchased from Invitro-

gen. Preparation of the antibody against the COOH-terminal

15 amino acids of rat PMP70 is described in Ref.30.

Synthesis of Photoreactive Palmitoyl Acid Analogue Bearing

Diazirine Moiety as a Photophore (Fig. 1)

N-Palmitoyl-3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-4-

[3-(trifluoromethyl)-3H-diazirin-3-yl]]-phenylalanine (5)—

N-(Diphenylmethylene)-3-[2-[2-[2-(2-tert-butoxylcarbon- Deprotection of compound 4 (0.20 g, 0.25 mmol) was per-

ylaminoethoxy)ethoxy]ethoxy]-4-[3-(trifluoromethyl)-3H-dia- formed with 50% TFA-CHCl₃(4 ml) at room temperature.

26316 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 34•AUGUST 20, 2010

Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

After stirring for 3 h, the solvent was removed. The product was mated sequencing on an ABI 310 DNA sequencer (PerkinElmer

extracted with CHCl₃three times from aqueous NaHCO₃solu- Life Science).

tion. The organic solution was combined, washed twice with

Site-directed Mutagenesis of MFE2

brine, and then dried over MgSO₄to afford compound 5 (0.16 g

quantity) as a pale yellow solid. Removal of the two tert-butyl

Site-directed mutagenesis was performed on pET21a/MFE2

groups was confirmed by NMR and the product was used with- using a QuikChange site-directed mutagenesis kit (Stratagene)

out further purification; ¹H NMR (CDCl₃) ␦ 7.21 (d, 1H, J ϭ 7.8 according to the manufacturer’s instructions. The oligonucleo-

Hz), 6.81 (br s, 1H), 6.69 (d, 1H, J ϭ 7.8 Hz), 6.51 (s, 1H), 4.52 (br tide primers used were designed on the basis of their sequences

s, 1H), 4.07–4.16 (m, 2H), 3.89–3.95 (m, 1H), 3.75–3.86 (m, (supplemental Table S1 ). The mutations in the constructions

2H), 3.64–3.75 (m, 5H), 2.91–3.17 (m, 4H), 2.00–2.09 (m, 4H), were confirmed by sequencing.

1.11–1.38 (m, 26H), 1.00–1.10 (m, 2H), 0.88 (t, 3H, J ϭ 7.1 Hz).

Purification of MFE2-His

N-Palmitoyl-3-[2-[2-[2-(2-biotinylaminoethoxy)ethoxy]-

ethoxy]-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]]-phenylala-

Overexpression and purification of recombinant MFE2 was

nine (1)—A solution of NHS-biotin (88 mg, 0.26 mmol) in performed as described by Haapalainen et al. (34) with some

N,N-dimethylformamide (3 ml) was added to a solution of com- modifications. Details are shown under the supplemental data.

pound 5 (160 mg, 0.24 mmol) and Et₃N (26 mg, 0.26 mmol) in

Photoaffinity Labeling

CHCl₃(3 ml), and the reaction mixture was stirred overnight at

room temperature under nitrogen. After removal of the solvent

The photoprobe (10 nmol) was dissolved in CHCl₃, and the

the residue was purified by column chromatography on silica solvent was evaporated by nitrogen gas stream to deposit the

gel (CHCl₃:methanol ϭ 5:1) to afford compound 1 (158 mg, probe on the wall of glass test tube as a thin film. Binding buffer

74%) as a pale yellow solid; ¹H NMR (5% CD₃OD-CDCl₃) ␦ 7.21 (25 m^MTris-HCl, pH 7.8, 300 mM NaCl, 5 mM EDTA, and 1 mM

(d, 1H, J ϭ 7.7 Hz), 6.74 (d, 1H, J ϭ 7.7 Hz), 4.63 (dd, 1H, J ϭ 5.1, DTT) containing 2 mg/ml of BSA was then added to make up a

9.4 Hz), 4.49–4.53 (m, 1H), 4.51 (dd, 1H, J ϭ 4.3, 7.7 Hz), 4.12– 20 ␮M probe solution and then mixed by vortex. In a typical

4.15 (m, 2H), 3.87–3.96 (m, 2H), 3.73–3.76 (m, 2H), 3.65–3.69 experiment, rat liver peroxisomes (100 ␮g of protein) or puri-

(m, 2H), 3.56–3.62 (m, 2H), 3.41–3.47 (m, 1H), 3.33–3.39 (m, fied MFE2-His (10 ␮g of protein) were suspended in a final

1H), 3.19 (dd, 1H, J ϭ 5.1, 13.7 Hz), 3.15 (dd, 1H, J ϭ 4.3, 7.3 Hz), volume of 0.1 ml of binding buffer. The photoprobe solution

3.02 (dd, 1H, J ϭ 9.4, 13.7 Hz), 2.92 (dd, 1H, J ϭ 5.1, 12.8 Hz), was then added to the suspension to a final concentration of 2

2.73 (dd, 1H, J ϭ 6.4, 12.8 Hz), 2.18 (t, 2H, J ϭ 6.8 Hz), 2.09 (t, ␮M, and the reaction mixtures were incubated at 4 °C for 2 h.

