Mass Spectrometric Identification of K210 Essential for Rat Malonyl

Dec 27, 2005 - When acetyl CoA was added to the reaction, K210 was protected from acetylation, indicating .... the cell, vibra cell sonicator (Sonics ...
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Mass Spectrometric Identification of K210 Essential for Rat Malonyl-CoA Decarboxylase Catalysis Hyung Wook Nam, Gha Young Lee, and Yu Sam Kim* Department of Biochemistry, College of Science, Protein Network Research Center, Yonsei University, Seoul, Korea 120-749 Received December 27, 2005

Proteomic technology provides useful tools to detect protein modification sites in vivo and in vitro. In this work, we applied proteomics to identify an essential amino acid residue involved in Malonyl-CoA Decarboxylase (MCD) catalysis. A reaction with acetic anhydride and MCD, under mild conditions without acetyl CoA as a substrate, resulted in the acetylation of six lysyl residues, K210, K58, K167, K316, K388, and K444. When acetyl CoA was added to the reaction, K210 was protected from acetylation, indicating a potential role for this residue in catalysis. In addition, K210 was the only lysyl residue, out of six, that was not endogenously acetylated. Because K210, K308, and K388 are conserved across species, they were site-specifically mutated to methionine which is size-wise similar to lysine but not protonated. The K308M and K388M MCD mutants retained 60% of their enzyme activities, whereas the K210M mutant was completely inactive. These results strongly suggest that K210 is an essential residue in rat MCD catalysis and is a likely proton donor to the alpha carbon of malonyl-CoA. Therapeutic inhibition of MCD may be a viable approach to treating various clinical pathologies associated with defective fatty acid metabolism. Keywords: malonyl-CoA decarboxylase (MCD) • chemical modification • acetic anhydride • propionic anhydride • mass spectrometric analysis • site-directed mutagenesis

Introduction The proteomics methods used to identify the post-translationally modified sites of proteins have been developed in accordance with the technical improvement in MS/MS of recent years. Analytical techniques such as precursor ion scanning, neutral loss scanning, and MS3 techniques are increasingly applied to proteins to study their biological functions.1,2 According to these basic technological improvements, a chemical proteomics technology called activity-based protein profiling (ABPP) has been successfully applied to the screening of novel enzymes based on active-site similarity from complex mixtures.3 If the 3D structure of a protein is known, its active site can be in silico predicted by analysis of the distances of the exposed residues from the centroid of the molecule.4 However, 3D structures of many enzymes including Malonyl-CoA Decarboxylase (MCD; EC 4.1.1.9) are not yet elucidated. MCD catalyzes the decarboxylation of malonyl-CoA to acetylCoA, which is a central intermediate in lipid metabolism. Malonyl-CoA decarboxylase has been studied in various model organisms, including the rat,5-7 goose,8-10 and bacteria.11-13 Recently, MCD was identified as a major regulator of cardiac fatty acid oxidation6,14-16 via its ability to modify intracellular * To whom correspondence should be addressed. Department of Biochemistry, College of Science, Yonsei University, 134 Sinchon-dong, Seoul, Korea 120-749. Tel: -82-2-2123-3448. Fax:-82-2-392-3488. E-mail: [email protected].

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malonyl-CoA levels. Pharmacological MCD inhibition may be a viable approach to treating clinical pathologies associated with myocardial ischemia.17 Hepatic expression of MCD also appears to reverse muscle, liver, and whole-animal insulin resistance.5 The severe consequences of human MCD deficiency is illustrated by such phenotypic consequences as malonic acid urea, developmental delay, seizure disorder, and mental retardation.8,15,18 These phenotypes overlap with genetic deficiencies of fatty acid oxidation enzymes, indicating that MCD plays a critical role in fatty acid metabolism. Vertebrate MCD is localized in different sub-cellular compartments, and is suggested to perform multiple functions.8,9,19 In the goose uropygial gland, cytoplasmic MCD may increase the synthesis of multi-methyl-branched fatty acids, by increasing the relative methylmalonyl-CoA abundance.14,15,20,21 In such nonadipogenic tissues as cardiac and skeletal muscle, cytoplasmic MCD may lower malonyl-CoA levels to permit fatty acid oxidation. In the rat liver, mitochondrial MCD may function to remove malonyl-CoA produced by the adventitious activity of propionyl-CoA carboxylase on acetyl-CoA.22 Peroxisomal MCD, which is localized at the site of dicarboxylic acid beta-oxidation, may eliminate malonyl-CoA, an end-product of odd chain length dicarboxylic acid oxidation.15 In Rhizobium leguminosarum bv. trifolii, MCD may play an important role in malonate metabolism in the clover nodules of bacteroids. The malonate metabolic mutant Rhizobium do not form bacteroids in the clover nodules, which causes a defect in symbiotic nitrogen fixation between R. trifolii and clover.23-25 10.1021/pr050487g CCC: $33.50

