Cloning and Functional Expression of a Mammalian Gene for a Peroxisomal Sarcosine Oxidase*

(Received for publication, April 29, 1996, and in revised form, October 29, 1996)

Bernadette E. Reuber Dagger , Christian Karl Dagger , Sylvia A. Reimann Dagger , Stephanie J. Mihalik § and Gabriele Dodt Dagger

From the Dagger  Institut für Physiologische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Federal Republic of Germany and § The Kennedy Krieger Research Institute, and the Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain. An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney. A corresponding cDNA was cloned from rabbit liver and the gene designated sox. This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme. Sequence analysis revealed an N-terminal ADP-beta alpha beta -binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1. Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases. Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver. Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney. Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.


INTRODUCTION

In mammals a variety of H2O2-producing oxidases including D-amino-acid oxidase, D-aspartate oxidase, L-hydroxy-acid oxidase, acyl-CoA oxidase, and L-pipecolic acid oxidase are compartmentalized in peroxisomes. The H2O2 generated from these reactions is then converted to H2O and O2 by the peroxisomal matrix enzyme catalase (1). Several disorders have been described in which there is a defect in peroxisomal assembly that results in a partial or total absence of peroxisomal functions (for a review see Ref. 2). Patients with these peroxisomal disorders such as Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and hyperpipecolatemia all have elevated levels of L-pipecolic acid, an imino acid, which in human and monkey liver is oxidized by a peroxisomal L-pipecolic acid oxidase (3). Indeed, L-pipecolic acid oxidase activity was not detected in liver samples from patients with Zellweger syndrome (4). Primates dehydrogenate L-pipecolic acid to delta -piperideine-6-carboxylate which is spontaneously converted to alpha -aminoadipic acid gamma -semialdehyde.

The subcellular localization of this pathway seems to differ in other mammalian species. In rabbits, guinea pigs, dogs, and sheep L-pipecolic acid oxidation is primarily mitochondrial (5). However, during our studies examining the subcellular distribution of L-pipecolic acid oxidation in rabbits, a considerable amount of L-pipecolic acid oxidation was detected in the peroxisomes, in addition to the previously reported mitochondrial activity (6). Interestingly, this peroxisomal enzyme showed a high specific activity for sarcosine and also oxidized L-pipecolic acid and L-proline. This finding raised the question whether the purified enzyme is also a sarcosine oxidase.

In mammals, the oxidative removal of the methyl group from sarcosine is catalyzed by sarcosine dehydrogenase (EC 1.5.99.1) and dimethylglycine dehydrogenase (EC 1.5.99.2) in mitochondria. These enzymes were characterized as the two main folate-containing enzymes in rat liver mitochondria. Both enzymes are closely linked to the electron transport chain and form 5,10-methylenetetrahydrofolate. This "active" formaldehyde is predominantly used for the formation of serine from glycine by serine hydroxymethylase (7-11).

The enzyme investigated in this study differs from the classical mammalian sarcosine dehydrogenase described earlier. The reaction takes place in peroxisomes rather than in mitochondria. The reaction mechanism is an H2O2-generating oxidation and not an electron transport chain-linked dehydrogenation. Although no mammalian sarcosine oxidases are known, several sarcosine oxidases from bacteria have been purified and characterized. They can be classified as monomeric enzymes (e.g. Arthrobacter sp. TE 1826 (12), Bacillus sp. NS-129 (13), Bacillus sp. B-0618 (14), Streptomyces sp. KB210-8SY (15), and Cylindrocarpon didymum M-1 (16)) and as tetrameric enzymes (e.g. Arthrobacter ureafaciens (17), from Corynebacterium sp. U-96 (18) and Corynebacterium sp P-1 (19)). The monomeric enzymes have a molecular mass of 42-45 kDa which is similar to the size of the beta -subunit of the tetrameric enzymes. All enzymes contain a covalently attached flavin (beta -subunit of the tetrameric enzymes); the tetrameric enzymes also have a noncovalently bound flavin and at least the enzyme from Corynebacterium sp. P-1 probably contains an additional tightly bound NAD (20). While the covalent attachment is not unusual for bacterial oxidases, it has been rarely found in mammals. The list of mammalian enzymes with covalently attached flavins includes the mitochondrial sarcosine dehydrogenase and dimethylglycine dehydrogenase from rat and the peroxisomal L-pipecolic acid oxidase from monkey liver (3).

Like the mitochondrial dehydrogenases, the bacterial tetrameric sarcosine oxidases (21) can bind tetrahydrofolate and then yield 5,10-methylenetetrahydrofolate instead of formaldehyde. It is not known whether the monomeric enzymes react with tetrahydrofolate.

A mammalian sarcosine oxidase from rabbit kidney has now been purified to investigate the association between the oxidation of the imino acid L-pipecolic acid and the methyl group acceptor sarcosine. Subsequently, the sarcosine oxidase gene was cloned from rabbit liver and expressed in Escherichia coli and in mammalian cells.


EXPERIMENTAL PROCEDURES

Materials

Sarcosine, L-pipecolic acid, 4-hydroxyphenylacetic acid, protease inhibitors, protein molecular weight standards, and protein standards for isoelectric focusing were from Sigma (Deisenhofen, Germany). L-Pipecolic acid was also purchased from Bachem (Heidelberg, Germany). FAD was obtained from Boehringer Mannheim (Mannheim, Germany).

Bacterial Strains, Libraries, and Cells

The E. coli strains K802, DH5alpha , and TG1 were used for all molecular screening and cloning procedures. To obtain the sox sequence, a rabbit liver cDNA library constructed in lambda gt10 (kindly provided by Dr. M. Kilimann) and a rabbit liver genomic library cloned into EMBL3 SP6/T7 (Clontech, Palo Alto, CA) were screened.

The human cell lines were obtained and cultured as described by Moser et al. (22). The transformed derivatives were kindly provided by Dr. S. J. Gould. African Green Monkey CV-1 cells were obtained from ATCC (Rockville, MD; ATCC number CCL70).

Sarcosine Oxidase Assay

Sarcosine oxidase activity was determined by measuring the hydrogen peroxide formation in a horseradish peroxidase-coupled fluorometric assay, as described by Poosch and Yamazaki (23). The reaction mixture contained in a total volume of 550 µl, 55 mM Tris, pH 8.4, 1 mM 4-hydroxyphenylacetic acid, 4 units of horseradish peroxidase (Boehringer Mannheim, types I or II), 9 mM sodium azide, 1 mM FAD, 100 µl of enzyme solution (protein 1-80 µg), and 9.8 mM sarcosine. The reaction was started with sarcosine, proceeded for various periods (15-60 min) at 37 °C in the dark, and was stopped by the addition of 1.5 ml of 0.2 M glycine/sodium carbonate buffer, pH 10.5. The fluorescence at 415 nm (excitation 318 nm) was determined with the spectrofluorometer JY 3D (Jobin-Yvon, France) or with the spectrofluorometer LS 50 (Perkin-Elmer, Weiterstadt, Germany).