2H, J ϭ 7.7 Hz), 1.55–1.70 (m, 4H), 1.37–1.45 (m, 4H), 1.18– After UV irradiation with 30 W/m²of 360 nm light for 20 min at

1.32 (m, 24H), 1.09–1.16 (m, 2H), 0.88 (t, 3H, J ϭ 6.8 Hz).

0 °C, samples were subjected to SDS-PAGE and transferred

onto a PVDF membrane. The labeled proteins were detected by

a chemiluminescent method using streptavidin-conjugated

Preparation of Rat Liver Peroxisomes

The livers of male Wister rats were fractionated by the meth- horseradish peroxidase.

ods of de Duve et al. (33). The livers were homogenized in

MALDI-TOF Analysis of the Labeled Protein

After photoaffinity labeling of purified MFE2-His (100 ␮g of

4 volumes of SVEH (0.25 ^Msucrose, 1 mM EDTA, 0.1% eth-

anol, and 5 m^MHepes-KOH, pH 7.4), and the supernatant

(post-nuclear supernatant) was recovered by centrifugation at protein), the reaction product was applied onto SoftLink Soft

1,000 ϫ g for 10 min at 4 °C. The heavy and light mitochondrial Release Avidin Resin equilibrated with the binding buffer. After

fraction was sedimented from the post-nuclear supernatant incubation of the suspensions for 2 h at 4 °C, the beads were

fraction by centrifugation at 20,000 ϫ g for 22 min. About 0.7 collected by centrifugation and washed five times with 250 ␮l of

ml of the light mitochondrial fraction was applied onto a 10-ml the binding buffer. Labeled proteins were eluted by 2 ϫ sample

linear Nycodenz gradient (density span from 1.15–1.25 g/ml in buffer for SDS-PAGE (4% (w/v) SDS, 19.8% (v/v) sucrose, 0.08 ^M

SVEH), and centrifuged 193,000 ϫ g for 2 h at 4 °C. Fractions of Tris-HCl, pH 6.8, 20 mM DTT, and 0.002% (w/v) bromphenol

ϳ1.0 ml were collected in preweighed microtubes, and the den- blue), and separated on a 5–10% SDS-polyacrylamide gradient

sity of each fraction was determined by refractometry. The per- gel. After electrophoresis, labeled proteins were transferred

oxisomal fraction was identified by the density and distribution onto a PVDF membrane, stained with Coomassie Brilliant Blue,

of catalase (33).

destained, and then the bands corresponding to the labeled

proteins were excised from the membrane. The membrane

fragments were incubated for 1 h at 27 °C with reduction buffer

Plasmid Construction

The full-length MFE2 cDNA was obtained from the plasmid (0.5 ^MTris, pH 8.5, 8 M guanidinium HCl, 0.3%(w/v) EDTA, and

pUC18/rat MFE2 (34) by PCR amplification using the primer 5% acetonitrile) containing 10 mg/ml of DTT and then for 20

pair: 5Ј-GCTAGCCCTCTGAGGTTCGACGGG-3Ј (the NheI min in the same buffer containing 30 mg/ml of iodoacetamide.

site is underlined) and 5Ј-CTCGAGCTTGGCATAGTCTTT- The S-carboxyamidomethylated sample was soaked in 100 ␮l of

CAG-3Ј (the XhoI site is underlined). The PCR-generated frag- digestion buffer (50 m^MNH₄HCO₃, pH 8.0, and 70% acetoni-

ment containing NheI and XhoI restriction sites was inserted at trile), and digested with 2 ␮g of sequencing-grade modified

the corresponding sites of pET21a, a C-terminal histidine tag trypsin for 12 h at 27 °C. The digestion was terminated by the

fusion protein expression vector, to construct pET21a/MFE2. addition of PMSF to 10 ␮M of the final concentration and the

The identity of the construction was confirmed by semiauto- sample was concentrated in a SpeedVac concentrator (Savant,

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Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

Other Methods

Protein was assayed as described

previously (29). Western blot analy-

sis was performed using primary

antibodies and a secondary anti-

body, donkey anti-rabbit or anti-

mouse IgG antibody-conjugated

horseradish peroxidase (GE Health-

care). Antigen-antibody complex

was visualized with ECLϩ Western

blotting detection reagent (GE

Healthcare).