 2006 American Chemical Society

K210 Essential for MCD Catalysis

Protein sequence alignment analyses revealed a high homology among different vertebrate MCD sequences. Bacterial MCDs exhibit approximately 25% sequence identity to vertebrates in comparison of the total amino acid sequence, whereas they show almost 90% identity in the region of 194∼338 and 420∼457 amino acids. Moreover, MCD does not share any detectable sequence homology to other known decarboxylases, indicating that the MCD gene may be evolutionarily distinct. There is an increasing interest among researchers to develop treatments for MCD-based diseases. However, the 3-dimensional structure of MCD is not solved, and details of its catalytic mechanism remain unknown. We obtained MCD crystals from R. trifolii, but failed to obtain sufficient diffraction.13 In enzyme catalysis, decarboxylation stereochemistry retains the alpha methyl group configuration. 26 Acetic anhydride acetylation inactivated MCD in geese, indicating that the epsilon amine of the lysyl group maybe involved in catalysis.27 However, the exact acetylation site has not been identified, even though mass spectrometry provides direct evidence of structural changes in the amino acid residue by post-translational modification. In this paper, we modified MCD chemically with acetic anhydride and analyzed acetylation site by proteomics technique using mass spectrometry, and found K210 to be essential. It was also confirmed by site-directed mutagenesis experiments on K210 which is homologous across species.

Experimental Section Materials. Acetic anhydride (97%), propionic anhydride (99%), urea, hydroxylamine, ammonium hydroxide, isopropylβ-D-thiogalactopyranoside (IPTG), ammonium bicarbonate, iodoacetamide, dithiothreitol (DTT), formic acid, malonyl CoA, acetyl CoA, CoA, malic dehydrogenase, citrate synthase, NAD, NADH, and malate were all purchased from Aldrich (St. Louis, MO). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI). pGEX-4T vector, thrombin, Gluthathione Sepharose 4 Fast flow, and Sephadex G-25 resin were all purchased from GE Healthcare (GE Healthcare Bio-Science AB, Uppsala, Sweden). For the dephosphorylation experiments, we purchased the Lambda protein phosphatase from New England Biolabs (Ipswich, MA). Water was purified with a Milli-Q system (Bedford, MA). Methanol and acetonitrile, HPLC grade, were purchased from J.T. Baker (Phillipsburg, NJ). The Bradford protein assay kit was purchased from Bio-Rad (Hercules, CA). The pfu polymerase purchased form Stratagene (La Jolla, CA) and Primers such as BamHI and EcoRI purchased from Takara Bio Inc. (Kyoto, Japan). For harvest and sonication of the cell, vibra cell sonicator (Sonics & Materials. Inc., CT) and Supra 22K centrifuge (Hanil, Inchon, Korea) are used. Preparation of Rat MCD. The MCD gene was prepared by PCR using pfu polymerase21,28,29 and two primers that contained BamHI and EcoRI at each end, respectively: forward primer: (5′-GATGGATCCATGCGAGGCTTGGGGCCAAG-3′) and reverse primer: (5′-AGAATTCTAGAGTTTGCTGTTGCTCTG-3′). The amplicons were digested with BamHI and EcoRI, and were sizechecked with electrophoresis on 1% agarose. The pGEX-4T vector was digested with BamHI and EcoRI. The insert and vector were ligated, and the recombinant plasmid was introduced into the E. coli BL21 competent cells. The cells were induced with 1 mM IPTG, harvested, and sonicated. The recombinant GST-MCD protein was mostly soluble, with a limited portion occurring in the insoluble precipitate. We purified GST-MCD from the soluble portion with glutathioneaffinity chromatography.9 Briefly, we loaded the cell lysates onto

