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Lysosomal Prenylcysteine Lyase Is a FAD-dependent Thioether Oxidase*

William R. TschantzDagger §, Jennifer A. DigitsDagger , Hyung-Jung Pyun, Robert M. Coates, and Patrick J. CaseyDagger ||**

From the Departments of Dagger  Pharmacology and Cancer Biology and || Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and the  Department of Chemistry, University of Illinois, Urbana, Illinois 61801

Received for publication, September 5, 2000, and in revised form, November 2, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached via a thioether bond to a cysteine residue at or near their C terminus. As prenylated proteins comprise up to 2% of the total protein in eukaryotic cells, and the thioether bond is a stable modification, their degradation raises a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that cleaves prenylcysteines to cysteine and an unidentified isoprenoid product. Here we show that the isoprenoid product of prenylcysteine lyase is the C-1 aldehyde of the isoprenoid moiety (farnesal in the case of C-15). The enzyme requires molecular oxygen as a cosubstrate and utilizes a noncovalently bound flavin cofactor in an NAD(P)H-independent manner. Additionally, a stoichiometric amount of hydrogen peroxide is produced during the reaction. These surprising findings indicate that prenylcysteine lyase utilizes a novel oxidative mechanism to cleave thioether bonds and provide insight into the unique role this enzyme plays in the cellular metabolism of prenylcysteines.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Covalent modifications by lipids play important roles in the subcellular localization and biological activity of a multitude of proteins in eukaryotic cells (1). One major class of lipid modification is termed prenylation, in which either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is covalently attached via a thioether bond to cysteine residue(s) at or near the C terminus of the protein (2, 3). The majority of prenylated proteins contain the so-called CaaX motif, which is defined by the presence of a cysteine residue fourth from the C terminus of the protein to which the isoprenoid is attached (4). CaaX proteins are subject to additional modifications after prenylation in that the three C-terminal residues are proteolytically removed, and the new C-terminal prenylcysteine is subject to carboxyl methylation (4, 5).

Prenylation is a stable modification, and these proteins can comprise up to 2% of total cellular protein (6, 7). The quantity of prenylated protein in cells, as well as the stability of the modification, raises a metabolic challenge to the cell in the disposal of prenylcysteines resulting from the normal turnover of these proteins. S-Acylation, another lipid post-translational modification, raises a similar metabolic challenge to the cell. An enzyme termed palmitoyl protein thioesterase removes the palmitoyl group from thiol-containing compounds arising from the degradation of palmitoylated proteins (8), and this enzyme has been implicated in a neurodegenerative lysosomal storage disorder, infantile neuroceroid lipofuscinosis (9). These findings in particular have highlighted an important role for enzymes involved in the metabolism of lipidated proteins.

In a continuing effort to understand the metabolic fate of prenylcysteines produced during turnover of prenylated proteins, we identified and cloned a lysosomal enzyme that catalyzes the degradation of prenylcysteines (10, 11). This enzyme, dubbed prenylcysteine lyase (PCLase),1 degrades prenylcysteines to yield free cysteine and an unidentified isoprenoid product (10). The production of free cysteine (i.e. containing a reduced sulfhydryl) and the lack of a NAD(P)H requirement suggested that PCLase utilizes a mechanism distinct from enzymes such as the carbon-sulfur beta -lyase and cytochrome P450- and flavin-containing monooxygenases known to cleave carbon-sulfur bonds (10). Additionally, PCLase has essentially no sequence similarity to known enzymes, suggesting that it might possess a novel mechanism for carbon-sulfur bond cleavage (11). In the present study, the unusual mechanism of the reaction catalyzed by PCLase was investigated.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Materials-- Farnesal was synthesized with modification using farnesol as described (12). [35S]Farnesylcysteine (FC) and [1-14C]FC were synthesized as described (10). Native PCLase was purified from bovine brain as described (10). Diphenyleneiodonium chloride (DPI) was obtained from Sigma. 7-Diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin was obtained from Molecular Probes Inc.

