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J. Biol. Chem., Vol. 278, Issue 49, 48674-48683, December 5, 2003

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Mechanistic Characterization of a Bacterial Malonate Semialdehyde Decarboxylase

IDENTIFICATION OF A NEW ACTIVITY IN THE TAUTOMERASE SUPERFAMILY^*

Gerrit J. Poelarends, William H. Johnson, Jr., Alexey G. Murzin, and Christian P. Whitman¶

From the Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, Texas 78712-1074 and the MRC Centre for Protein Engineering, Cambridge CB2 2QH, United Kingdom

Received for publication, June 24, 2003 , and in revised form, September 3, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Malonate semialdehyde decarboxylase (MSAD) has been identified as the protein encoded by the orf130 gene from Pseudomonas pavonaceae 170 on the basis of the genomic context of the gene as well as its ability to catalyze the decarboxylation of malonate semialdehyde to generate acetaldehyde. The enzyme is found in a degradative pathway for the xenobiotic nematocide trans-1,3-dichloropropene. MSAD has no sequence homology to previously characterized decarboxylases, but the presence of a conserved motif (Pro¹-(X)₈ -Gly-Arg¹¹-X-Asp-X-Gln) in its N-terminal region suggested a relationship to the tautomerase superfamily. Sequence analysis identified Pro¹ and Arg⁷⁵ as potential active site residues that might be involved in the MSAD activity. The results of site-directed mutagenesis experiments confirmed the importance of these residues to activity and provided further evidence to implicate MSAD as a new member of the tautomerase superfamily. MSAD is the first identified decarboxylase in the superfamily and is possibly the first characterized member of a new and distinct family within this superfamily. Malonate semialdehyde is analogous to a -keto acid, and enzymes that catalyze the decarboxylation of these acids generally utilize metal ion catalysis, a Schiff base intermediate, or polarization of the carbonyl group by hydrogen bonding and/or electrostatic interactions. A mechanistic analysis shows that the rate of the reaction is not affected by the presence of a metal ion or EDTA while the incubation of MSAD with the substrate in the presence of sodium cyanoborohydride results in the irreversible inactivation of the enzyme. The site of modification is Pro¹. These observations are consistent with the latter two mechanisms, but do not exclude the first mechanism. Based on the sequence analysis, the outcome of the mutagenesis and mechanistic experiments, and the roles determined for Pro¹ and the conserved arginine in all tautomerase superfamily members characterized thus far, two mechanistic scenarios are proposed for the MSAD-catalyzed reaction in which Pro¹ and Arg⁷⁵ play prominent roles.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The trans-1,3-dichloropropene catabolic pathway is elaborated by the soil bacterium Pseudomonas pavonaceae 170 and enables the organism to use trans-1,3-dichloropropene (Scheme 1, 1) as a sole source of carbon and energy (1). The compound, a key ingredient in the nematocides Shell D-D and Telone II, is first converted in three enzymatic steps to trans-3-chloroacrylate (2), which, in turn, is processed to acetaldehyde (4). The evolution of this pathway has been the subject of several recent studies because it may be a newly evolved pathway assembled by the bacterium in response to repeated exposure to the manmade compound, 1,3-dichloropropene (1–3).

View larger version (6K): [in this window] [in a new window]   SCHEME 1

Although the decarboxylation of 3 to generate 4 is quite facile and proceeds non-enzymatically, it is presumably accelerated by an enzyme in this catabolic pathway. In this regard, the two genes coding for the - and -subunits of CaaD (caaD1 and caaD2, respectively in Fig. 1) are found within a cluster of genes that includes two additional open reading frames located immediately downstream (4). One is a gene that codes for a protein having 130 amino acids (designated orf130) and the second one is a partial gene that codes for a 178-amino acid segment of a protein (designated orf4). The partial orf4 sequence has a high degree of sequence identity with putative alcohol dehydrogenases in Bacillus subtilis (47%) and Escherichia coli (56%) (4), indicating that the protein encoded by orf4 is not likely to function as a decarboxylase. Thus, it was hypothesized that orf130 might encode a malonate semialdehyde decarboxylase (MSAD) that converts 3 to 4.