Protein concentrations were estimated by the method of Bradford (24) using the Coomassie Protein Plus reagent (Pierce). Bovine serum albumin was used as standard.

Purification of Sarcosine Oxidase

All purification steps were performed at 4 °C under conditions that minimize light exposure of the enzyme. Kidneys from New Zealand White rabbits were cut in half to isolate the kidney cortex. The cortex (10 g) was minced and homogenized in 5 volumes (v/w) of homogenization buffer (250 mM sucrose, 6.8 mM HEPES, 1 mM EGTA, pH 7.5, containing 0.5 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, and 1 µM leupeptin) with 3 up-and-down passages of a loose-fitting pestle in an Potter-Elvehjem homogenizer at 800 rpm (Braun, Melsungen, Germany). The homogenate was centrifuged at 600 × g for 10 min. The resulting pellet was resuspended in 3.5 volumes (v/w) homogenization buffer and again centrifuged at 600 × g for 10 min. Both supernatants were combined and further centrifuged at 5,100 × g for 15 min to pellet the M fraction which contained most of the mitochondria and peroxisomes. This pellet was gently rehomogenized in 6 volumes of homogenization buffer. To release the enzyme from the organelles, the M fraction was frozen in a thin layer at -70 °C for at least 15 min, quickly thawed at 37 °C, chilled, and centrifuged at 26,000 × g for 15 min. The supernatant containing the solubilized enzyme was saved.

Heat Denaturation

The enzyme solution was brought to 46 °C, stirred continuously for 10 min, and then chilled immediately. The denatured proteins were removed by centrifugation at 26,600 × g for 15 min. The resulting supernatant was used for CM-cellulose chromatography.

CM-52 Cellulose Batch Chromatography

CM-52 cellulose (Whatman), equilibrated in 1 mM potassium phosphate buffer, 1 mM EGTA, pH 6.0, was mixed with 2 volumes of enzyme solution (~70 ml) and shaken at 4 °C for 30 min. Afterward, the gel suspension was transferred onto a glass frit (porosity G3), and a light vacuum was applied to remove the filtrate. The remaining gel was washed 5 times with 20 ml of equilibration buffer, and the enzyme was eluted in 7 fractions of 15 ml each (200 mM potassium phosphate buffer, 1 mM EGTA, pH 8.3). The enzymatically active fractions were combined and concentrated by diafiltration in an Amicon chamber (PM 30 membrane, Amicon, Witten, Germany) to about 22 ml.

Butyl-Sepharose Chromatography

Subsequently, the enzyme solution was slowly mixed with 1 volume of 1.72 M potassium phosphate buffer, pH 7.8, and subjected to butyl-Sepharose chromatography.

Butyl-Sepharose CL 4B (Pharmacia, Freiburg, Germany), equilibrated with 1 M potassium phosphate buffer, 0.1 mM EGTA, pH 7.8 (column 1 × 2 cm, 1.6-ml bed volume) was loaded with the enzyme solution (0.47 ml/min) and washed with 15-bed volumes of equilibration buffer containing 1 µM pepstatin and 1 µM leupeptin. The enzyme was eluted with a linear decreasing gradient of 1 M to 300 mM potassium phosphate in 0.1 mM EGTA, pH 7.8, 1 µM pepstatin and 1 µM leupeptin (95 ml of 1 M and 95 ml of 300 mM buffer). Enzymatically active fractions were combined and concentrated in an Amicon chamber equipped with a PM30 membrane (Amicon). The enzyme was stored at -70 °C in the dark.

Organelle Separation in Nycodenz Gradients

To investigate the subcellular distribution of sarcosine oxidase, freshly prepared M fractions were separated in a Nycodenz gradient. One ml of M fraction was incubated with 1 ml of 50% (w/v) Nycodenz (Nycomed AS, Oslo, Norway) in 2 mM MOPS,1 1 mM EGTA, pH 7.5, for 30 min to increase the density of the peroxisomal fraction and then loaded on top of a linear 30-ml Nycodenz gradient 18-50% (w/v) in 2 mM MOPS, 1 mM EGTA, pH 7.5. The 18% Nycodenz solution contained 3% (w/v) sucrose. The sample was overlaid with homogenization buffer. Centrifugation was performed at 33,000 × g for 90 min in a SS90 vertical rotor (Sorvall). Thirty fractions of approximately 1 ml were collected from the bottom of the gradient with a peristaltic pump. Peak fractions of mitochondria and peroxisomes were identified by marker enzyme analysis. Succinate cytochrome c dehydrogenase, a mitochondrial marker, was determined according to Parkes and Thompson (25) and catalase, a peroxisomal marker, was measured as described by Hübl and Bretschneider (26).

Gel Electrophoresis, Isoelectric Focusing, and Immunoblotting

Protein samples were submitted to SDS-polyacrylamide gel electrophoresis in 10% gels as described by Laemmli (27), except that piperazinediacrylamide replaced bisacrylamide as the cross-linking agent. The gels were silver-stained as described by Wray et al. (28). Isoelectric focusing of purified proteins was performed with the Pharmacia Phast system. Five percent acrylamide gels were rehydrated in 9 M urea, 50 mM dithioerythritol, 10% sorbitol, 10% Nonidet P40, containing 20 µl each of ampholytes pH 3-10, 5-8, and servalyte 4-9 per ml buffer. Samples were diluted with the same volume of buffer containing 9 M urea, 50 mM dithioerythritol, 10% Nonidet P-40, and 20 µl of ampholytes pH 3-10 per ml. Focusing proceeded at 540 V for approximately 410 V-h at 15 °C. The gels were fixed in 20% trichloroacetic acid for 15 min and silver-stained with the Phast system following the instructions of the manufacturer.

After SDS-PAGE the proteins were transferred onto nitrocellulose with a Trans-Blot Semidry Electrophoretic Transfer Cell (Bio-Rad) or with a Bio-Rad mini blot apparatus, according to the manufacturer. Methanol (20% final concentration) and SDS (1.3 mM final concentration) were added to the transfer buffer described by Bjerrum et al. (29). The nitrocellulose was blocked for 2 h in TBS (100 mM NaCl, 100 mM Tris-HCl, pH 7.4) with 5% (w/v) bovine serum albumin and subsequently washed with TBS containing 0.1% (v/v) Tween 20. Antibodies directed against FAD (kindly provided by Dr. M. Barber) (30) were used in a 1:500 dilution in TBS, 0.1% Tween. Antiserum against L-pipecolic acid oxidase from monkey liver was either diluted 1000-fold and used directly for immunoblotting or affinity purified against purified L-pipecolic acid oxidase from monkey liver and then used without further dilution. The procedure for affinity purification followed the protocol of Höhfeld et al. (31). Detection was performed with a 1:15 000 dilution of alkaline phosphatase-coupled goat anti-Ig from rabbit (Sigma) in a nitro blue tetrazolium/4-bromo-5-chloro-3-indolyl phosphate-coupled reaction, as described (32). Alternatively, the enhanced chemiluminescence kit (Amersham, Braunschweig, Germany) was used for immunodetection.