research articles a Gluthathione Sepharose 4 Fast Flow column. The fusion protein, bound to the matrix, was cleaved with thrombin for 18 h at 22 °C. Purified MCD was active with similar characteristics, as reported previously.28 Chemical Modification of MCD with Acetic Anhydride or Propionic Anhydride. Acetic or propionic anhydride (3 nmol) was added directly to MCD (100 µg). The pH of the reaction mixture was maintained at pH 8.0 by the careful addition of 10% ammonium hydroxide (5 µL). After the reaction proceeded at 30 °C for 30 min., hydroxylamine (1 M, 50 µL) was added to de-esterify any tyrosine residues, and to destroy excess reagents. For the protection experiment, 50 mM of acetyl-CoA or CoA was added to the reaction mixture. Under the acetylation reaction conditions described above, acylated MCD was run on SDS-PAGE gels. The MCD band was cut precisely for in-gel-tryptic digestion. Subsequently, the peptides were extracted for further analysis. Gel Filtration Chromatography. To measure the enzyme activity, acetylated MCD mixtures (10 µg proteins) were chromatographically separated on Sephadex G-25 columns (5 cm × 5 mm I. D.) packed to Tricorn 5/50 empty column (GE Healthcare Bio-Science AB, Uppsala, Sweden), using Agilent 1100 HPLC systems (Palo alto, CA). Using this process, it could be monitored the chromatographic behavior of proteins due to denaturation in addition to desalting. Malonyl-CoA Decarboxylase Assay. The enzyme activity was monitored by measuring the absorbance at 340 nm, caused by NADH generation from coupling the reaction product, acetyl-CoA, with the malate dehydrogenase/citrate synthase system.30 For these readings, we used the following reagents in a 0.4 mL quartz cuvette with 1 cm path length: Tris-HCl (1 M, pH 8.0), 50 µL; DTT (50 mM), 5 µL; malate (1 M), 10 µL; NAD (50 mM), 3 µL; NADH (10 mM), 2 µL; malic dehydrogenase (1 unit/µL), 5 µL. The contents were mixed and incubated for 1min., followed by the addition of 5 µL of citrate synthase (1 unit/µL). After an incubating for 1min., we combined 3 µL of the malonyl-CoA decarboxylase preparation and 3 µL of malonyl-CoA. Water was used to bring the total volume to 350 µL. Amino Acid Sequence Analysis by Mass Spectrometry. MCD was electrophoresed on 12% SDS-PAGE. The MCD band was excised from the gel matrix, and 100 µg of the protein was digested with 1 µg trypsin at 37 °C for 18 h. The peptides were separated and analyzed by nano RP-LC-ESI-MS/MS to determine their amino acid sequences. Ultimate nanoLC systems, combined with the FAMOS autosampler and Switchos column switching valve (LC-Packings, Amsterdam, The Netherlands) was used. The samples were loaded onto precolumn (2 cm × 200 µm I. D.; Zorbax 300SB-C18, 5 µm, Agilent, CA), and washed with the loading solvent (H2O/0.1% formic acid, flow rate: 4 µL/min.) for 10 min. to remove salts. Subsequently, a Switchos II column switching device transferred flow paths to the analytical column (15 cm × 75 µm I. D.; Zorbax 300SBC18, 5 µm, Agilent, CA). The nano-flow eluted at a flow rate of 200 nl/min. using a 110 min. gradient elution from 0% solvent A to 32% solvent B, where solvent A was 0.1% formic acid with 5% acetonitrile and solvent B was 0.1% formic acid with 90% acetonitrile. The column outlet was coupled directly to the high voltage ESI source, which was interfaced to the QSTAR Mass spectrometer (Applied Biosystems, Foster city, CA). The nanospray voltage was typically 2300V in the nanoLC-ESI MS/MS mode. The nano RP-LC-ESI-MS/MS running on the QSTAR instrument was acquired in ‘Information Dependent AcquisiJournal of Proteome Research • Vol. 5, No. 6, 2006 1399

research articles tion’ mode (IDA), which allows the user to acquire MS/MS spectra based on an inclusion mass list and dynamic assessment of relative ion intensity. The data acquisition time was set to 3s per spectrum over m/z range of 400-1500 Da for nanoLC-MS/MS analyses. We performed searches on a National Center for Biotechnology Information (NCBI) nonredundant database using the MASCOT software package (Matrix Sciences, UK). In silico, we subtracted 190 peptides from MCD. The sequence coverage was 80%. Mass Spectrometric Analysis of Acylation Sites. The peptide sequences were analyzed using the nano RP-LC-ESI-MS/MS methods described above. We focused our database analysis on acylated peptides, and searched NCBI using the acetylation or propionylation modification parameter queries. For the quantification procedure, we used the acquisition software Analyst QS (Applied Biosystems, Foster city, CA), which has tools for extracting single extracted ion chromatogram (XIC). The peak area represents the peptide quantity in the sample. Site-Directed Mutagenesis of MCD. For these experiments, we mutated each three lysyl residues in MCD using the Quick Change Kit (Stratagene, CA) and the following mutagenic primers (mutated sites are underlined): K210M, 5′-TGAGGTGCTTCAGATGATCAGCGATTGTGAG-3′, K308M, 5′-CACCTTCCTCATAATGCGAGTGGTCAAGGAG-3′, K388M, 5′-GCGAAGTCTGAGATGCTGGCACAGGCACTG-3′. The mutant MCDs were prepared by the same method described above and 100 ng of proteins were used to assay enzyme activity.