View larger version (4K): [in this window] [in a new window]   FIG. 1. Organization of the trans-3-chloroacrylate catabolic gene cluster of P. pavonaceae 170.2 Genes are shown as open boxes, and arrows indicate the direction of transcription. The SalI restriction sites are shown. The triangle indicates the location of a putative transcription terminator.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Materials—Chemicals and biochemicals were purchased from Fisher Scientific Inc., Fluka Chemical Co. (Milwaukee, WI), Sigma-Aldrich Chemical Co., or EM Science (Cincinnati, OH), unless stated otherwise. CaaD was purified by a published procedure (5). Tryptone, yeast extract, and agar were obtained from BD Biosciences (Franklin Lakes, NJ). Molecular biology enzymes, endoproteinase Glu-C (protease V8), DNA ladders, protein molecular weight standards, the high pure plasmid isolation kit, the high pure PCR product purification kit, multipurpose agarose, and the deoxynucleotide triphosphates (dNTPs) were purchased from F. Hoffmann-La Roche, Ltd. (Basel, Switzerland). The pET-3a vector was obtained from Promega Corp. (Madison, WI). Oligonucleotides for DNA amplification and sequencing were synthesized by Genosys (The Woodlands, TX). The Amicon concentrator and the YM3 and YM10 ultrafiltration membranes were obtained from Millipore Corp. (Bedford, MA). The membrane tubing was purchased from Spectrum Laboratory Products, Inc. (Gardena, CA). Pre-packed PD-10 Sephadex G-25 columns were obtained from Biosciences AB (Uppsala, Sweden).

Bacterial Strains and Plasmids—E. coli strain BL21(DE3) was obtained from Promega Corp., and was used for the cloning of the PCR products, plasmid DNA isolation, and the overproduction of MSAD and the mutant enzymes. The pET3a expression vector (Promega Corp.) was used for the expression of orf130 and the mutant genes. The recombinant cosmid pPS41 contains the trans-3-chloroacrylate catabolic gene cluster of P. pavonaceae 170 and was used as the DNA template for the PCR amplification of the orf130 gene.² Its construction is described elsewhere (4).

General Methods—Techniques for restriction enzyme digestion, ligation, transformation, and other standard molecular biology manipulations were based on methods described by Sambrook et al. (7). The PCR was carried out in a PerkinElmer DNA thermocycler Model 480 obtained from PerkinElmer Inc. (Wellesley, MA). DNA sequencing was performed by the DNA Core Facility in the Institute for Cellular and Molecular Biology (The University of Texas at Austin). HPLC was performed on a Waters (Milford, MA) 501/510 system using either a TSKgel DEAE-5PW (anion exchange) column or a TSKgel Phenyl-5PW (hydrophobic interaction) column (Tosoh Bioscience, Montgomeryville, PA). Protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions or by PAGE under native conditions on gels containing 15% polyacrylamide. The gels were stained with Coomassie Brilliant Blue. Protein concentrations were determined with the Bio-Rad protein assay or by measuring the absorbance of the protein in 20 mM NaH₂PO₄ buffer (pH 6.5) containing 6.0 M guanidium hydrochloride, at 280 nm (for wild-type MSAD, A_0.1% = 0.775). Absorbance data were obtained on a Hewlett Packard 8452A Diode Array spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded in 100% H₂O on a Varian Unity INOVA-500 spectrometer using selective pre-saturation of the water signal with a 2-s presaturation interval. The lock signal is dimethyl-d₆ sulfoxide (DMSO-d₆). Chemical shifts are standardized to the DMSO-d₆ signal at 2.49 ppm. The N-terminal amino acid sequencing was performed by Eurosequence BV (Groningen, The Netherlands) using chemicals, reagents, and a sequenator (Model 477A) obtained from Applied Biosystems (Warrington, UK). The native molecular masses of the purified MSAD and its mutants were determined by gel filtration chromatography using a Superose 12 (Amersham Biosciences) column connected to the Waters 501/510 HPLC system.

Sequence Analysis—BLAST and iterative PSI-BLAST searches of the National Center for Biotechnology Information (NCBI) databases were performed using the translated amino acid sequence of the orf130 gene from P. pavonaceae 170 as the query sequence (8). The databases were searched with the "NR" option (all non-redundant GenBank^TM CDS translations + PDB + SwissProt + PIR + PRF). Amino acid sequences were aligned using a version of the ClustalW multiple sequence alignment routines available in the computational tools at the EMBL-EBI Web site (9).