Spectophotometric Characterization of the Flavoprotein

Sarcosine oxidase dissolved in 10 mM potassium phosphate buffer, 0.1 mM EGTA, pH 7.8, was initially scanned in a Shimadzu double beam dual wavelength recording spectrophotometer UV 300. One ml of the enzyme solution was then precipitated in the dark with 100 µl of 3 M trichloroacetic acid at -20 °C for 1 h and then centrifuged at 14,000 × g for 5 min. The yellow pellet was resupended in 1 ml of 5 M guanidine hydrochloride. Additional spectra were recorded for the supernatant and the redissolved pellet against corresponding blanks. The protein dissolved in M guanidine hydrochloride was concentrated using a Centricon 30 microconcentrator (Amicon). The concentrated protein solution was diluted again with 5 M guanidine hydrochloride, and the recorded spectrum was compared with the spectrum of the corresponding filtrate.

Sequencing and RT-PCR

Nucleotide sequences were determined by primer walking using the Sequenase Version 2.0 and T7 DNA Polymerase (U. S. Biochemical Corp.) following the method of Sanger et al. (33). Alternatively, samples were sequenced on a DNA sequencer 373A (Applied Biosystems, Weiterstadt, Germany) utilizing the Prism Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit.

The complete cDNA for sarcosine oxidase was constructed by RT-PCR. 1 µg of total rabbit liver RNA was reverse transcribed with 2 pmol of T3-1 primer (5'-GCGAGAAAGTAGTTGTGA-3') and 200 units of Superscript RT (Life Technologies, Inc., Eggenstein, Germany). The synthesized cDNA was used as template for a PCR with two specially constructed primers A (5'-TAGAGCCTCGAGATGGCGGCTCAGAAAGAT-3') and B (5'-CCGTCTAGAATATCTCAGGGACACTCC-3'), which annealed at bp 1-18 and bp 1245-1262, respectively, and contained either a XhoI or a XbaI restriction site. The major part of the PCR product, the fragment between base pairs 189 and 1182, was exchanged against the corresponding fragment from the cDNA clone obtained by library screening to avoid Taq polymerase errors. The rest of the PCR fragment was confirmed by sequencing. This PCR fragment was cloned into the XhoI and XbaI sites of pKS+ (Stratagene, La Jolla, CA) to yield the pBR1 construct.

Plasmids

In addition to pBR1, the constructs pBR2-4 were used to express full-length sarcosine oxidase in E. coli and for transfection experiments in mammalian cell cultures. Cloning of the XhoI/NotI fragment from pBR1 into the SalI/NotI sites of pGEX4T-3 (Pharmacia) resulted in the pBR2 construct which encodes a N-terminal fusion with glutathione S-transferase (GST). To obtain the pBR3 construct, coding for an N-terminal fusion with maltose binding protein (MBP), the XhoI/XbaI fragment of pBR1 was cloned into the SalI/HindIII sites of pMALc2 (Invitrogen, NVLeek, The Netherlands). The XbaI and HindIII sites were filled in with Klenow enzyme (Boehringer Mannheim) to yield blunt ends. The XhoI/XbaI fragment of pBR1 was also cloned into the corresponding sites of the mammalian expression vector pcDNA3 (Invitrogen) to yield pBR4.

Plaque and Northern Hybridization

For library screening phage plaques were transferred to nylon filters. Prehybridization and hybridization with a digoxigenin (DIG)-labeled probe (a 1500-bp long cDNA fragment of a human pipecolic acid oxidase clone2 labeled with DIG-dUTP following Feinberg et al. (34)) was carried out at 42 °C in 7% SDS, 2% blocking reagent (Boehringer Mannheim), 50% formamide, 0.1% N-lauroylsarcosine, and 5 × SSC in 50 mM sodium phosphate buffer, pH 7.0. The filters were washed twice with 2 × SSC, 0.1% SDS at room temperature for 10 min and then twice with 0.1 × SSC, 0.1% SDS at 60 °C for 30 min. For chemiluminescent detection of positive clones, the filters were processed with alkaline phosphatase-coupled DIG antibodies and Lumigen PPD (Boehringer Mannheim) and exposed to x-ray film. The genomic library was screened with a probe (bp 1-188) from a pipecolic acid oxidase cDNA clone2, labeled with [alpha -32P]dATP. Hybridization and washing followed the procedure described in Sambrook et al. (35) for high stringency conditions. Phage DNA was isolated (36) and the inserts subcloned into pKS+.

Total RNA from rabbit kidney and liver was isolated by guanidine thiocyanate/phenol/chloroform extraction (37), separated with a glyoxal agarose gel (38), and transferred onto a nylon membrane. The membrane was probed with an alpha -32P-labeled human L-pipecolic acid oxidase cDNA2 (39) for 24 h at 42 °C and washed under the same conditions already explained for the plaque hybridization.

Expression of Sarcosine Oxidase in E. coli

The plasmids pBR2 or pBR3, respectively, were used to transform E. coli TG1 cells. Expression was induced with 0.3 mM IPTG at 37 °C for pBR3 and with 0.1 mM IPTG at 30 °C for pBR2 for 7 h. Lysis of bacteria which expressed the MBP fusion protein is described in Sambrook et al. (35). The protein was purified by affinity chromatography with an amylose resin (New England BioLabs, Schwalbach, Germany). For purification of the GST fusion protein cells were lysed by sonication followed by an incubation in lysis buffer (phosphate-buffered saline pH 7.5/1% Triton X-100) according to the instructions of Pharmacia. The fusion protein was further purified with a glutathione-Sepharose 4B resin as described by the manufacturer (Pharmacia).

Expression of sox in Mammalian Cells

CV-1 cells and human fibroblast cell lines were transfected with 3 µg of pBR4 plasmid in the presence of 30 µg of lipofectamine (Life Technologies, Inc.) according to the manufacturer's suggestions. After 48 h cells were analyzed by indirect immunofluorescence as described by Slawecki et al. (40). Best results were obtained at a dilution of 1:400 for affinity-purified alpha -pipecolic acid oxidase antibodies and 1:100 for alpha -catalase antibodies (The Binding Site, Heidelberg Germany). The fluorescein isothiocyanate- and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies (Dianova, Hamburg, Germany) were diluted 1:100. Micrographs were taken with Tmax 400 and Ektachrome 400 films (Kodak).