Results and Discussion Post-translational Modification of Rat MCD Expressed in E. coli. To determine the sequence coverage and MCD modification state, we purified this enzyme from E. coli, digested it with trypsin, and then analyzed it via nano RP-LC-ESI-MS/ MS analysis. Using the MS/MS technique, each amino acid residue is revealed, along its any potential post-translational modification (PTM). Negatively charged PTMs, such as phosphorylation and sulfation, occasionally suppress ionization in the positive ionization mode, making these modifications difficult to detect by MS. But acylation is a chemically neutral modifications, it does not hamper MS detection. Rather, acylation increases the hydrophobic property of peptides. Previous reports suggest that peptide propionylation increases hydrophobicity, causing variable retention times in reversedphase HPLC.31 Theoretical trypsin digests of recombinant rat MCD (54 kDa) released approximately 190 tryptic peptides. The SDS-PAGE-gels revealed no noticeable size differences between the un-acetylated and the acetylated MCD proteins. The number of peptides analyzed in the nano RP-LC-ESI-MS/MS experiment depends on the chromatographic peak widths and the instrument’s duty cycle. With chromatographic peak widths of 10 s and scan times of 3 s per CID spectrum, three ions can be monitored with several spectra for each of the selected ions. From the sequencing results, the MCD generated 220 peptides with high score and 80% sequence coverage. Almost all peptide residues were identified with reliable scores, even though there were some unspecific cleaved peptides to trypsin. Generally, tryptic mis-cleavages occur adjacent to proline. However, other reasons for mis-cleavages include protein structure, amino acid arrangement and contamination with other proteases. In addition to any mis-cleavages encountered herein, it was discovered that the E. coli-expressed MCD was phosphorylated at seven seryl/threonyl residues, and was acetylated at 11 lysyl residues (K58, K145, K167, K182, K227, K316, K375, K385, K388, 1400

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Figure 1. Relative quantification of five peptides in the endogenously acetylated K176 peptide using extracted ion chromatogram (XIC). The peak area of XIC was used to calculate relative amounts of (A) artificially propionylated peptide (m/z: 1008.05(+2); 105 min.), (B) endogenously acetylated peptide (m/z: 1001.06(+2); 103 min.), (C) nonreacted and mis-cleaved peptide (m/z: 979.00(+2); 88.8 min.), (D and E) trypsin digested peptides (m/z: 453.76(+2); 22.5 min. and m/z: 536.20(+2); 41.35 min.) in 158ADLLEAQALKxLVEGPHVR175 residue. KAC: acetyl on epsilon amine of lysine, KPRO: propionyl on epsilon amine of lysine.

K441, K474). However, only five lysyl residues (K58, K167, K316, K388, K444) possessed higher sequencing scores than the protein threshold score (Mowse scoring algorithm) and reliability, as determined by manual de-novo sequencing. These results indicate that some E. coli acetyltransferase 32-34 may transfer acetyl groups randomly to five lysyls located on the protein’s surface. These results also require to determine the degree of acetylation on MCD in E. coli for further experiments on in vitro acetylation. To quantify the acetylation: nonacetylation ratio, MCD protein was propionylated with propionic anhydride which shows similar reaction properties on primary amine group of the target peptide. Propionylation of nonendogenously acetylated lysyl residues is demonstrated by two ion chromatograms for one peptide separated by 14 mass units. By this experiment, it was found that the propionylation of MCD was not completed. For instance, five different peptides were identified in the endogenously acetylated (K167) peptide: (A)propionylated-ADLLEAQALKPROLVEGPHVR(m/z: 1008.05(+2); 105 min.), (B) endogenously acetylated-ADLLEAQALKACLVEGPHVR (m/z: 1001.06(+2); 103 min.), (C) nonacylated-ADLLEAQALKLVEGPHVR (m/z: 979.00(+2); 88.8 min.), (D) tryptic fragment LVEGPHVR (m/z: 453.76(+2); 22.5 min.) and (E) tryptic fragment ADLLEAQALK (m/z: 536.20(+2); 41.35 min.). To relative quantification, +2 charged ion of each peptide was extracted and overlaid (Figure 1). The elution time of each peptide was well correlated to their hydrophobic properties. Similar retention time between propionyl- and acetyl-containing peptides was obtained, indicating that their chromatographic behavior is similar. However, based on XIC of dominant propionylated peptide in Figure 1, the quantity of endogenously acetylated peptide was roughly calculated to be less than 2%, indicating that this enzyme preparation could be used for