Polymerase Chain Reaction—The amplification reaction mixtures (100 µl) contained the standard Taq amplification buffer, 200 µM of each dNTP, 100 ng of each primer, 100 ng of template DNA, and 2 units of TaqDNA polymerase. The cycling parameters were 94 °C for 5 min followed by 30 cycles of 94 °C for 60 s, 58 °C for 60 s, and 72 °C for 90s, with a final elongation step of 72 °C for 10 min. The reaction mixtures were subjected to electrophoresis in 0.8% agarose gels, and PCR products were stained with ethidium bromide.

Construction of Expression Vector—The orf130 gene was amplified by the PCR using the cosmid pPS41 as the template and the forward and reverse primers 5'-ATACATATGCCACTTCTCAAGTTC-3' (designated primer F) and 5'-CATGGATCCTCAGACGAGGTCCCCAGT-3' (designated primer R), respectively. The forward primer contains a NdeI restriction site (in bold) and the reverse primer has a BamHI restriction site (in bold). The PCR reaction was carried out as described above and the PCR product was purified using the high pure PCR product purification kit. The restriction sites NdeI and BamHI, introduced during the amplification reaction, were used to clone orf130 into the plasmid pET3a, yielding plasmid pET(orf130), for overexpression under control of the T7 promoter. The cloned orf130 gene was sequenced in order to confirm that no mutations had been introduced in the amplification reaction.

Site-directed Mutagenesis—The P1A and R11A mutants of MSAD were generated by the PCR using the primers 5'-ATACATATG-GCACTTCTCAAGTTC-3' and 5'-ATACATATGCCACTTCTCAAGTTCGACATCTTCTACGGGGCAACCGACGCT-3', where the NdeI restriction sites are shown in bold and the codons for the desired mutations are underlined. These primers anneal to the 5'-end of the wild-type coding sequence and were used in combination with primer R. The R75A mutant was generated by overlap extension PCR as described elsewhere (10) using primers F and R as the external primers. The internal PCR primers were oligonucleotides 5'-GTGATATCTCGACCTGCATCAGAAGAGCAGAAGGTC-3' and 5'-GACCTTCTGCTCTTCTGATGCAGGTCGAGATATCAC-3' (the mutated codon is underlined). The PCR reactions were carried out as described above, the products purified and cloned into plasmid pET3a for expression of the mutant genes. The mutant genes were sequenced in order to verify that only the intended changes had been introduced.

Expression and Purification of MSAD and the Mutant Enzymes— Bacterial cells were grown at 37 °C in Luria-Bertani (LB) medium supplemented with ampicillin (Ap) (100 µg/ml) when appropriate. E. coli BL21(DE3) transformants containing the desired plasmid were collected from a plate by resuspending them in LB medium (1 ml), which was then used to inoculate 50 ml of LB/Ap medium so that an initial OD₆₀₀ of about 0.05 was obtained. After overnight growth at 37 °C, the culture was used to inoculate 1 liter of LB/Ap medium. This culture was grown at 37 °C for 45 min to an OD₆₀₀ of 0.6, at which time 0.5 mM isopropyl--D-thiogalactoside (IPTG) was added to induce expression of the genes encoding MSAD and its mutants. Cells were harvested 5 h after induction by centrifugation (10 min at 10,000 x g), washed with 0.1 volume of 10 mM Tris-SO₄ buffer, pH 8 (Buffer A), and stored at –20 °C until used.