RESULTS

Chemistry—To identify and char-

acterize long-chain fatty acid (LCFA)-

binding proteins in peroxisomes, we

synthesized a novel photoreactive

LCFA probe 1 (Fig. 1A). The syn-

thetic probe consists of a LCFA, a

diazirine-based photophore, and a

biotin moiety with a hydrophilic

linker. The (3-trifluoromethyl)phe-

nyldiazirine is one of the most

promising photophores. By pho-

toirradiation, it produces a carbene

species, a highly reactive intermedi-

FIGURE 1. Synthesis of photoreactive LCFA probe. A, structure of the photoreactive LCFA probe. B, synthetic

scheme for the preparation of the photoreactive LCFA probe.

ate, and immediately makes a covalent bond with spatially close

molecules. A biotin moiety was directly anchored to the photo-

phore via a hydrophilic linker as a tag for the detection and

isolation of labeled proteins utilizing biotin-avidin interaction,

and is also useful for identifying the labeled protein and its

ligand-interacting site. For preparation of a photoreactive fatty

acid analogue, a phenylalanine analogue has been employed to

introduce a diazirine photophore with an accompanying car-

boxylate at the end of palmitic acid (16:0). Preparation of the

photoreactive phenylalanine derivative was achieved in excel-

lent yield according to the previous method (35). The coupling

reaction with palmitic acid and the subsequent biotinylation to

give the corresponding photoreactive fatty acid probe 1 is out-

lined in Fig. 1B.

Photoaffinity Labeling of Rat Liver Peroxisomes—Using the

photoreactive LCFA analogue as a probe, we performed pho-

toaffinity labeling experiments on purified rat liver peroxi-

somes. Purified rat liver peroxisomes were incubated with the

photoreactive fatty acid analogue, and the labeled proteins were

separated on a 7–15% SDS-polyacrylamide gradient gel, and

then detected by streptavidin-HRP. As shown in Fig. 2, an

80-kDa protein was specifically labeled by the probe upon

activation with UV irradiation. The efficiency of the labeling

of the 80-kDa protein increased with the irradiation time. In

addition to the 80-kDa protein, two bands with molecular

masses of 110 and 70 kDa, respectively, were also detected by

streptavidin-HRP. However, these two bands were detected

even in the non-irradiated sample, suggesting that these two

proteins were not photolabeled products, but nonspecifically

reacted with streptavidin-HRP.

FIGURE 2. Photoaffinity labeling of rat liver peroxisomes. Purified rat liver

peroxisomes (100 ␮g of protein) were incubated with the photoreactive LCFA

probe for 2 h at 4 °C. After UV irradiation at 360 nm for 0–30 min at 0 °C, labeled

proteins were separated on a 7–15% SDS-polyacrylamide gradient gel, and

detected by streptavidin-HRP. The arrowhead indicates the 80-kDa protein

labeled by the photoaffinity probe. Asterisks indicate nonspecific bands.

Farmingdale, NY). The resulting residue was resuspended in 10

␮l of 0.1% TFA. The tryptic peptide mixture was desalted with

Zip Tip (Millipore, Billerica, MA) and eluted in 2 ␮l of 33.3%

acetonitrile with 0.06% TFA. The recovered tryptic peptides (1

␮l) were mixed with 1 ␮l of a 10 mg/ml solution of ␣-cyano-4-

hydroxycinnamic acid in 33.3% acetonitrile with 0.06% TFA

and analyzed by MALDI-TOF mass spectrometry on a Bruker

MALDI-TOF AutoFlex mass spectrometer (Bruker-Daltonics,

Billerica, MA).

26318 JOURNAL OF BIOLOGICAL CHEMISTRY

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Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

Ligand : palmitoyl-CoA

-UV

1 : 0

1 : 1

1 : 5 1 : 10 1 : 25 1 : 50

kDa

97.4

66.2

*

kDa

66.2

PMP70

Ligand : palmitic acid

-UV

1 : 0

1 : 1

1 : 5 1 : 10 1 : 25 1 : 50

kDa

97.4

66.2

kDa

66.2

FIGURE 4. Purification of the photolabeled 80-kDa protein. Purified rat

liver peroxisomes (1 mg of protein) were labeled by the photoreactive LCFA

probe. After solubilization with 0.1% Triton X-100, labeled proteins were puri-

fiedbySoftLinkSoftReleaseAvidinResin. Purifiedproteinswereseparatedon

a 5–10% SDS-polyacrylamide gradient gel, and stained with silver staining.

The arrowhead and asterisk indicates the 80- and 65-kDa proteins labeled by

the photoaffinity probe, respectively.