RESULTS

Purification of Sarcosine Oxidase

Rabbit kidney sarcosine oxidase catalyzes the demethylation of sarcosine to glycine, formaldehyde, and H2O2. The H2O2 formation can be quantified in a fluorescence coupled assay with 4-hydroxyphenylacetic acid as fluorophore. The purification procedure for sarcosine oxidase from rabbit kidney is summarized in Table I. We obtained the M fraction containing peroxisomes, mitochondria, and lysosomes by differential centrifugation. Although preliminary experiments determining the subcellular distribution of sarcosine oxidase suggested a peroxisomal localization for sarcosine oxidase, an additional centrifugation step to purify the peroxisomes resulted in a lower yield of sarcosine oxidase, probably due to breakage of the organelles and loss of the enzyme into the supernatant. Sarcosine oxidase was purified 666-fold with an overall yield of 12.8% (Table I). The purest fraction separated into two closely migrating bands by SDS-PAGE (Fig. 1). These bands appear to have nearly identical pI and molecular mass in two-dimensional gel electrophoresis. The molecular mass of sarcosine oxidase was estimated at 44 kDa under denaturing conditions (Fig. 1). Isoelectric focusing in 6 M urea and 10% Nonidet P40 gave a pI of 7.8. The optimal enzyme activity in Tris buffer at 37 °C was found at a pH of 8.6.

Table I.

Partial purification of sarcosine oxidase from rabbit kidney


Fraction (step) Protein Total activity Specific activity Purification Recovery, % homogenate

mg nmol H2O2/min nmol H2O2/mg protein/min -fold
Homogenate 1369 121.7 0.089 1 100
M fraction 229.5 178.9 0.780 8.8 147
Supernatant of M fraction 42.2 100.0 2.37 26.7 82.1
Heat treatment 32.8 96.6 2.95 33.1 79.3
CM 52 batch chromatography 8.5 85.8 10.2 114 70.5
Butyl-Sepharose chromatography 1.5 32.9 22.5 253 27.0
peak fraction Butyl-Sepharose 0.26 15.6 58.9 666 12.8


Fig. 1. SDS-PAGE of fractions obtained during purification of sarcosine oxidase. Electrophoresis was performed in 10% acrylamide gels, and proteins were silver-stained. Approximately 0.5-1 µg of protein was loaded in each lane. Gel lanes are as follows: lane 1, M fraction; lane 2, supernatant from the M fraction after a freeze-thaw cycle; lane 3, supernatant after heat treatment; lane 4, pool after CM52 batch chromatography; lane 5, butyl-Sepharose pool; lane 6, peak fractions after butyl-Sepharose; lane 7, marker proteins with indicated molecular mass.
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Sarcosine Oxidase Oxidizes Sarcosine, L-Pipecolic Acid, and L-Proline

Substrate studies at a fixed concentration of 9.8 mM, which are summarized in Table II, identify sarcosine as the major substrate. The catalytic constants of each substrate are compared with the catalytic constant for sarcosine. Other prominent substrates are L-pipecolic acid and L-proline with rates of 30 and 21%, respectively, compared with sarcosine. The D-isomers of aspartic acid, alanine, proline, and pipecolic acid reacted to a minor extent, but we could not rule out a small contamination of the protein preparation with D-aspartate oxidase. No H2O2 was formed with dimethylglycine. This substrate pattern is quite different from that for D-amino acid oxidase and D-aspartate oxidase (41) but similar to that of mammalian L-pipecolic acid oxidase isolated from monkey liver (3). L-pipecolic acid oxidase predominantly catalyzes the oxidation of L-pipecolic acid but also reacts with L-proline and sarcosine with rates of 23 and 10%, respectively.

Table II.

Relative activity of sarcosine oxidase toward different substrates

Purified sarcosine oxidase was incubated with different substrates at a concentration of 9.8 mM. The H2O2 formation was measured in 55 mM Tris, pH 8.4 at 37 °C as described under "Experimental Procedures." Results were expressed as percent activity relative to sarcosine. For this the catalytic constants for every substrate were compared with the catalytic constant for sarcosine.
Substrate Relative activity

%
Sarcosine 100
L-Pipecolic acid 30
L-Proline 21
L-Alanine 2.2
L-Lysine 1.6
D-Aspartic acid 4.3
D-Alanine 3.3
D-Proline 3.3
D-Pipecolic acid 1.6
DL-Pipecolic acid 18
Dimethylglycine 0

The catalytic efficiency, the ratio of kc/Km, is more suitable to determine which substrate is predominantly metabolized when several competing substrates are present (42). As shown in Table III, the kc for sarcosine is about 7 times higher than the kc for L-pipecolic acid, but the Km value for L-pipecolic acid (5.4 mM) is much lower than that for sarcosine (66.7 mM). Thus, the calculated catalytic efficiencies of 0.158 mM-1 min-1 for L-pipecolic acid and 0.093 mM-1 min-1 for sarcosine suggest that L-pipecolic acid is a slightly favored substrate.

Table III.

Kinetic values for purified sarcosine oxidase

The H2O2 formation was measured in 55 mM Tris, pH 8.4 at 37 °C as described under "Experimental Procedures."
Km Kc kc/Km

mM min-1 min-1 mM-1
Sarcosine 66.7 0.20 0.093
L-Pipecolic acid 5.88 0.846 0.158

The effect of benzoate, a known competitive inhibitor of other peroxisomal oxidases (D-amino-acid oxidase and L-pipecolic acid oxidase) was investigated with L-pipecolic acid and sarcosine as substrates. When L-pipecolic acid was the substrate, a Ki of 2.04 mM was calculated for benzoate, with competitive inhibition (Fig. 2B). This value is similar to the Ki of 0.75 mM previously reported for L-pipecolic acid oxidase (3). The Ki value for benzoate with sarcosine as substrate was estimated at 5.46 mM. However, in this case the inhibition type was noncompetitive (Fig. 2A).


Fig. 2. Inhibition of sarcosine oxidase by benzoic acid using sarcosine or L-pipecolic acid as substrate. H2O2 formation by purified sarcosine oxidase was measured with sarcosine at substrate concentrations between 5.9 and 29.8 mM (A) and L-pipecolic acid at different substrate concentrations between 1.4 and 17.7 mM (B). The concentration of the inhibitor benzoic acid varied between 0 and 5 mM. The Ki using sarcosine as substrate was estimated at 5.46 mM and for L-pipecolic acid as substrate at 2.04 mM.
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Sarcosine Oxidase Contains a Covalently Bound Flavin

Purified sarcosine oxidase had a yellow color, and its sarcosine oxidizing activity was not dependent on FAD addition. Its absorption spectrum with a peak at 450 nm was typical for a flavoprotein (data not shown). When the purified enzyme was precipitated with 5% trichloroacetic acid, the protein pellet was yellow, indicating that the flavin was tightly associated with the protein. After resuspending the pellet in 5 M guanidine HCl, the purified enzyme absorbed at 356, 372, and 450 nm with a shoulder at 480 nm (Fig. 3B), a pattern typical for tightly bound flavins (43). After subsequent ultrafiltration (cut off, 30 kDa) the filtrate did not exhibit a typical flavin spectrum, but the retained enzyme, resuspended in 5 M guanidine HCl, had the same spectrum as the original solution. The fact that the flavin remained bound to the enzyme, even after precipitation with trichloroacetic acid or after resolubilizing in 5 M guanidine HCl, strongly suggested a covalent association.