In a typical purification procedure, cells from three 1-liter cultures were thawed, combined, and suspended in 15 ml of Buffer A. Cells were disrupted by sonication for 30 s per ml of suspension at a 60 watts output using a W385 sonicator from Heat systems-Ultrasonics, Inc. (Farmingdale, NY), after which unbroken cells and debris were removed by centrifugation (30 min at 20,000 x g). The supernatant was filtered through a 0.2-µm pore diameter filter and applied to the TSKgel DEAE-5PW column (150 x 21.5 mm), which had previously been equilibrated with Buffer A. The column was washed with 50 ml of Buffer A, and retained proteins were eluted with a 300-ml increasing linear gradient of 0–0.5 M Na₂SO₄ in Buffer A at a flow rate of 5 ml/min. Fractions (10 ml) that showed the highest decarboxylase activity were pooled and concentrated to about 10 ml using an Amicon-stirred cell equipped with a YM3 (3,000 MW cutoff) or a YM10 (10,000 MW cutoff) ultrafiltration membrane. Subsequently, solid (NH₄)₂SO₄ was added to a concentration of 1 M and the resulting solution was stirred for 60 min at 4 °C. After centrifugation (30 min at 20,000 x g), the supernatant was loaded onto the TSKgel Phenyl-5PW column (150 x 21.5 mm), which had previously been equilibrated with Buffer B (1 M (NH₄)₂SO₄ in Buffer A). The column was washed with 50 ml of Buffer B, and retained proteins were eluted with a 250-ml decreasing linear gradient of 1.0–0 M (NH₄)₂SO₄ in Buffer A at a flow rate of 5 ml/min. Fractions (10 ml) with the highest decarboxylase activity were pooled and concentrated to about 3 ml. The concentrate was loaded onto a Sephadex G-75 column (100 x 2.5 cm), previously equilibrated with Buffer A. The protein was eluted with Buffer A at a flow rate of 1 ml/min. Fractions (9 ml) with the highest decarboxylase activity were analyzed by SDS-PAGE, and those that contained purified enzyme were pooled and concentrated to a protein concentration of about 5 mg/ml. The purified enzymes were filtered through a 0.2-µm pore diameter filter and stored at 4 °C. The mutant enzymes exhibited sufficient activity such that fractions containing them could be identified by an activity assay.

Mass Spectrometric Characterization of MSAD, MSAD Modified by 3 or 4, and the MSAD Mutants—The masses of MSAD, MSAD modified by 3 or 4, and the three mutants of MSAD were determined using an LCQ electrospray ion trap mass spectrometer (ThermoFinnigan, San Jose, CA), housed in the Analytical Instrumentation Facility Core in the College of Pharmacy at the University of Texas at Austin. The protein samples were made up as described elsewhere (5). The observed monomer mass for MSAD was 14,106 Da (calc. 14,109 Da). The observed monomer mass for the P1A mutant was 14,080 Da (calc. 14,080 Da), that of the R11A mutant was 14,022 Da (calc. 14,021 Da), and that of the R75A was 14,021 Da (calc. 14,021 Da). The masses for the modified MSAD samples are reported below.

¹H NMR Spectroscopic Assay for MSAD Activity Using 3—The ¹H NMR spectra monitoring the enzymatic decarboxylation of 3 by MSAD were generated as follows. An amount of 2 (4 mg, 0.04 mmol) dissolved in DMSO-d₆ (30 µl) was added to 100 mM Na₂HPO₄ buffer (0.6 ml; pH 9) in an NMR tube. The pH of the buffer was adjusted to 8.5. Subsequently, an aliquot of CaaD (50 µl of a 11 mg/ml solution made up in 20 mM Na₂HPO₄ buffer, pH 7.3) was added to the reaction mixture. ¹H NMR spectra were recorded 1.5, 6, and 10 min after mixing. After 14 min, an aliquot of MSAD (100 µl of a 9.1 mg/ml solution made up in 20 mM Na₂HPO₄ buffer, pH 7.3) was added to the reaction mixture. ¹H NMR spectra were recorded 1.5 and 4.75 min after the addition of MSAD. The reaction was completed after 4.75 min. The final pH of the reaction mixture was 7.20.

In a second experiment, an identical reaction mixture was placed in an NMR tube along with an aliquot of MSAD (100 µl of a 9.1 mg/ml solution made up in 20 mM Na₂HPO₄ buffer, pH 7.3). The pH of the buffer was adjusted to 8.5. The reaction was initiated by the addition of the same quantity of CaaD. ¹H NMR spectra were recorded 1.5, 6 and 10 min after the addition of CaaD. The final pH of the reaction mixture was 7.13. In a third experiment, the same reaction mixture was placed in an NMR tube along with a smaller quantity of MSAD (2.5 µl of a 0.91 mg/ml solution made up in 20 mM Na₂HPO₄ buffer, pH 7.3). The reaction was started by the addition of the same quantity of CaaD. ¹H NMR spectra were recorded every 3 min after the addition of CaaD until the reaction was completed (27 min). The signals for 2, 3, and 4 (and the hydrates of 3 and 4) are reported elsewhere (5).