PMP70

Ligand : CoA

-UV

1 : 0

1 : 1

1 : 5

1 : 10

kDa

97.4

PMP70, a peroxisomal membrane protein, but resembled those

of catalase, a peroxisomal matrix protein, implying that the

80-kDa protein is located in the peroxisomal matrix. To identify

the 80-kDa protein, peroxisomes were solubilized with 0.1%

Triton X-100 after photoaffinity labeling, and the labeled pro-

teins were isolated by streptavidin-agarose resin. As shown in

Fig. 4, the 80-kDa protein was successively purified. The puri-

fied probe-incorporated 80-kDa protein was digested with

trypsin, and the resulting tryptic peptide mixture was analyzed

by MALDI-TOF mass spectrometry. As a result, several specific

tryptic peptide fragments of the labeled protein were obtained,

and the MASCOT search engine revealed that almost all of the

major fragments correspond to MFE2-specific tryptic frag-

ments (supplemental Fig. S1 ). In addition to these fragments, a

66.2

*

kDa

66.2

PMP70

FIGURE 3. Photoaffinity labeling of rat liver peroxisomes in the presence

of palmitoyl-CoA, palmitic acid, and CoA. Purified rat liver peroxisomes

were photoaffinity labeled in the presence of increasing amounts of palmi-

toyl-CoA (A), palmitic acid (B), and CoA (C), respectively. After UV irradiation,

labeled proteins were separated on a 7–15% SDS-polyacrylamide gradient

gel, and detected by streptavidin-HRP (upper panels). Western blot analysis

for PMP70 is used as a loading control (lower panels). The arrowheads and

asterisks indicate the 80-kDa protein and the 65-kDa protein labeled by the

photoaffinity probe, respectively.

Next, to characterize the binding specificity of the probe to characteristic peak was observed at m/z 1345.81. We assigned

the 80-kDa protein, we examined the competitive effects of the fragment as a photoadduct fragment (details are shown

palmitoyl-CoA, palmitic acid, and CoA on the photoaffinity below).

labeling of the 80-kDa protein. As shown in Fig. 3A, the probe

Together with MFE2, a 65-kDa protein was also purified by

photoincorporation within the 80-kDa protein was suppressed streptavidin-agarose resin (Fig. 4). The 65-kDa protein was

by the presence of palmitoyl-CoA in a concentration-depen- indeed labeled by the LCFA probe and the labeling of the

dent manner. On the other hand, the labeling was not inhibited 65-kDa protein was competitively inhibited by the presence of

even in the presence of an excess amount of palmitic acid or palmitoyl-CoA with almost the same extent as that of MFE2

CoA (Fig. 3, B and C). These data indicate that the 80-kDa (Fig. 3). Accompanying with intact MFE2, the 65-kDa protein

protein has a potent capacity to interact with palmitoyl-CoA, was also detected in the purified rat liver peroxisomes by immu-

and the photoreactive LCFA probe could bind to the 80-kDa noblotting using anti-MFE2 antibody (data not shown). These

protein in the region responsible for its palmitoyl-CoA-binding data indicate that MFE2 is partially degraded to the 65-kDa

site.

protein and the 65-kDa region of MFE2 still possesses a potent

Identification of the 80-kDa Protein—In preliminary experi- capacity to interact with palmitoyl-CoA and the photoreactive

ments, the 80-kDa protein labeled by the photoaffinity probe LCFA probe.

was susceptible to extraction with sodium carbonate, and was

MFE2, also called multifunctional protein 2 or ^D-bifunctional

protected from digestion with trypsin in the absence of Triton protein, is a 79-kDa enzyme that possesses a typical peroxiso-

X-100, but degraded by trypsin in the presence of Triton X-100 mal matrix protein targeting signal (COOH-terminal Ala-Lys-

(data not shown). These behaviors were not similar to those of Leu). MFE2 consists of a 3R-hydroxyacyl-CoA dehydrogenase

AUGUST 20, 2010•VOLUME 285•NUMBER 34

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Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

tion with trypsin, the resulting

peptide mixture was analyzed by

MALDI-TOF mass spectrometry.

As shown in Fig. 6, various tryptic

fragments with observed molecular

masses that were compatible with

the calculated molecular mass of

MFE2 tryptic fragments were iden-

tified in samples prepared from

both labeled and unlabeled MFE2.

However, a peak at m/z 1345.39

was detected only in the sample pre-

pared from labeled MFE2. The frag-

ment was also detected in the cross-

linked MFE2 purified from rat liver

peroxisomes as described above

(supplemental Fig. S1 A). The

molecular mass of the fragment

did not agree with any of the theo-

retical MFE2 fragments digested

with trypsin, instead, it matched

exactly with the mass of the tryptic

(A)

(B)

MFE2-His His-Pex19p

Ligand

- + - +

kDa

200

116

97.4

116

97.4

MFE2-His

66.2

45.0

66.2

45.0

*

His-Pex19p

31.0

21.5

14.4

21.5

14.4

CBB staining

Avidin-HRP

FIGURE5. Photoaffinity labelingofpurifiedMFE2-His. PurifiedMFE2-His and His-Pex19p(A)were incubated

with the photoreactive LCFA probe for 2 h at 4 °C. After UV irradiation at 360 nm for 30 min at 0 °C, labeled

proteins were separated on a 7–15% SDS-polyacrylamide gradient gel, and detected by streptavidin-HRP (B).