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Figure 2. Gel filtration chromatograms of acetylated MCD on Sephadex G-25 column. The column was calibrated with BSA (66 kDa) as a standard (A). (B) The purified rat MCD (54 kDa), (C) Acetylated MCD with 100 fold molar excess acetic anhydride, (D) Acetylated MCD with 3-fold molar excess acetic anhydride. The black line (214 nm) and dot line (280 nm) mean the wavelength of protein specific absorption.

Figure 3. Reaction time dependence on acetylation of MCD. (A) Gel filtration chromatogram of acetylated MCD (at 214 nm) obtained at different reaction times. (B) Specific activity of acetylated MCD obtained at different reaction times. 10 µg MCD was taken and incubated with 3-fold excess acetic anhydride. Samples were taken at each time point and fractionated immediately by Sephadex G-25 to measure enzyme activity. Each specific activity (S. A.) is the mean value of three times measurements.

further study for in vitro modification with acylating reagents in high sequence coverage. Inactivation of MCD by Acetylation with Acetic Anhydride. The MCD catalytic mechanism involves carbanion protonation generated by decarboxylation, whereby the catalytic site configuration is retained.26 Rainwater and Kolattukudy27 demonstrated that goose MCD is inactivated by acetylation with acetic anhydride, suggesting that lysyl residue(s) may be involved in the carbanion protonation generated by decarboxylation. Previously, a histidine residue was suggested to be the proton donor,26 because diethylpyrocarbonate, rather than pyridoxal5′-phosphate, inhibited enzymatic activity. They claimed that the lysine modification inhibits enzymatic activity and that inactivation by diethylpyrocarbonate is spontaneously reversible, but is not reversed by hydroxylamine treatment.27 Together with the high pH optimum, they speculated that the histidine residue is probably not involved in the reaction as a proton donor, and that the readily acetylated epsilon-amino group of the lysyl residue might participate in the reaction as the proton donor. However, the identity of the participating lysyl residue is unknown. Artificial protein modifications, having small stericeffects, may in some cases impact protein structure less. Acetic anhydride is the smallest acylation reagent to primary amine residues in lysine. Generally, lysine acetylation increases the hydrophobicity and decreases the positively charged properties in the modified residue. Carbeck and co-workers35 successfully used acetylation of lysyl groups to decrease the net charge on lactalbumin by -8. Whitesides and co-workers36,37 demonstrated there was no significant influence on the refolding of acetylated carbonic anhydrase, which did not contain the positively charged lysyl residues, following SDS denaturation.

In some cases, an excess of acetic anhydride or reaction conditions can denature certain enzymes. When we loaded the acetylated MCD onto the Sephadex G-25 column for remove reaction chemicals, detected a distorted peak compared with BSA standard (Figure 2A) and nonreacted MCD (Figure 2B). These results indicated that MCD might be denatured by excess acetyl reaction, 100-fold acetic anhydride input (Figure 2C). To avoid protein denaturation, only a 3-fold excess acetic anhydride was added. When we applied a 3-fold excess of acetic anhydride, a nativelike peak resulted (Figure 2D). To ensure that the protein did not denature over the course of the assay, acetylated MCD was analyzed by size exclusion chromatography at various time points. These data showed identical chromatograms, indicating that no significant denaturation occurred over the 120 min. (Figure 3A). However, analysis of acetylated MCD activity revealed a time-dependent loss of specific activity (SA), whereby 70% SA was lost by 30 min. (Figure 3B). Mapping Acetylation Sites of MCD by Acetic Anhydride. After the SDS-PAGE analysis of the acetylated MCD, the band was excised precisely. Acetylated MCD was digested “in gel” and was loaded on the nano RP-LC-ESI-MS/MS to identify acetylation sites. As shown in Figure 4, six acetylation sites were identified and manual confirmed. The reaction of MCD with propionic anhydride resulted in propionylation of the same six lysyl residues (Figure 5, data not shown for other peptides). Except for K210, only five acetylation sites were identified in the endogenously acetylated MCD, suggesting that K210 might be essential for enzyme catalysis. K210 is one of three conserved vertebrate lysyl residues (K210, K308, K388) in MCD (Figure 6). Because K210 is not endogenously acetylated, it may be Journal of Proteome Research • Vol. 5, No. 6, 2006 1401