Spectrophotometric Assays for MSAD Activity—Two assays were used to quantify MSAD activity. In the first assay, the activity of MSAD was monitored by following the production of NADH at 340 nm in a coupled assay using the -NAD⁺-dependent aldehyde dehydrogenase at 22 °C. The assay mixture (1 ml final volume) consisted of 50 mM Na₂HPO₄ buffer (pH 9.0), 5 mM -NAD⁺, 1–4 units/ml of aldehyde dehydrogenase (1 unit will oxidize 10 µmol of 4 to acetic acid per min at 25 °C at pH 8 in the presence of -NAD⁺), and 3 (11). The substrate 3 was generated in situ by the incubation of 2 with CaaD as follows. Varying amounts (1–10 µl) of 2 from a stock solution (10, 50, or 500 mM) made up in 100 mM Na₂HPO₄ buffer (pH 9.0) and CaaD (5 µl; 40–80 µg) were added to the assay mixture. When there is no further decrease in absorbance at 224 nm (indicating that 2 has been converted to 3) (5), a quantity of MSAD (1.5 µl; 0.3 µg) was added to the assay mixture and the increase in absorbance at 340 nm was followed. The initial rate of NADH formation is proportional to the concentration of MSAD at all substrate concentrations examined. Under these conditions, 3 is in equilibrium with the hydrate and the chemical decarboxylation of 3 is negligible as is the oxidation of 3 by aldehyde dehydrogenase. In the second assay, the activity of MSAD was monitored by following the depletion of NADH at 340 nm in a coupled assay using the -NADH-dependent alcohol dehydrogenase at 22 °C. The assay mixture consisted of 100 mM K₂HPO₄ buffer, pH 9.0, 0.3 mM -NADH, and 3. The substrate was generated as described above using varying amounts of 2 (1–20 µl) from a 1 M stock solution made up in 100 mM K₂HPO₄ buffer (pH 8.5) and CaaD (15 µl from a 16 mg/ml solution in 20 mM Na₂HPO₄ buffer, pH 7.3). When there is no further decrease in absorbance at 224 nm, a quantity of alcohol dehydrogenase (10 µl from of a 6 mg/ml stock solution made up in 100 mM K₂HPO₄ buffer, pH 9.1) was added to the assay mixture. The assay was initiated by the addition of a quantity of MSAD (5 µl of a 0.1 mg/ml stock solution in 20 mM Na₂HPO₄ buffer, pH 7.3) and the decrease in absorbance at 340 nm was followed. The initial rate of NADH consumption is proportional to the concentration of MSAD at all substrate concentrations examined (1–20 mM). In both assays, one unit of enzyme activity is defined as the amount of enzyme required to convert 1 µmol of substrate to product in 1 min.

Effect of Metal Ions on the MSAD Activity—The enzyme (1 mg/ml) was incubated for 1 h at 22 °C in 50 mM NaH₂PO₄ buffer (pH 6.5), which was made 5 mM in various metal ions (MgCl₂, CaCl₂, CoCl₂, NiCl₂, FeCl₂, ZnCl₂, CuCl₂, and MnCl₂). In a separate control experiment, the same concentration of MSAD was incubated without any metal ion under otherwise identical conditions. Subsequently, an aliquot (2 µl) was removed, diluted 500-fold into the assay buffer, and the specific activities were determined using the coupled assay described above. The MSAD activity was measured in the presence of the various metal ions (at 0.2 mM) as well as in the absence of these metal ions. Higher concentrations of some metal ions (CuCl₂, NiCl₂, and ZnCl₂) inhibited the aldehyde dehydrogenase. The FeCl₂ was maintained in the reduced state by storing the solution under argon. The enzyme was also dialyzed for 24 h at 4 °C with buffer (10 mM Tris-SO₄, pH 8.2 or 20 mM NaH₂PO₄, pH 7.3) made 5 mM in EDTA. The specific activity was determined using the coupled assay.