The asterisk indicates nonspecific bands.

peptide of MFE2 (residues Trp²⁴⁹

-

Glu²⁵⁰-Arg²⁵¹; m/z 489.23) modi-

fied with the probe (m/z 1345.69 ϭ

(HD) domain, a 2E-enoyl-CoA hydratase 2 (H2) domain, and a 489.23 ϩ 856.46). On the other hand, the tryptic fragment cor-

sterol carrier protein 2-like domain, and is known to catalyze responding to MFE2 sequence Trp²⁴⁹-Glu²⁵⁰-Arg²⁵¹(m/z

the second and third steps of the peroxisomal ␤-oxidation of 489.24) was absent from the labeled MFE2-His, but observed in

fatty acids and their derivatives (36).

the control experiment using tryptic digestion of the unlabeled

Purification and Photoaffinity Labeling of MFE2—COOH- MFE2-His. To confirm that the amino acid region (Trp²⁴⁹

-

terminal His₆-tagged rat MFE2 (MFE2-His) was expressed in Glu²⁵⁰-Arg²⁵¹) within MFE2 is involved in ligand binding, we

Escherichia coli and purified with Talon metal affinity resin. purified the MFE2 mutants by replacing these amino acids with

MFE2-His was expressed upon isopropyl-␤-D-thiogalactopy- Ala, and analyzed the ligand binding efficiency. As shown in Fig.

ranoside induction as a major protein and exclusively recovered in 7, wild type MFE2 was labeled by the photoaffinity probe. How-

a soluble form. MFE2-His purified from the soluble fraction was ever, MFE2(W249A) and MFE2(R251A) exhibited a decreased

Ͼ80% pure as judged by SDS-PAGE (supplemental Fig. labeling efficiency. The 1abeling efficiencies of MFE2(W249A)

S2 A ). It is suggested that rat MFE2 functions as a homodimer. and MFE2(R251A) with the probe decreased to ϳ30% com-

Therefore, we analyzed the oligomeric state of purified MFE2 pared with that of the wild type MFE2 (Fig. 7C). These three

using sucrose density gradient centrifugation. The molecular mass amino acid residues lie within the HD domain of MFE2, and a

of the purified MFE2-His was estimated to be 155 kDa on sucrose disease-causing mutation has been identified in this region (38).

density gradient centrifugation, which almost corresponded to a The mutant, MFE2(W249G), also exhibited a decreased label-

dimeric form of MFE2-His (79.4 kDa) (supplemental Fig. ing efficiency, whereas MFE2(N158D), another disease-causing

S2 B ). The purified MFE2-His was subjected to photoaffinity mutant in which the mutated residue is situated outside of the

labeling with the photoreactive LCFA probe. As shown in labeled region (38), exhibited only a slightly affected labeling

Fig. 5, MFE2-His was efficiently labeled by the probe. On the efficiency for the photoreactive LCFA probe under the same

other hand, purified His₆-tagged Pex19p (His-Pex19p), a conditions. The purified MFE2 HD domain was also labeled by

cytosolic protein involved in the targeting of peroxisomal the photoaffinity ligand, and MFE2 HD(W249A) exhibited a

membrane proteins (37), was not labeled by the probe under decreased labeling efficiency. However, the ligand was incorpo-

the same conditions. These findings indicate that the pho- rated in the purified HD domain very weakly compared with

toaffinity ligand indeed incorporates into MFE2 with high full-length MFE2 (data not shown). The ternary composition of

specificity.

the three domains of MFE2 is required for the efficient sub-

Identification of the Substrate-binding Site of MFE2—As strate binding of MFE2. These data indicate that the amino acid

mentioned above, the photoreactive LCFA probe bound to region within MFE2 (Trp²⁴⁹-Glu²⁵⁰-Arg²⁵¹) is important for

MFE2 at its palmitoyl-CoA-binding site. Therefore, we tried ligand binding.

to identify the individual amino acid residues labeled by the

Characterization of the Substrate Binding Mode of MFE2—

probe within MFE2 to characterize the substrate-binding As described above, the photophore of the photoreactive LCFA

site of MFE2. MFE2-His was incubated with the probe and probe cross-linked with MFE2 at the three amino acid regions

the labeled MFE2-His was purified by avidin resin. After diges- of the MFE2 HD domain (Trp²⁴⁹-Glu²⁵⁰-Arg²⁵¹). The crystal