Production and Purification of Recombinant His6-PCLase-- Recombinant PCLase was produced by infection of Sf9 cells with a recombinant baculovirus containing the PCLase cDNA using a variation of the previously published procedure (11). Specifically, histidine-tagged PCLase was produced by insertion of six histidines between residues 496 and 497 in the C-terminal region of PCLase using the Gene Editor kit (Promega) following the manufacturer's instructions; the previously described pFastBac/PCLase construct (11) was used as a template for mutagenesis. Recombinant baculovirus containing this His6-PCLase variant was produced using the Bac-to-Bac system (Life Technologies, Inc.) and was used to infect Sf9 cells as described previously (11) except that serum-free media was used, and the cells were harvested at 48 h post-infection.

Recombinant His6-PCLase was purified from extracts of infected Sf9 cells by a combination of nickel-affinity and anion-exchange chromatography. The cell pellet from a 4-liter culture of infected Sf9 cells was resuspended in 40 ml of 50 mM Tris-Cl, pH 7.7, 0.2 mM EGTA, 0.2 mM EDTA, containing a mixture of protease inhibitors (13). Cells were disrupted, and the homogenate was centrifuged at 100,000 × g for 1 h. PCLase was extracted from the resulting pellet with 20 ml of 50 mM Tris-Cl, pH 7.7, containing 100 mM NaCl, 0.5% Triton X-100, and the protease inhibitor mixture, followed by centrifugation at 100,000 × g for 1 h. The supernatant was loaded onto a 1-ml column of nickel-nitrilotriacetic acid resin (Qiagen), the column was washed with 5 ml of 50 mM Tris-Cl, pH 7.7, containing 100 mM NaCl, 0.2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 5 mM imidazole, and PCLase eluted with a 40-ml linear gradient of imidazole (5-500 mM) in the above buffer. Fractions containing PCLase activity were pooled, dialyzed against 20 mM Tris-Cl, pH 7.7, containing 0.2% CHAPS (Buffer A), and the dialysate loaded onto a Mono Q HR 5/5 column equilibrated with Buffer A. PCLase was eluted with a 20-ml linear gradient of NaCl from 0 to 500 mM in Buffer A. Fractions containing PCLase were pooled, dialyzed as above, flash-frozen in aliquots, and stored at -80 °C until use.

Identification of the Isoprenoid Product of the PCLase Reaction-- Purified bovine PCLase (120 ng) was incubated with 6.66 µM [14C]FC (~50 mCi/mmol) in 15 mM Tris-Cl, pH 7.7, 15% glycerol, 0.02% CHAPS, and 10 mM Zwittergent 3-14 for 30 min at 37 °C. In some reactions, semicarbazide was added to a final concentration of 40 mM after the enzymatic reaction, and the incubation was continued for 30 min at 37 °C. Prior to HPLC analysis, 10 nmol of unlabeled farnesylcysteine, farnesal, and the semicarbazide adduct of farnesal were added to provide internal standards. Samples were chromatographed on a C18 reverse-phase column with a 45-min gradient from 10 to 100% acetonitrile containing 0.1% triflouroacetic acid. Radioactivity was quantified by scintillation spectroscopy. The elution positions of the isoprenoid standards were determined by monitoring absorbance at 210 nm.

Determination of the Oxygen Requirement of PCLase-- PCLase (300 nM) and reaction buffer (50 mM Tris-Cl, pH 7.7, containing 5 µM [35S]FC) were deoxygenated by bubbling argon through the solutions. The solutions were then mixed and incubated at 37 °C under argon. Aliquots (20 µl) were removed and quenched with the solvent used for thin-layer chromatography (n-propanol:NH4OH:H2O (6:3:1); 10 µl) at indicated times. At the time indicated in Fig. 2, the reaction chamber was opened to air, and additional aliquots were taken and reactions quenched as described above. Accumulation of the product [35S]cysteine was analyzed by thin-layer chromatography as described (10).