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Figure 4. MS/MS spectra of six acetyl peptides in rat MCD. (A-E) endogenous acetylation at K58, K167, K316, K388, K444, and (F) artificial acetylation at K210. (A) TPAYELREKACTpPAPAEGQCADFVSFYGGLAEAAQR; m/z: 942.43(+3), (B) ADLLEAQALKACLVEGPHVR; m/z: 667.37(+3), (C) ELQKACEFPHLGAFSSLSPIPGFTK; m/z: 857.77(+3), (D) SEKACLAQALQGPLMR; m/z: 791.94(+2), (E) SpSLKACGLTSSCGLMVNYRYYLEETGPNSISYLGSK; m/z: 1280.93(+3), (F) VTWHSPCEVLQKACISECEAVHPVK; m/z: 935.13(+3), AC: acetyl, PRO: propionyl, p: phophoryl. Journal of Proteome Research • Vol. 5, No. 6, 2006 1403

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Figure 5. MS/MS spectra of VTWHSPCEVLQK210PROISECEAVHPVK; m/z: 939.80(+3). K210 residue is propionylated with propionic anhydride.

Figure 6. Sequence alignments of MCDs from the different species. Lysine residue, K210 and K308 of rat MCD are conserved in all MCDs, whereas K388 residue is conserved in vertebrate species.

Figure 7. Overlaid extracted ion chromatograms(XIC) of VTWHSPCEVLQKACISECEAVHPVK (m/z: 935.17(+3)) and VTWHSPCEVLQK (m/z: 749.36(+2)): (A) XIC for the two peptides obtained from acetylation of MCD by acetic anhydride followed by tryptic digestion, (B) XIC by the method in the presence of CoA, (C) XIC by the method in the presence of acetyl CoA. 1404

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Figure 8. Relative enzyme activity of mutant MCDs. K210M MCD lost completely its enzyme activity, while K388M and K308M MCD sustained their activity in about 60%. To measure the relative activity, 100 ng of wild type and mutant MCDs respectively were used.

located in the active site crevice where it is not readily available to cellular acetyltransferase. In the crevice, K210 may play dual key functional roles, including stabilizing the transition state enol intermediate generated by decarboxylation of malonylCoA and transferring a proton to the intermediate to form acetyl-CoA. However, the transition state oxyanion could be stabilized by other residues, including hydrogen on backbone peptide bonds. Protection of Inactivation by Acetyl-CoA. The inactivation reaction of MCD by acetic anhydride was performed in the presence of acetyl-CoA or CoA, to determine whether an essential lysyl residue is located on either the substrate or product binding site. The inactivation reaction was performed in three different ways; (A) the reaction of MCD by acetic anhydride as control, (B) the reaction in the presence of CoA, and (C) the reaction in the presence of acetyl-CoA. After tryptic digestion of MCD and MS analysis of peptides, ion chromatograms of two peptides, VTWHSPCEVLQKACISECEAVHPVK, and VTWHSPCEVLQK were extracted and overlaid (Figure 7). As shown in Figure 7, the acetylation of MCD by acetic anhydride was completely protected in the presence of acetyl-CoA, whereas the reaction was not inhibited by the addition of CoA. These results suggest clearly that the binding of the acyl moiety of acyl-CoA to the active site crevice prevents the entry of the acetylating agent to the active site lysyl residue. Sequence Analysis and Site-Directed Mutagenesis. Twelve different MCD genes from various eukaryotic and prokaryotic species, including Homo sapiens, have been identified in the GeneBank Database. The vertebrate MCD amino acid se-

quences are similar to each other. However, the bacterial MCD sequences only show 25% homology to their vertebrate counterparts. Focused on lysyl residues, K210 and K308 of rat MCD were conserved in all MCDs. Additionally, the K388 lysylresidues are homologous among vertebrate species and often endogenously acetylated. These results indicate that K210, K308, and K388 may play specific roles in catalysis (Figure 6). In the present study, we mutated K210, K308, and K388 to methionine, which is size-wise similar but not protonated. For standardization of enzyme assay, 100 ng of wild type and mutant MCDs respectively were used. Figure 8 illustrates that K210M mutant MCD was enzymatically inactive, whereas K308M and K388M possessed roughly 60% activities. Especially, K388 residue was modified with acetyl group endogenously, but there was no activity difference between K308M and K388M, indicating that acetylation at K388 does not directly relate to the enzyme activity. Although predicting the roles of the K308 or the K388 residues in MCD catalysis is difficult, the results herein clearly show that K210 is essential for MCD catalysis.