Sodium Cyanoborohydride Treatment of MSAD in the Presence of Substrate and Product—In these experiments, a solution containing 12.5 µM enzyme (based on a subunit molecular mass of 14,106) in a final volume of 100 µl of 100 mM Na₂HPO₄ buffer (pH 9.0) and 25 mM NaCNBH₃ was placed on ice. Subsequently, 3 or 4 was added to a final concentration of 1 mM, and the mixture was allowed to react for 4 h. A stock solution of 3 (10 mM) was generated in situ by the action of CaaD on 2 and the 100 mM stock solution of 4 was made up by the addition of the appropriate amount of 4 to water. The reaction was quenched by the addition of 1 ml of ice-cold 10 mM Tris-SO₄ buffer (pH 8.2), which reacts with the remaining 3 or 4. The resulting imine is reduced by the NaCNBH₃. In order to remove these products and the excess NaCNBH₃, and to establish the irreversibility of the reaction, the reaction mixtures were dialyzed for 24 h with 20 mM Na₂HPO₄ buffer (pH 9.0). The dialyzed enzymes were assayed for residual activity using the coupled assay. The samples treated with 3 or 4 in the presence of NaCNBH₃ did not regain activity.

Control reactions containing enzyme, buffer, and substrate, or enzyme, buffer, and NaCNBH₃ were carried out under identical conditions. In addition, two structurally similar ketones and one aldehyde (acetopyruvate, acetoacetate, and succinate semialdehyde) were incubated with MSAD under similar conditions. These mixtures did not lead to inactivation of MSAD.

Mass Spectral Analysis of the Modified MSAD and Peptide Mapping—Three samples were made up as follows. A quantity of MSAD (20 µl from a 9 mg/ml solution resulting in 180 µg) and a sufficient volume of 100 mM Na₂HPO₄ buffer (pH 9) containing 25 mM NaCNBH₃ were added to an Eppendorf tube to produce a final volume of 1 ml. One sample was treated with 3 (100 µl from a 10 mM solution of 3 generated by the action of CaaD on 2 in 100 mM Na₂HPO₄ buffer, pH 9.0) and the second sample was treated with 4 (10 µl from a 100 mM solution of 4 dissolved in H₂O). A third sample was not treated with either compound and was used as the control sample. The three samples were incubated for 4 h at 0 °C. Subsequently, the samples were loaded onto separate PD-10 Sephadex G-25 gel filtration columns, which had previously been equilibrated with 100 mM (NH₄)₂CO₃ buffer (pH 8.0). The proteins were eluted by gravity flow using the same buffer. Fractions (0.5 ml) were analyzed for the presence of protein by UV absorbance at 214 nm. The appropriate fractions containing the purified protein samples were concentrated (4-fold) under vacuum, analyzed by ESI-MS, and used in the following peptide mapping experiments.

Two additional samples, containing MSAD, NaCNBH₃ and 3, were made up identically but were only incubated for 1 or2hat0 °C. The two samples were purified as described above and analyzed by ESI-MS. These samples were not used in the peptide mapping experiments.

For the peptide mapping experiments, a quantity ( 50 µg) of unmodified MSAD and MSAD modified by 3 or 4 was dried under vacuum. The individual protein pellets from the three samples were dissolved in 10 µl of 10 M guanidine HCl and incubated for 2 h at 37 °C. Subsequently, the protein samples were diluted 10-fold with 100 mM (NH₄)₂CO₃ buffer (pH 8.0) and incubated for 48 h at 37 °C with sequencing grade protease V-8 (2.5 µl of a 10 mg/ml stock solution made up in water) (12). These V-8 treated samples were made up and analyzed on the delayed extraction Voyager-DE PRO MALDI-TOF instrument (PerSeptive Biosystems, Framingham, MA) as previously described (5). The ions in the samples were also subjected to MALDI-PSD analysis using the protocol described elsewhere (5).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Expression, Purification, and Characterization of the Orf130 Gene Product—The orf130 gene was overexpressed in E. coli BL21(DE3) using the T7 expression system. After SDS-PAGE of crude extract prepared from these BL21(DE3) cells, a dominant protein band with the expected subunit molecular mass of 14 kDa was observed (data not shown). To establish that this overproduced protein indeed represents the product of the orf130 gene, it was subjected to N-terminal sequencing. The N-terminal amino acid sequence was established as PLLKFD, which is identical to that predicted from the orf130 gene sequence if the initiating methionine is removed during posttranslational processing.