Identification of the Flavin Moiety in PCLase-- The visible spectrum of a 12 µM solution of PCLase in 20 mM Tris-HCl, pH 7.7, 0.2% CHAPS was recorded on a Hewlett Packard diode-array spectrophotometer. The spectrum of the buffer alone was also determined and was subtracted from the enzyme spectrum. The identification of the flavin as flavin adenine dinucleotide (FAD) was performed as described (14). Briefly, PCLase (240 µg in 100 µl) was denatured by addition of an equal volume of 20% trichloroacetic acid and incubation for 15 min on ice. The sample was then centrifuged, and the supernatant divided into two equal portions, with one-half neutralized immediately by addition of 25 µl of 4 M K2HPO4, followed by determination of its fluorescence (lambda ex = 450 nm; lambda em = 535 nm). The remaining half was incubated at 37 °C for 3 h in the dark, neutralized as above, and its fluorescence was determined. The ratio of FAD to protein was determined using the molar extinction coefficient at 454 nm of 12.5 mM-1 cm-1 (15).

Analysis of the inhibition of PCLase by DPI was performed by adding increasing concentrations of DPI (see Fig. 3C) to PCLase (200 nM) in 50 mM Tris-HCl containing 5 µM [35S]FC. Reactions were conducted for 15 min at 37 °C, and product formation was determined by thin-layer chromatography as described above.

PCLase Activity Assays-- PCLase activity was routinely determined by following the conversion of [35S]FC (~50,000 cpm/time point) to [35S]cysteine by thin-layer chromatography as described (10). In the studies comparing cysteine and hydrogen peroxide production, a fluorimetric assay of cysteine production was also employed. This assay involved reacting the cysteine formed during the reaction with the fluorogenic compound 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin to yield a fluorescent product. PCLase (300 nM) was mixed with saturating FC (100 µM) in 50 mM sodium phosphate, pH 7.4, at 25 °C. At time intervals ranging to 30 min, 50-µl samples were removed and mixed with 50 µl of 0.2 mM 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin in the assay buffer. A control reaction without FC was processed in parallel. The intensity of fluorescence was determined (lambda ex = 384 nm; lambda em = 470 nm) in a 100-µl cuvette, and cysteine formed was calculated from a cysteine standard curve.

Measurement of Hydrogen Peroxide Production by PCLase-- Hydrogen peroxide produced in the PCLase reaction was measured using the Amplex Red kit (Molecular Probes). PCLase (10 ng/µl) was incubated at 23 °C in the buffer provided, which also contained horseradish peroxidase and Amplex Red. After the base line was established, the PCLase substrate FC was added to a final concentration of 5 µM. The action of horseradish peroxidase on Amplex Red in the presence of hydrogen peroxide converts the Amplex Red into a fluorogenic product (16). Fluorescence analysis of the reaction mixture was conducted (lambda ex = 563 nM, lambda em = 587 nM).


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Identification of the Isoprenoid Product of the PCLase Reaction-- In the previous study, the PCLase action on FC was shown to produce free cysteine and an unidentified hydrophobic moiety derived from the isoprenoid constituent (10). Because in that study the isoprenoid product appeared to be less polar than farnesol (10), we hypothesized that this product might be the isoprenoid aldehyde, i.e. farnesal. To test this possibility, FC that had 14C incorporated in the isoprenoid moiety was incubated with purified bovine PCLase. Analysis of the reaction mixture by reverse-phase HPLC revealed the expected loss of radioactivity in the substrate FC because of digestion by PCLase, as well as the appearance of a peak of radioactivity that comigrated with an authentic farnesal standard (Fig. 1, A and B). When the reaction mixture was subjected to derivatization with semicarbazide, a compound that forms an imine adduct with aldehydes (17), the radiolabeled product of the reaction now comigrated with the authentic farnesal-semicarbazone standard (Fig. 1C). Similar results were seen when reaction mixtures were analyzed using a normal-phase thin-layer chromatography system (data not shown). These data revealed that the isoprenoid product of the PCLase reaction was the aldehyde of the isoprenoid and indicated an oxidative type of mechanism for the enzyme.



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Fig. 1.   Identification of the isoprenoid product of PCLase. [1-14C]FC was incubated with buffer alone (panel A) or with purified bovine PCLase (120 ng; panels B and C) for 30 min at 37 °C. For the reaction shown in panel C, semicarbazide (SCA; 40 mM) was added after the enzymatic reaction, and the incubation was continued for an additional 30 min at 37 °C. Reactions were terminated and analyzed by C18 reverse-phase HPLC as described under "Experimental Procedures." Radioactivity in each fraction was quantified by liquid scintillation spectroscopy. The data shown are from a single experiment that is representative of three such experiments.FCHO, farnesal.