Fig. 3. Absorption spectrum of purified and recombinant sarcosine oxidase. A, the recombinant GST-sarcosine oxidase fusion was precipitated with 10% (w/v) trichloroacetic acid, and the pellet was diluted to a protein concentration of 0.2 mg/ml with phosphate-buffered saline, pH 7.5, and the spectrum was recorded against the same buffer. B, sarcosine oxidase pooled after butyl-Sepharose chromatography was precipitated with 10% (w/v) trichloroacetic acid, dissolved in 5 M guanidine hydrochloride, and ultrafiltrated. The retenate was diluted in 5 M guanidine hydrochloride to the original volume, and the spectrum was recorded against 5 M guanidine hydrochloride in a Shimadzu UV 300 spectrophotometer. Protein concentration was 0.17 mg/ml.
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Subsequently, sarcosine oxidase from rabbit kidney, along with pipecolic acid oxidase from monkey liver (covalently bound flavin), and D-amino-acid oxidase from pig kidney (noncovalently bound FAD) were separated by SDS-PAGE, blotted onto nitrocellulose, and examined with antibodies directed against flavins (30). Both, sarcosine oxidase and L-pipecolic acid oxidase reacted with the antibodies, suggesting that their flavin was covalently bound, whereas D-amino-acid oxidase, which has a noncovalently bound flavin, did not react with the antibodies (Fig. 4). The same samples were analyzed with antibodies raised against L-pipecolic acid oxidase from monkey liver (3). Sarcosine oxidase from rabbit kidney was recognized by the antibodies, but the antibodies did not cross-react with D-amino-acid oxidase (Fig. 4). Sarcosine oxidase migrated slightly faster than L-pipecolic acid oxidase during SDS-PAGE.


Fig. 4. Immunodetection of different peroxisomal oxidases with antibodies against L-pipecolic acid oxidase and FAD. L-Pipecolic acid oxidase from monkey liver (lane 1), sarcosine oxidase from rabbit kidney (lane 2), and D-amino-acid oxidase from pig kidney (lane 3) (Sigma) were separated by SDS-PAGE. Proteins were silver-stained or blotted onto nitrocellulose and incubated with anti-L-pipecolic acid oxidase antiserum or anti-FAD antiserum. One µg of protein was loaded for silver staining and immunodetection with anti-L-pipecolic oxidase antiserum and approximately 10 µg for immunodetection with the anti-FAD antibodies.
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Sarcosine Oxidase Is a Peroxisomal Enzyme

Because the antibodies against L-pipecolic acid oxidase cross-reacted with sarcosine oxidase, they could be used to determine the subcellular localization of sarcosine oxidase. For these studies, a heavy mitochondrial fraction (M fraction) from rabbit kidney cortex was further separated in a Nycodenz gradient. Peak fractions of peroxisomes, mitochondria, and the supernatant fractions, identified by marker enzyme analysis, were separated by SDS-PAGE and blotted onto nitrocellulose. When the blot was analyzed with anti-L-pipecolic acid oxidase antibodies, a single protein band with a molecular mass of 44 kDa was identified in the peroxisomal fractions (Fig. 5). All of the faint cross-reacting bands in the mitochondrial fractions and in the supernatant fractions also appeared in control blots incubated with preimmune serum from rabbits.


Fig. 5. Immunodetection of sarcosine oxidase in subcellular fractions after Nycodenz gradient centrifugation. Purified antiserum against L-pipecolic acid oxidase from monkey liver was used to detect sarcosine oxidase from rabbit kidney in gradient fractions after SDS-PAGE and blotting onto nitrocellulose. P, peroxisomal peak fractions lanes 1-3; lane 4, purified L-pipecolic acid oxidase from monkey liver; M, mitochondrial peak fractions lanes 5 and 6; lane 7, marker proteins, no immunodetection with the antibodies; and S, supernatant fractions lane 8 and 9. Approximately 1 µg of protein was loaded in each lane.
[View Larger Version of this Image (43K GIF file)]


Molecular Cloning of Rabbit Liver Sarcosine Oxidase

The similarity between L-pipecolic acid oxidase from monkey liver (3) and sarcosine oxidase from rabbit kidney, especially the substrate specificity and the immunological cross-reactivity with L-pipecolic acid oxidase antibodies, encouraged us to investigate the sarcosine oxidase gene from rabbit liver.

When we probed a Northern blot with RNA from rabbit kidney and liver with a partial cDNA clone for human pipecolic acid oxidase,2 hybridizing RNAs (approximately 2.3 kilobases (Fig. 6)) were detected in both kidney and liver with a higher expression level in kidney.


Fig. 6. Expression of the sox gene in rabbit kidney and liver. Total RNA from rabbit kidney (lanes 1 and 2) and rabbit liver (lanes 3 and 4) were separated with a glyoxal agarose gel and blotted on nitrocellulose. Each lane contained 20 µg of RNA. The positions of the 18 S and 28 S rRNAs are indicated. The arrow marks a 2.3-kilobase long mRNA expressed in kidney and in liver.
[View Larger Version of this Image (27K GIF file)]


This same partial cDNA clone from human pipecolic acid oxidase was chosen to screen a rabbit liver cDNA library. A distinct cDNA clone (bp 157-2083) including the polyadenylation signal but lacking a putative start codon was obtained. Since we were not able to identify the 5'-end of the gene applying the rapid amplification of cDNA ends protocol (44), we probed a genomic rabbit liver library with a cDNA fragment (188 bp) from the very 5'-end of the human liver L-pipecolic acid oxidase gene. Several clones containing the putative translation start site and additional 5' bp were isolated. A full-length cDNA was compiled by RT-PCR. We designated the rabbit sarcosine oxidase gene as sox. The sequence of the complete cDNA and the deduced amino acid sequence are shown in Fig. 7. The cDNA consists of a 12-bp 5'-untranslated region, an open reading frame of 1170 bp, and 913 bp of 3'-untranslated region and encodes for a protein Sox with 390 amino acids and a molecular mass of 44 kDa. The length of the cDNA is consistent with both the size of the native enzyme and with the size of the detected mRNA (Fig. 6), allowing 150-200 bp for the poly(A) tail. The N terminus of the protein has sequence homology to an ADP-beta alpha beta -binding fold, typical for proteins that bind FAD, NAD+, or NADP+ (45, 46). The last three amino acids, AHL, represent a peroxisomal targeting signal 1 (PTS1), characteristic for mammalian proteins that are translocated into peroxisomes (47).