Conclusion Although MCD was first inactivated by acetylation two decades ago, the mechanism for inactivation remains unsolved. Until now, the location and the precise conversion of post-translationally modified residues in MCD were difficult to identify.27 Proteomics technologies, such as nano RPESI-MS/MS, provide useful tools for pinpointing precise residue modifications in vivo or in vitro. In this work, we applied this technology to identify an essential amino acid residue involved in MCD catalysis. Moreover, we identified the K210 lysyl site as being artificially acetylated, and resulting in the loss of MCD enzymatic activity when mutated by site directed mutagenesis. We propose that this lysyl residue is located deep within the MCD active site crevice, and plays a key role in the proton transfer to alpha carbon of malonyl-CoA to form acetyl-CoA as a mode of configuration retention (Figure 9). Thus, the design of MCD inhibitors that target such lysyl residues may ultimately provide therapeutic benefits to patients with disorders of fatty acid metabolism.

Acknowledgment. This work was supported by a grant from The Korea Science and Engineering Foundation through Protein Network Research Center (PNRC 2003-2-0086) and also in part by The Korea Research Foundation (KRF-2000-015DP0288).

Figure 9. Proposed mechanism of malonyl-CoA decarboxylase catalysis. Journal of Proteome Research • Vol. 5, No. 6, 2006 1405

research articles References (1) Mann, M.; Jensen, O. N. Nat. Biotechnol. 2003, 21 (3), 255-261. (2) Gronborg, M.; Kristiansen, T. Z.; Stensballe, A.; Andersen, J. S.; Ohara, O.; Mann, M.; Jensen, O. N.; Pandey, A. Mol. Cell. Proteomics 2002, 1 (7), 517-527. (3) Okerberg, E. S.; Wu, J.; Zhang, B.; Samii, B.; Blackford, K.; Winn, D. T.; Shreder, K. R.; Burbaum, J. J.; Patricelli, M. P. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (14), 4996-5001. (4) Ben-Shimon, A.; Eisenstein, M. J. Mol. Biol. 2005, 351 (2), 309326. (5) An, J.; Muoio, D. M.; Shiota, M.; Fujimoto, Y.; Cline, G. W.; Shulman, G. I.; Koves, T. R.; Stevens, R.; Millington, D.; Newgard, C. B. Nat. Med. 2004, 10 (3), 268-274. (6) Sambandam, N.; Steinmetz, M.; Chu, A.; Altarejos, J. Y.; Dyck, J. R.; Lopaschuk, G. D. Eur. J. Biochem. 2004, 271 (13), 28312840. (7) Roduit, R.; Nolan, C.; Alarcon, C.; Moore, P.; Barbeau, A.; Delghingaro-Augusto, V.; Przybykowski, E.; Morin, J.; Masse, F.; Massie, B.; Ruderman, N.; Rhodes, C.; Poitout, V.; Prentki, M. Diabetes 2004, 53 (4), 1007-1019. (8) FitzPatrick, D. R.; Hill, A.; Tolmie, J. L.; Thorburn, D. R.; Christodoulou, J. Am. J. Hum. Genet. 1999, 65 (2), 318-326. (9) Voilley, N.; Roduit, R.; Vicaretti, R.; Bonny, C.; Waeber, G.; Dyck, J. R.; Lopaschuk, G. D.; Prentki, M. Biochem. J. 1999, 340 (Pt 1), 213-217. (10) Mulder, H.; Lu, D.; Finley, J. t.; An, J.; Cohen, J.; Antinozzi, P. A.; McGarry, J. D.; Newgard, C. B. J. Biol. Chem. 2001, 276 (9), 64796484. (11) Chohnan, S.; Akagi, K.; Takamura, Y. Biosci. Biotechnol. Biochem. 2003, 67 (1), 214-217. (12) Hugler, M.; Krieger, R. S.; Jahn, M.; Fuchs, G. Eur. J. Biochem. 2003, 270 (4), 736-744. (13) Jung, J. S.; Baek, D. J.; Lee, G. Y.; Kim, Y. S.; Oh, B. H. Acta Crystallogr. D Biol. Crystallogr. 2003, 59 (Pt 1), 166-167. (14) Young, M. E.; Goodwin, G. W.; Ying, J.; Guthrie, P.; Wilson, C. R.; Laws, F. A.; Taegtmeyer, H. Am. J. Physiol. Endocrinol. Metab. 2001, 280 (3), E471-479. (15) Sacksteder, K. A.; Morrell, J. C.; Wanders, R. J.; Matalon, R.; Gould, S. J. J. Biol. Chem. 1999, 274 (35), 24461-24468. (16) Dyck, J. R.; Barr, A. J.; Barr, R. L.; Kolattukudy, P. E.; Lopaschuk, G. D. Am. J. Physiol. 1998, 275 (6 Pt 2), H2122-2129. (17) Dyck, J. R.; Lopaschuk, G. D. J. Mol. Cell Cardiol. 2002, 34 (9), 1099-1109.