Analysis of Molecular Oxygen Requirement for PCLase Action-- The finding that PCLase produced an oxidized isoprenoid suggested that the enzyme utilized molecular oxygen as a cosubstrate. To examine this possibility, the PCLase reaction was studied in the absence and presence of oxygen. Solutions of the enzyme and assay components were made anaerobic by degassing with argon, placed in a sealed chamber, and aliquots were removed for analysis of product formation. Essentially no product formation was detected under anaerobic conditions (Fig. 2). However, when the reaction chamber was opened to the atmosphere to allow oxygen entry, product formation ensued in a linear fashion with a rate constant of 0.25 min-1 (Fig. 2). These results indicated that PCLase requires molecular oxygen for product formation and are consistent with a reaction proceeding via an oxidative mechanism that forms a prenyl aldehyde.



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Fig. 2.   Dependence of the PCLase reaction on molecular oxygen. Deoxygenated PCLase (300 nM; see "Experimental Procedures") was incubated in deoxygenated buffer containing 5 µM [35S]FC in a sealed chamber under argon. Aliquots were removed at the indicated time points, reaction mixtures were resolved by thin-layer chromatography, and cysteine product formation was visualized by fluorography and quantified by liquid scintillation spectroscopy. The arrow indicates the time when the chamber was opened. The data shown are the average of duplicate determinations from a single experiment that is representative of three such experiments.

Detection of a Flavin Cofactor Associated with PCLase-- Many enzymes that use oxygen also utilize a flavin and/or NAD(P)H as cofactors (18). However, PCLase shows no NAD(P)H requirement for catalysis (10). To investigate the possibility for other cofactors, the visible spectrum of recombinant PCLase was measured. This spectroscopic analysis revealed the presence of characteristic peaks of a flavoenzyme at 360 and 454 nm (Fig. 3A) (18).



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Fig. 3.   Identification of a flavin associated with PCLase. Panel A, spectroscopic analysis of PCLase. The visible spectrum of a 12 µM solution of PCLase is shown. Panel B, fluorescence analysis of the flavin released from PCLase. The supernatant obtained from acid denaturation of 240 µg of recombinant PCLase was analyzed by fluorescence spectroscopy as described under "Experimental Procedures." Panel C, inhibition of PCLase by DPI. Purified recombinant PCLase was incubated with the indicated concentration of DPI for 15 min at 37 °C, and enzyme activity was then determined as described under "Experimental Procedures." For all panels, the data shown are from single experiments that are representative of at least two such experiments.

To determine whether the apparent flavin cofactor was flavin mononucleotide (FMN) or FAD, we took advantage of the facts that FAD has a much lower intrinsic fluorescence than FMN because of the pi -pi stacking of the isoalloxazine ring system with the adenine ring of FAD and that FAD can be converted to FMN by acid hydrolysis of the diphosphate bond, thereby releasing this pi -pi interaction (14). Therefore, PCLase was subjected to acid denaturation, and the denatured enzyme was pelleted. The resultant supernatant was divided with one-half being immediately neutralized and the other half incubated in the acid for 3 h at 37 °C prior to neutralization. Analysis of the fractions by fluorescence spectroscopy revealed an emission signature consistent with the presence of a flavin (Fig. 3B). This fluorescence markedly increased in the sample subjected to acid hydrolysis (Fig. 3B), indicating the identity of the flavin as being FAD. Furthermore, this experiment indicated that the FAD cofactor was not covalently attached to the enzyme, because it was released upon denaturation. In addition, the absorption intensity at 454 nm corresponded to a ratio of FAD to protein of 1.3:1 (data not shown; see "Experimental Procedures"), indicating that the enzyme contained a stoichiometric amount of FAD.

To demonstrate that the bound FAD was required for catalysis, PCLase was treated with DPI, a compound that blocks the reoxidation of FAD to the reduced state (19). DPI treatment inhibited the PCLase reaction in a dose-dependent fashion with an IC50 of ~100 µM (Fig. 3C). These results provided compelling evidence that FAD participates in a catalytic manner in the enzyme by cycling between its oxidized and reduced forms, a result again completely consistent with an unusual oxidative mechanism for the enzyme.