Fig. 7. Nucleotide and amino acid sequence of the sox gene and the corresponding gene product from rabbit liver. The coding region is written with capital letters and the untranslated regions with lowercase letters.
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Rabbit Sarcosine Oxidase Shows High Homology to Bacterial Sarcosine Oxidases

This mammalian sarcosine oxidase shows high homologies to monomeric bacterial sarcosine oxidases from Streptomyces sp. KB210-8SY (15), Bacillus sp. NS-129 (13), Bacillus sp. B-0618 (14), and Arthrobacter sp. TE 1826 (12). A sequence alignment with several different sarcosine oxidases obtained after a BLAST search (38) is shown in Fig. 8.


Fig. 8. Sequence alignment between Sox from rabbit liver and four monomeric sarcosine oxidases from microorganisms. The alignment was performed with Lasergene (DNASTAR, London, UK) by the Clustal method using a PAM250 residue weight table. Amino acids identical in four out of five sequences are overlaid with black boxes. The PTS1 motif at the C terminus of rabbit Sox is marked by asterisks. The ADP-beta alpha beta -binding fold and three other distinct sequence motifs, unique for sarcosine oxidases, are numbered 1-4 and overlined.
[View Larger Version of this Image (106K GIF file)]


The amino acid identities over the whole protein between rabbit Sox and the four monomeric bacterial sarcosine oxidases are between 25 and 28%. Apart from the ADP-beta alpha beta -binding fold, three other almost identical regions were identified among the proteins. These segments are labeled 1-4 in Fig. 8. While the binding fold is characteristic for many enzymes, the three other homology regions are unique for monomeric sarcosine oxidases. Lower identities (14.9, 14.6, and 12.3%) were found with the beta -subunit of the heterotetrameric sarcosine oxidase from Corynebacterium sp. P-1 (49), with the N terminus of dimethylglycine dehydrogenase from rat liver (50), and with the amino acid deaminase from Proteus mirabilis (51). High identities occurred with a not yet identified gene product (accession number U23529[GenBank]) from Caenorhabditis elegans. Interestingly, the encoded protein of C. elegans showed an N-terminal duplication of the first 300 amino acids of Sox. The identities to rabbit Sox are 25.1% for the first part and 27.6% for the C-terminal complete part (Fig. 9). As with the rabbit sarcosine oxidase, the C. elegans protein contains a PTS1 signal at the C terminus (AHL for rabbit Sox and SKI for the C. elegans gene product).


Fig. 9. Homologies between Sox from rabbit liver and a yet unidentified gene product from C. elegans. The 703-amino acid long gene product of C. elegans seems to be duplicated as it shows homologies to rabbit Sox as well in the N-terminal half (25.1%) as in the C-terminal half (27.6%) of the protein.
[View Larger Version of this Image (13K GIF file)]


Comparison of Purified Sarcosine Oxidase to the Recombinant Gene Product of the Rabbit Liver sox Gene

To ensure that the right gene had been cloned, three characteristics of the purified sarcosine oxidase, the covalently attached flavin, the unusual substrate specificity, and the peroxisomal localization were investigated with recombinant sarcosine oxidase synthesized in E. coli.

Bacterial Expression of sox Revealed That Sarcosine Oxidase Has a Covalently Attached Flavin

Several approaches were used to express sox in E. coli. After transformation of E. coli with the pBR2 plasmid, a GST-fusion protein with a molecular mass of 80 kDa could be purified with a glutathione-Sepharose column. The recombinant protein had a slightly yellowish color. The spectrum of the recombinant protein showed absorption maxima at 380 and 450 nm (Fig. 3A) which were similar to those of the purified enzyme (Fig. 3B) and consistent with a bound flavin (43). When the protein was trichloroacetic acid-precipitated as described for the purified enzyme, no flavin was detected in the supernatant. The spectrum recorded for the recombinant enzyme (Fig. 3A) is similar to the spectra described for sarcosine oxidases (52). Interestingly, the shoulder at 480 nm, which has been described for the monomeric bacterial sarcosine oxidases, was more pronounced with the purified enzyme (52) (Fig. 3B).

Although the fusion protein could be synthesized with the covalently bound flavin, we were not able to isolate the protein in an enzymatically active form. Even after induction of the protein expression at a low IPTG concentration and at a temperature of 30 °C, the formation of inclusion bodies (53) could not be prevented. The GST-fusion protein was only solubilized and purified after treatment with detergents.

Sarcosine Oxidase Synthesized as Fusion with Maltose-binding Protein Has Enzymatic Activity toward Sarcosine, L-Pipecolic Acid, and L-Proline

A different fusion protein was used to isolate enzymatically active sarcosine oxidase. After transformation of E. coli with the pBR3 construct, an MBP-fusion protein with a molecular mass of 86 kDa was partially purified with an amylose resin. After separation by SDS-PAGE the protein levels observed for the sarcosine oxidase fusion were lower than for the fusion protein found when the pMALc2 plasmid alone was used for expression in E. coli, even if the induction was carried out for 7 h (Fig. 10). Immunoblotting with the previously used antibodies against L-pipecolic acid oxidase revealed an 86-kDa fusion protein cross-reacting with the antibodies, as well as a lower migrating protein band, likely due to partial degradation of the MBP-Sox protein (Fig. 10). Fractions purified by affinity chromatography with an amylose resin were investigated for oxidase activity using different substrates. The fusion protein oxidized the substrates sarcosine, L-pipecolic acid, and L-proline with different kinetics as shown by their Km and kc values (Table IV). Interestingly, the lowest Km was determined for L-pipecolic acid as substrate (1.9 mM), but kc was lower than that for sarcosine and L-proline. Sarcosine and L-proline had Km values of 6.7 and 8.0 mM, respectively. A marked inhibition was noted with sarcosine at higher substrate concentrations (above 13 mM). Catalytic efficiencies were best for L-pipecolic acid, followed by L-proline and sarcosine. When the pMALc2 vector alone was expressed in E. coli, the similarly processed protein product showed no activity with any of the three substrates.


Fig. 10. Expression of rabbit sox in E. coli. The following samples were separated by 8% SDS-PAGE and stained with Coomassie Blue: protein standard (lane 1); crude extract (1.5 µg of protein) from E. coli transformed with pMALc2 (lane 2) or with pBR3 (lane 3); purified protein (0.3 µg) after affinity chromatography of pMalc2 (lane 4) or pBR3 (lane 5) -transformed cells. Expression of pMALc2 yielded a MBP-beta -Gal-alpha -fusion with a molecular mass of 50 kDa (lane 2 and 4), which showed no cross-reactions with antibodies raised against pipecolic acid oxidase (lane 6). In contrast, expression of pBR3 resulted in an MBP-Sox fusion with a molecular mass of 86 kDa cross-reacting with anti-pipecolic acid oxidase antibodies (lane 7).
[View Larger Version of this Image (45K GIF file)]


Table IV.