1406

Journal of Proteome Research • Vol. 5, No. 6, 2006

Nam et al. (18) Surendran, S.; Sacksteder, K. A.; Gould, S. J.; Coldwell, J. G.; Rady, P. L.; Tyring, S. K.; Matalon, R. J. Neurosci. Res. 2001, 65 (6), 591594. (19) Saha, A. K.; Ruderman, N. B. Mol. Cell Biochem. 2003, 253 (1-2), 65-70. (20) Razeghi, P.; Young, M. E.; Abbasi, S.; Taegtmeyer, H. Biochem. Biophys. Res. Commun. 2001, 287 (1), 5-10. (21) Lee, G. Y.; Kim, N. H.; Zhao, Z. S.; Cha, B. S.; Kim, Y. S. Biochem. J. 2004, 378 (Pt 3), 983-990. (22) Touma, E.; Suormala, T.; Baumgartner, E. R.; Gerbaka, B.; Ogier de Baulny, H.; Loiselet, J. J. Inherit. Metab. Dis. 1999, 22 (2), 115122. (23) Lee, H. Y.; An, J. H.; Kim, Y. S. Eur. J. Biochem. 2000, 267 (24), 7224-7230. (24) An, J. H.; Lee, H. Y.; Ko, K. N.; Kim, E. S.; Kim, Y. S. Mol. Cells 2002, 14 (2), 261-266. (25) Lee, H. Y.; Kim, Y. S. J. Biochem. Mol. Biol. Biophys. 2002, 6 (4), 283-288. (26) Kim, Y. S.; Kolattukudy, P. E. J. Biol. Chem. 1980, 255 (2), 686689. (27) Rainwater, D. L.; Kolattukudy, P. E. Int. J. Biochem. 1982, 14 (7), 609-614. (28) Lee, G. Y.; Bahk, Y. Y.; Kim, Y. S. J. Biochem. Mol. Biol. 2002, 35 (2), 213-219. (29) Lee, G. Y.; Cho, J. W.; Lee, H. C.; Kim, Y. S. Biochim. Biophys. Acta 2002, 1577 (1), 133-138. (30) Kolattukudy, P. E.; Poulose, A. J.; Kim, Y. S. Methods Enzymol. 1981, 71 Pt C, 150-163. (31) Nam, H. W.; Simpson, R.; Kim, Y. S. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005, 826 (1-2), 91-107. (32) Weier, D.; Muller, C.; Gaspers, C.; Frentzen, M. Biochem. Biophys. Res. Commun. 2005, 334 (4), 1127-1134. (33) Starai, V. J.; Escalante-Semerena, J. C. J. Mol. Biol. 2004, 340 (5), 1005-1012. (34) Pompeo, F.; Mushtaq, A.; Sim, E. Protein Expr. Purif. 2002, 24 (1), 138-151. (35) Carbeck, J. D.; Negin, R. S. J. Am. Chem. Soc. 2001, 123 (6), 12521253. (36) Gudiksen, K. L.; Gitlin, I.; Yang, J.; Urbach, A. R.; Moustakas, D. T.; Whitesides, G. M. J. Am. Chem. Soc. 2005, 127 (13), 47074714. (37) Gao, J.; Whitesides, G. M. Anal. Chem. 1997, 69 (4), 575-580.

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