Determination of Hydrogen Peroxide Production by PCLase-- Many flavoenzymes generate hydrogen peroxide during catalysis by reduction of oxygen (18). To determine whether PCLase produced hydrogen peroxide, a peroxidase-linked assay was used that employed a nonfluorogenic substrate (Amplex Red) that is converted to a fluorogenic compound by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (16). Addition of the substrate FC to a mixture of PCLase in the presence of HRP and Amplex Red resulted in a biphasic time course of hydrogen peroxide formation (Fig. 4), the first phase apparently being due to equilibration of the reaction, with the second phase representing the steady state rate of hydrogen peroxide production. The rate of the linear increase in hydrogen peroxide formation in the second phase (kobs = 0.27 ± 0.02 min-1) correlated well with the rate of cysteine formation determined for aliquots of the same reaction mixture (kobs = 0.18 ± 0.02 min-1, indicating stoichiometric production of hydrogen peroxide during turnover by the enzyme.



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Fig. 4.   Determination of hydrogen peroxide production by PCLase. Purified recombinant PCLase (10 ng/µl) was incubated in the presence of horseradish peroxidase and Amplex Red reagent. The substrate FC (5 µM) was added to initiate the reaction. Hydrogen peroxide was detected as described under "Experimental Procedures." The data shown are from a single experiment that is representative of three such experiments.

Based on the data reported in this and the previous study (10), we propose the mechanism for the PCLase reaction shown in Fig. 5. Although the mechanism depicted is not the only one that could be drawn for PCLase, we believe that it is the most likely mechanism, because it is the simplest one that can be envisioned that accounts for all three products of this reaction, i.e. free cysteine, the prenyl aldehyde, and hydrogen peroxide. In this mechanism, the reaction is initiated by FAD-mediated hydride abstraction from C-1 of the isoprenoid moiety, producing a reduced flavin and an allylic (i.e. resonance-stabilized) carbocation intermediate. Attack of a water molecule on the carbocation results in formation of a hemithioacetal, which then collapses to the prenyl aldehyde concomitant with C-S bond breakage in which cysteine acts as the leaving group. Meanwhile the hydride of the reduced flavin is transferred to molecular oxygen, resulting in hydrogen peroxide formation and reoxidation of the flavin.



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Fig. 5.   Proposed mechanism of PCLase. The mechanism proposed for the reaction catalyzed by PCLase is shown. R, the homogeranyl portion of the farnesyl unit.

In summary, this study provides evidence for a novel mechanism in the catabolism of prenylcysteines by PCLase. These findings are particularly intriguing in light of the fact that PCLase has essentially no homology to other known proteins (11). The uniqueness of PCLase may be a result of uniqueness of its substrates; thioethers are rare in mammalian organisms, and prenylcysteines are the only known thioethers that contain an allylic carbon in the C-S bond. Characterization of structure-function relationships of PCLase are likely to yield important findings as to how it achieves this type of catalysis. These and other ongoing studies should provide insight into this unusual enzyme that will allow a better understanding of the cellular role of PCLase in the metabolism of prenylcysteines.


    ACKNOWLEDGEMENTS

We thank Eric Furfine, Irwin Fridovitch, and Kendra Hightower for helpful discussions, Ted Meigs for comments on the manuscript, and Alan Embry for assistance with the figures.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants GM46372 (to P. J. C.) and GM13956 (to R. M. C.).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.

§ Supported by a National Institutes of Health postdoctoral fellowship.

** To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710-3686. Tel.: 919-613-8613; Fax: 919-613-8642; E-mail: casey006@mc.duke.edu.

Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.C000616200


    ABBREVIATIONS

The abbreviations used are: PCLase, prenylcysteine lyase; FC, farnesylcysteine; HPLC, high pressure liquid chromatography; DPI, diphenyleneiodonium chloride; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NAD(P)H, reduced nicotinamide adenine dinucleotide; HRP, horseradish peroxidase; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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