26320 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 34•AUGUST 20, 2010

Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

-ligand

3000

2000

1000

0

473.25

640-643

489.24

249-251

1011.36

24-32

1250.38

702-712

1352.40

494-505

729.43

252-258

1155.43

11-23

794.40

126-132

1389.38

111-121

1206.40

1282.46

93-104

669-680

400

500

900

m/z

1000

1100

1200

1300

1400

600

700

800

+ligand

3000

2000

1000

0

1345.39

400

500

900

1000

1100

1200

1300

1400

600

700

800

FIGURE 6. Identification of the substrate-binding site of MFE2. Purified MFE2-His (100 ␮g of protein) was incubated with the photoreactive LCFA probe. The

probe-incorporated MFE2-His was isolated by avidin resin followed by trypsin digestion. The resulting tryptic peptide mixture was analyzed by MALDI-TOF

mass spectrometry (lower panel). As a control, unlabeled MFE2-His was similarly digested with trypsin and the resulting tryptic peptide mixture was subjected

onto MALDI-TOF mass spectrometry (upper panel). The diagonal line indicates the mass difference between the peak in control sample corresponding to

unmodified MFE2-peptide (amino acids 249–251) and the peak corresponding to an adduct of the MFE2-peptide (amino acids 249–251) with the LCFA probe

in the sample treated with the probe.

structure of the HD domain of rat MFE2 HD has been solved by tion with the photoreactive LCFA probe. These data indicate

Haapalainen et al. (34). Based on the three-dimensional struc- that the hydrophobic cavity leading to the active site of the

ture, these residues lie on the ␤ strand located on top of the MFE2 HD domain can be assigned as one of the substrate-

dimerization interface of the subunits (Fig. 8A). The location is binding sites of MFE2.

apart from the catalytic triad of MFE2 HD domain (Ser¹⁵¹

,

Tyr¹⁶⁴, Lys¹⁶⁸). However, these residues also locate at the tip

DISCUSSION

of the hydrophobic cavity leading to the active site (Fig. 8B).

Peroxisomes play important roles in fatty acid metabolism.

Therefore, we speculated that MFE2 could anchor the Therefore, they contain various enzymes involved in fatty acid

photophore of the LCFA derivative ligand on the top of the hydro- metabolism and transport. However, because of the highly

phobic cavity (Trp²⁴⁹-Glu²⁵⁰-Arg²⁵¹) and bury the hydrophobic hydrophobic nature of the substrates, the precise mechanisms

probe along with the hydrophobic cavity directing the fatty-acyl of their substrate recognition and binding has been poorly

tail of the ligand toward the active site. To challenge this hypo- understood. In this study, we adopted a novel photochemical

thetical substrate binding mode of MFE2 HD, we replaced the approach. We synthesized a photoreactive palmitic acid ana-

hydrophobic residues (Ile¹⁸⁰, Ile²⁸⁸) and the aromatic residue logue bearing a diazirine moiety as a photophore, and charac-

(Tyr¹⁵⁶) forming the hydrophobic cavity of the MFE2 HD terized the molecular mechanisms especially in the substrate

domain, and examined the photoaffinity labeling of these binding of peroxisomal ␤-oxidation enzymes by photoaffinity

mutant proteins. As shown in Fig. 9, wild type MFE2 was labeling using a novel photoreactive LCFA probe.

labeled by the photoaffinity probe. However, MFE2(I180N),

At first, we identified MFE2 as a ligand-binding protein based

MFE2(I288N), and MFE2(Y156S) were impaired in the interac- on the following observations. 1) The probe used in this study

AUGUST 20, 2010•VOLUME 285•NUMBER 34

JOURNAL OF BIOLOGICAL CHEMISTRY 26321

Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

indicate that MFE2 could bind LCFA derivatives as substrates.

Why was only MFE2 specifically labeled by the photoreactive

LCFA probe among the peroxisomal proteins supposed to

interact with fatty acid derivatives such as ACOX1? We do not

know the exact reason at present. However, we presume a pos-

sible explanation as follows. The photoprobe used in this study

is composed of LCFA moiety, a diazirine-based photophore,

and a biotin moiety with a hydrophilic linker. The structure of

the photoprobe is different from that of CoA, LCFA, and also

LCFA-CoA. However, amphipathic characteristics of the pho-

toprobe seem to resemble somehow that of LCFA-CoA. In

addition, as mentioned above MFE2 shows broad substrate

specificity to hydrophobic molecules. MFE2 can also accept

medium chain fatty acyl-CoA, 17␤-estradiol, and 5-andro-

stene-3␤,17␤-diol (12, 16, 19, 44–46). Therefore, MFE2 could

have a potent capacity to interact with the photoprobe at its

substrate-binding site. On the other hand, acyl-CoA-binding

proteins such as AOX1 did no bind the photoprobe because

they probably recognize acyl-CoA more in a rigorous manner.