Kinetic values for recombinant MBP-sarcosine oxidase

The H2O2 formation was measured in 55 mM Tris, pH 8.4 at 37 °C as described under "Experimental Procedures."
Substrate Km kc kc/Km

mM min-1 min-1 mM-1
Sarcosine 6.7 0.542 0.081
L-Pipecolic acid 1.9 0.432 0.227
L-Proline 8.0 1.23 0.153

Rabbit Sox Is a Peroxisomal Protein in Mammalian Cells

To investigate the localization of Sox in mammalian cells, the cDNA was cloned into the mammalian expression vector pcDNA3 creating pBR4. Three different human skin fibroblast cell lines, which were already transformed with SV40 large T antigen3 and CV-1 cells (monkey kidney epithelial cells), were transfected with pBR4, using Lipofectamine. Two days after transfection, the cells were analyzed by indirect immunofluorescence. All cell lines expressed the gene with transfection rates of 10-20%. Because rabbit sarcosine oxidase has a PTS1 signal at the C terminus, a peroxisomal localization of the enzyme was expected. After normal fibroblasts (GM5756) were transformed with pBR4 and subjected to indirect immunofluorescence using the antibodies against L-pipecolic acid oxidase (3), a punctate staining pattern was obtained (Fig. 11A). Double staining to include antibodies against the peroxisomal matrix protein catalase revealed that Sox colocalized with catalase, suggesting that it is found in or at the peroxisomes (Fig. 11B). The second investigated cell line 005-T is a transformed fibroblast cell line from a patient lacking a PTS1 receptor (complementation group 2 of the peroxisomal biogenesis disorders (40, 54)) that results in a peroxisomal import defect for PTS1 and PTS2 proteins. When pBR4 was transfected into these cells, Sox was synthesized, but the protein was found throughout the cytoplasm of the cells (Fig. 11C). Again, this protein gave the same staining pattern as catalase, which is not imported into the peroxisomes of 005-T cells (Fig. 11D). The third cell line, which was from a patient with classical rhizomelic chondrodysplasia punctata (RCDP), has an isolated peroxisome import defect for the PTS2 protein thiolase but normal PTS1 import (40, 55). Transfection of pBR4 resulted in a peroxisomal localization (Fig. 11E) of sarcosine oxidase (note the same subcellular distribution for catalase in Fig. 11F) in this cell line. These results indicate that sarcosine oxidase is a peroxisomal protein imported into the organelles by the PTS1-dependent pathway. The same results were obtained when different plasmids encoding an N-terminal histidine-tagged sarcosine oxidase (pBR5) or a protein fusion with maltose-binding protein (pBR6) were transfected. Expression of sarcosine oxidase cDNA in CV-1 cells also resulted in a peroxisomal localization of the gene product.


Fig. 11. Subcellular localization of recombinant Sox from rabbit liver and catalase in normal transformed human fibroblasts and in fibroblasts from patients with peroxisomal biogenesis disorders. Normal fibroblasts (GM5756) (A and B), 005-T cells (no import of PTS1 and PTS2 proteins) (C and D), and cells from a patient with RCDP (no import of PTS2 proteins) (E and F) were transfected with pBR4 in the presence of lipofectamine. For indirect immunofluorescence the cells were double-stained with antibodies raised against pipecolic acid oxidase (A, C, and E) and catalase (B, D, and F) followed by fluorescein isothiocyanate- and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Sox as well as catalase are imported into the peroxisomes in normal fibroblasts (A and B) and cells from the RCDP patient (E and F) but remained in the cytoplasm of 005-T cells (C and D). The bar indicates 10 µm.
[View Larger Version of this Image (112K GIF file)]



DISCUSSION

During our studies of L-pipecolic acid oxidase, we discovered that rabbits have a similar enzyme, but this enzyme also oxidizes sarcosine. When the gene for this protein was cloned, we found that its amino acid sequence showed the most homology to the monomeric sarcosine oxidases.

Previously, it had been reported that while in human and monkey liver, L-pipecolic acid is oxidized in peroxisomes (3); in rabbit liver and kidney L-pipecolic acid is primarily oxidized in mitochondria (6). However, our studies with rabbit kidneys suggested that there might be L-pipecolic acid oxidation in peroxisomes as well. When we reexamined the oxidation of 14C-radiolabeled L-pipecolic acid in subcellular fractions of a Nycodenz gradient by measuring the formation of aminoadipic acid (6), approximately 80% was attributable to mitochondrial activity, and 20% could be accounted for as peroxisomal activity4. When oxidase activity was determined by H2O2 formation from L-pipecolic acid, almost all activity was found in the peroxisomal fraction, confirming two different systems for the oxidation of L-pipecolic acid in rabbit kidney. Like L-pipecolic acid, sarcosine has been reported to be oxidized by mitochondria in certain mammals (7, 9).

Since antibodies raised against pipecolic acid oxidase from monkey liver (3) cross-reacted with rabbit sarcosine oxidase, as well as with the recombinant enzyme, we were able to use them in immunoblot analysis of fractionated rabbit kidney to show that rabbit Sox was solely peroxisomal. This peroxisomal localization was further supported by the finding that the rabbit sarcosine oxidase sequence encoded for a C-terminal PTS1 (tripeptide AHL), as well as the sequence for the hypothetical C. elegans protein (tripeptide SKI) (accession number U23529[GenBank]), but the sequences for the bacterial sarcosine oxidases (12-15) did not contain a peroxisomal targeting signal.

Even if skin fibroblasts are not typical cells for the expression of amino-acid oxidases, the availability of cell lines with different peroxisomal import defects made them an excellent tool to study the intracellular localization of Sox in mammalian cells. Expression of cDNA for the rabbit sox gene resulted in an exclusively peroxisomal localization of the corresponding gene product in normal fibroblasts and in kidney CV-1 cells. In contrast, the peroxisomal biogenesis disorder cell line 005-T, which has no detectable PTS1 receptor, synthesized the Sox when transfected with the corresponding plasmid but did not import it into the peroxisomes. Instead, it remained in the cytoplasm like the peroxisomal matrix protein catalase. This finding indicated that the import of sarcosine oxidase into peroxisomes was dependent on an intact PTS1 receptor. In contrast, when sox was expressed in a cell line from a patient with RCDP, with a distinct defect in the import of PTS2 proteins, the protein was targeted to peroxisomes as in normal fibroblasts, further suggesting that rabbit sarcosine oxidase behaves like a typical PTS1 protein.

When the amino acid sequence for rabbit sarcosine oxidase was submitted to a BLAST search (48), the highest homology was seen with an unknown gene product from C. elegans, followed by the monomeric bacterial sarcosine oxidases. The gene for heterotetrameric enzyme from Corynebacterium sp. P-1 is organized as an operon containing genes that encode for all four subunits and the glyA gene, which encodes serine hydroxymethyltransferase (14). The beta -subunit of this sarcosine oxidase shows less homology to Sox from rabbit than the monomeric enzymes but has a close relationship to the N terminus of mitochondrial dimethylglycine dehydrogenase from rat liver. The relationship to this mitochondrial dehydrogenase is supported by the finding that the alpha -subunit of the tetrameric enzyme has additional homology to the C terminus of dimethylglycine dehydrogenase (49). The tetrameric sarcosine oxidases share more similarities with the mitochondrial dimethylglycine dehydrogenases (the sequence for sarcosine dehydrogenase is not known), whereas the monomeric sarcosine oxidases are closer to the peroxisomal enzyme in mammalian cells.

The close relationship between purified and recombinant rabbit sarcosine oxidases, L-pipecolic acid oxidase from monkey liver and the bacterial sarcosine oxidases is further reflected in their substrate specificity. The tetrameric sarcosine oxidase from Corynebacterium sp. P-1 is able to metabolize L-proline and L-pipecolic acid but at turnover rates 220-fold less than that for sarcosine (56). No information is available as to whether L-proline or L-pipecolic acid are substrates for monomeric sarcosine oxidases.

When the substrate specificities of native rabbit kidney sarcosine were compared with that of the recombinant fusion protein investigated in this study and with that of pipecolic acid oxidase from monkey liver (Table V), all three enzymes utilized the same amino acids as substrates but the kinetics were different. The common link between these substrates is the imino moiety (Fig. 12) and the similarity of the reaction mechanisms. Oxidation of these substrates by sarcosine oxidase or pipecolic acid oxidase would yield glycine and formaldehyde for sarcosine, Delta -piperideine-6-carboxylate for L-pipecolic acid, and Delta -pyrroline-5-carboxylate for L-proline. In contrast, the oxidation by D-amino-acid oxidase would result in the formation of Delta -piperideine-2-carboxylate from D-pipecolic acid.

Table V.

Comparison of relative catalytic constants of different oxidases

The catalytic constants for the three main substrates of different oxidases were compared. Note that these are not catalytic efficiencies, because the Km values were not known in each case.
Recombinant sarcosine oxidasea Rabbit kidney sarcosine oxidaseb Monkey liver L-pipecolic acid oxidasec

% % %
Sarcosine 46 100 10
L-Pipecolic acid 53 30 100
L-Proline 100 23 23

a Recombinant sarcosine oxidase (fusion with maltose-binding protein), this study, substrate concentration 9.8 mM.
b Purified sarcosine oxidase from rabbit kidney, this study, substrate concentration 9.8 mM.
c Purified L-pipecolic acid oxidase from monkey liver, Mihalik et al. (3), substrate concentration 5 mM.


Fig. 12. Sarcosine, L-pipecolic acid, and L-proline have similar chemical structures.
[View Larger Version of this Image (16K GIF file)]


Sarcosine oxidase from rabbit liver exhibits an ADP-beta alpha beta -binding fold satisfying the 11 consensus sequence requirements postulated by Wierenga et al. (57). The aspartate in position 1 does not fit the consensus, but in several well characterized FAD-binding sites an aspartate has been observed in this position (49, 58). The absorption spectrum of the GST-Sox fusion protein and of the native protein suggests that sarcosine oxidase has a tightly attached flavin as coenzyme. The flavin attachment site has not yet been identified. As in the monomeric sarcosine oxidases, the rabbit enzyme lacks the DHVA tetrapeptide identified as the flavin attachment site (His-175) in Corynebacterium sp. U-96 and Corynebacterium sp. P-1 (both tetrameric enzymes) (49, 59). However, in all monomeric sarcosine oxidases, in the C. elegans protein, and in rabbit Sox, a conserved histidine (His-49 for rabbit Sox) aligns with the flavin attachment site (His-84) of dimethylglycine dehydrogenase (50, 60). Newer results from Willie and Jorns (20) and Willie et al. (52) indicate that in the case of the enzyme from Corynebacterium sp. P-1 (19) this flavin is FMN, whereas two monomeric sarcosine oxidases contain FAD.

It might be remarkable that the fourth fragment of high homology at the C terminus (Fig. 2) shows high similarity to the N-terminal ADP-binding fold but did not fit the consensus of Wierenga et al. (57) exactly.

Several lines of evidence indicate that the purified rabbit enzyme is identical to the sox gene product. Both the native enzyme from rabbit kidney and the recombinant enzyme were localized in or targeted to peroxisomes. The calculated size from the sox sequence was identical to that estimated by SDS-PAGE of the native enzyme. The same polyclonal antibodies recognized both the purified and the recombinant enzyme. Both enzymes utilized the same substrates with similar catalytic efficiencies for sarcosine and L-pipecolic acid. A covalently attached flavin was found in both enzyme preparations. Some features such as the peroxisomal localization and the involvement of a nucleotide could be predicted from the primary sequence of the sox gene and were confirmed with the native protein.

In summary, L-pipecolic acid, L-proline, and sarcosine can be degraded by the same enzyme via an identical reaction mechanism. This enzyme belongs to a family of sarcosine oxidases which are all characterized by a flavin moiety which is covalently bound. This common type of flavin binding to the protein suggests that it may be required for this reaction mechanism. In contrast, the D-amino-acid oxidases, which have a similar but chirally opposite mechanism, do not contain a covalently bound flavin. Future studies of this reaction mechanism should help in elucidating just how flavin binding to enzymes is associated with particular reaction mechanisms.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Institut für Physiologische Chemie, Ruhr-Universität Bochum, Universitätstr. 150, 44780 Bochum, Germany. Tel.: (49)-234-700 4938; Fax: (49)-234-709 4266; E-mail: gabriele.dodt{at}rz.ruhr-uni-bochum.de.
1   The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PTS1, peroxisomal targeting signal 1; DIG, digoxigenin; IPTG, isopropylthio-beta -D-galactoside; GST, glutathione S-transferase, MBP, maltose binding protein; RCDP, rhizomelic chondrodysplasia punctata; bp, base pair(s); RT-PCR, reverse transcription-polymerase chain reaction.
2   S. J. Mihalik and G. Dodt, manuscript in preparation.
3   S. J. Gould and G. Dodt, unpublished data.
4   G. Dodt, unpublished data.

Acknowledgments

We are grateful to Prof. Dr. A. W. Holldorf for his generous advice and support. We thank Prof. Dr. S. J. Gould for providing the opportunity to B. R. to perform studies in his lab and for his support. We also thank U. Freimann for her skillful technical assistance and Dr. W. B. Schliebs for helpful discussions.


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