To improve the limited utility of this ligand as LCFA-CoA

probe, we are now trying to modify the structure and the posi-

tion of the photophore and detection tag not to affect the nature

of LCFA-CoA.

MFE2-His

CBB staining

W249G N158D

W249A R251A

wild

kDa

Ligand

labeled MFE2-His

- +

97.4

66.2

kDa

97.4

MFE2-His

66.2

120

85.6

100.0

100

80

60

40

20

0

MFE2 is a matrix protein existing inside of peroxisomes.

Therefore, the photoreactive LCFA probe must be imported

into peroxisomes. We assessed the transport of the photoreac-

tive LCFA probe across the peroxisomal membrane by limited

trypsin digestion. The ligand-incorporated MFE2 was pro-

tected from trypsin digestion after photoaffinity labeling. How-

ever, photoaffinity labeling of MFE2 did not occur when ligand

was incubated with peroxisomes pretreated with trypsin.

Under this condition, peroxisomal membrane proteins were

degraded but peroxisomal matrix proteins were not (data not

shown). These data indicate that the ligand is transported into

peroxisomes by peroxisomal membrane proteins. However, at

present, we have not identified the peroxisomal membrane pro-

teins that interact with this ligand.

29.5

28.1

25.3

FIGURE 7. Photoaffinity labeling of mutant MFE2-His. A, comparison of the

purities of mutant MFE2-His (10 ␮g of protein). B, wild type and mutant MFE2-

His (3.9 ␮g of protein) were labeled by the photoreactive LCFA probe. The

labeled proteins were separated on a 7–15% SDS-polyacrylamide gradient

gel, and detected by streptavidin-HRP. C, the amount of probe-incorporated

MFE2s shown in B was quantified with a LAS4000 luminoanalyzer (Fuji Film,

Tokyo, Japan) and the relative labeling percentage was expressed as a ratio of

the labeling percentage of each mutant MFE2 with that of wild type MFE2.

Next, we characterized the substrate binding mode of MFE2.

We suggest that MFE2 anchors its substrate around the region

consisting of Trp²⁴⁹to Arg²⁵¹and buries the substrate along the

was specifically incorporated into an 80-kDa peroxisomal hydrophobic cavity in the proper direction toward the catalytic

matrix protein upon activation with UV irradiation (Fig. 2), center of the MFE2 HD domain based on the following obser-

and the MALDI-TOF mass analysis identified the 80-kDa vations. 1) MFE2 was labeled by the photoreactive LCFA probe,

protein as peroxisomal MFE2 (supplemental Fig. S1 ). 2) and the labeling was competitively inhibited in the presence

Recombinant MFE2-His was specifically labeled by the pho- of palmitoyl-CoA (Fig. 3). 2) MALDI-TOF mass analysis iden-

toreactive LCFA probe (Fig. 5). MFE2 catalyzes the second tified three amino acid residues of the MFE2 HD domain

and third steps of peroxisomal ␤-oxidation of straight chain (Trp²⁴⁹-Glu²⁵⁰-Arg²⁵¹) as ligand adduct fragment (supple-

and branched chain fatty acids and bile acid intermediates. mental Fig. S1 A and Fig. 6), and the MFE2 mutants bearing

The patients with MFE2 deficiency and MFE2 knock-out these residues (W249A, W249G, R251A) exhibited a decreased

mice showed accumulations of VLCFA, branched chain fatty binding efficiency (Fig. 7). 3) Based on the three-dimensional

acids, and bile acid intermediates (39–43). Jiang et al. (44) structure of rat MFE2 HD, these residues are located apart from

reported that MFE2 purified from rat liver exhibited hydratase/ the catalytic center, but located on the tip of the hydrophobic

dehydrogenase activities on hexadecenoyl-CoA and 2-methyl- cavity leading to the active site (Fig. 8). 4) The MFE2 mutants

hexadecenoyl-CoA. In addition, although LCFA is largely disrupting the aromatic and hydrophobic residues forming the

degraded through the mitochondrial ␤-oxidation pathway, hydrophobic cavity of the MFE2 HD domain (Y156S, I180N,

LCFA oxidation of MFE2-deficient human fibroblasts was I288N) were impaired in the interaction with the photoreactive

lower than control fibroblasts under a condition in which the LCFA probe (Fig. 9). The HD domain of MFE2 belongs to the

mitochondrial LCFA oxidation was inhibited (40). These data short-chain alcohol dehydrogenase/reductase (SDR) super-

26322 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 34•AUGUST 20, 2010

Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

estradiol and R-phenylethanol

were shown to locate in the hydro-

phobic cavities of human 17␤-hy-

droxysteroid dehydrogenase 1 and

Lactobacillus brevis R-specific alco-

hol dehydrogenase, respectively (49,

50). These observations suggest that

the hydrophobic cavity leading to

the active site is important for sub-

strate binding of short-chain alco-

hol dehydrogenase/reductase pro-

teins. In the case of MFE2 HD,

Ylianttila et al. (51) recently per-

formed a docking study of 3R-hy-

(A)

droxydecanoyl-CoA to

a

het-

erodimer of yeast Candida

tropicalis MFE2 HD-A and MFE2

HD-B. They hypothesized the resi-

dues of the COOH-terminal

domain of MFE2 HD-B form hydro-

gen bonds with the phosphate

groups of the CoA, and the fatty acid

tail of the substrate could be guided

into the hydrophobic pocket. Our

studies also showed the diazirine

photophore covalently attached to 3

amino acid residues in the COOH-

terminal portion of MFE2 HD, and

suggest that the fatty acid tail pene-

trates into the hydrophobic pocket

of the other MFE2 HD (Fig. 9).

(B)

Y156

The three amino acid region

(Trp²⁴⁹-Glu²⁵⁰-Arg²⁵¹), which we

identified as a photoprobe anchor-

ing site, is located on the dimer

interface of the MFE2 HD domains.

Among them, Trp²⁴⁹of both mono-

mers is positioned in close proxim-

ity and the side chains of the two

tryptophan residues lie parallel with

each other. Therefore, Trp²⁴⁹is

suggested to be important for ho-

modimerization of the domains.

Ferdinandusse et al. (38) identified a

W249G mutation in a patient with

MFE2 deficiency, and they consid-

ered the mutation to be responsible

for dimerization based on structural

I180

I288

FIGURE 8. Three-dimensional structure of MFE2 HD domain. A, binary structure of rat MFE2HD (Protein Data

Bank code 1GZ6). The structure consists of two identical monomers colored blue and cyan, respectively. The

catalytic residue (Tyr¹⁶⁴) is colored yellow, and the probe-incorporated fragment (Trp²⁴⁹-Arg²⁵¹) is colored red.

The bound NAD^ϩare shown as a stick model and colored gray. B, zoom-in view of the molecular surface of the

putative substrate binding cavity. The catalytic residue (Y164) and the probe-incorporated fragment (W249-

R251) are colored as described in A. Aromatic residue (Y156) and hydrophobic residues (I160, I288) forming the

hydrophobic cavity are colored magenta. Images were generated using PyMOL.

ϩ

family, containing a typical Rossmann-fold scaffold for NAD

analysis. In this study, MFE2(W249A) and MFE2(W249G)

binding and a Ser-Tyr-Lys triad for catalysis (47). The hydro- indeed affected the ligand binding efficiency (Fig. 7). How-

phobic cavity leading to the active site also appears to be a ever, these mutants as well wild type MFE2 exhibited a

common feature among members of the short-chain alcohol molecular mass corresponding to a dimer on sucrose density

dehydrogenase/reductase family. Tanaka N. et al. (48) first gradient centrifugation (data not shown). These data indi-

reported a ternary structure of E. coli 7␣-hydroxysteroid dehy- cate that the tryptophan residue could be responsible for

drogenase, an short-chain alcohol dehydrogenase/reductase substrate binding and not solely for dimerization.

ϩ

protein, complexed with NAD and glycochenodeoxycholic

As for the photoaffinity studies on peroxisomal proteins, it

acid as a substrate. Based on the ternary structure, the substrate was useful to identify the fatty acid-binding proteins. Man-

was deposited in the hydrophobic cavity. In addition, 17␤- groo et al. (52) previously found that acyl-CoA oxidase was

AUGUST 20, 2010•VOLUME 285•NUMBER 34

JOURNAL OF BIOLOGICAL CHEMISTRY 26323

Photoaffinity Labeling of a Peroxisomal ␤-Oxidation Enzyme

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FIGURE 9. Photoaffinity labeling of mutant MFE2-His. Wild type and

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In this study, we have identified a peroxisomal ␤-oxidation

enzyme by photoaffinity labeling using a novel photoreactive

LCFA probe. Furthermore, we aimed to develop a photochem-

ical study to characterize the substrate binding mode of the

protein. This technique would be useful to identify and charac-

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Lipids:

Identification of a Substrate-binding Site in

a Peroxisomal β-Oxidation Enzyme by

Photoaffinity Labeling with a Novel

Palmitoyl Derivative

Yoshinori Kashiwayama, Takenori Tomohiro,

Kotomi Narita, Miyuki Suzumura, Tuomo

Glumoff, J. Kalervo Hiltunen, Paul P. Van