The recombinant product was purified using three column chromatography steps providing typically 15 mg of protein per liter of culture. The protein was at least 95% pure as assessed by SDS-PAGE. The purified enzyme was analyzed by ESI-MS and gel filtration chromatography. It generates one major peak in the mass spectrometer, which corresponds to a mass of 14,106 ± 1 Da. A comparison of this value to the expected mass of the orf130 gene product (14,240 Da) confirms that Pro¹ of the recombinant protein is not blocked by the initiating methionine. The native molecular mass of the protein was estimated to be about 70 kDa, suggesting that the native enzyme is a homopentameric protein.³

Assignment of Function for the Orf130 Gene Product—A nonredundant search of the NCBI data base using the BLASTP program (8) and the translated amino acid sequence of orf130 as the query yielded only two proteins (YodA and YrdN) with significant overall sequence similarity (51 and 53%, respectively). These results could not be used to assign a function for the orf130 gene product because both YodA and YrdN are hypothetical proteins from B. subtilis for which no physiological functions have been reported (13). However, the genetic context of the orf130 gene in the trans-3-chloroacrylate catabolic gene cluster (vide supra) provided strong evidence that the gene product might function as a MSAD.

¹H NMR Characterization of the MSAD-catalyzed Reaction—In order to determine the function of the orf130 gene product, the decarboxylation reaction was monitored by ¹H NMR spectroscopy using 3, which was generated by the action of CaaD on 2 (Fig. 2). Accordingly, incubation of 2 with CaaD generates a mixture of 3 (3.20 and 9.50 ppm) and the hydrate of 3 (2.31 and 5.14 ppm) (Fig. 2, top) (5). The spectrum recorded 1.5 min after the addition of the orf130 gene product to the NMR tube (Fig. 2, bottom), shows the loss of the signals corresponding to 3 (as well as the signals corresponding to the hydrate) and the appearance of signals readily assigned to 4 (2.04 and 9.47 ppm) and the hydrate of 4 (1.13 and 5.05 ppm) (5). The rapid decarboxylation of 3 to afford 4 in the presence of the gene product of orf130 clearly establishes its MSAD activity. Hence, the orf130 gene product is hereafter referred to as MSAD.

View larger version (8K): [in this window] [in a new window]   FIG. 2. 1H NMR (500-MHz, H2O) spectra monitoring the reaction catalyzed by the orf130 gene product. Top, the 1H NMR spectrum, acquired 10 min after the addition of CaaD to an NMR tube containing 2, showing signals corresponding to 2, 3, and the hydrate of 3 (2.31 and 5.14 ppm). Bottom, the 1H NMR spectrum, acquired 1.5 min after the addition of the orf130 gene product to the same NMR tube, showing signals corresponding to 4 and the hydrate of 4 (1.13 and 5.05 ppm).

Kinetic Characterization and Properties of MSAD—Although two coupled assays to measure MSAD activity were developed, attempts to measure kinetic parameters for MSAD using these assays were not successful. In these assays, MSAD activity was linked to either the oxidation of 4 to acetate by including aldehyde dehydrogenase in the reaction mixture or to the reduction of 4 to ethanol by including alcohol dehydrogenase in the reaction mixture (11, 14). Oxidation of 4 results in the concomitant reduction of NAD⁺, which is indicated by an increase in absorbance at 340 nm. The reduction of 4 results in the concomitant oxidation of NADH, which is indicated by a decrease in absorbance at 340 nm. In the coupled assay using aldehyde dehydrogenase, a plot of various concentrations of 3 (estimated from the concentration of 2) versus the initial rates measured at each concentration remained linear up to 5 mM. At concentrations above 5 mM, CaaD and aldehyde dehydrogenase are inhibited. For this reason, alcohol dehydrogenase was tested in the coupled assay. However, a plot of various concentrations of 3 versus the initial rates measured remained linear up to 20 mM. Hence, MSAD could not be saturated, as assessed by either of these assays. Accordingly, the activity of MSAD is expressed in units of specific activity using a substrate concentration of 1 mM (Table I).

Several substrate analogs were tested as potential inhibitors of MSAD. In the presence of 3 (at 1 mM), the enzyme was not inhibited by 10 mM acetoacetic acid, acetopyruvic acid, 3-chloropropionic acid, propiolic acid, malonate, and succinic semialdehyde (data not shown). Neither malonate nor succinic semialdehyde was a substrate for MSAD.

Identification of MSAD as a New Tautomerase Superfamily Member—Sequence analysis of MSAD using two iterations of PSI-BLAST resulted in 22 sequences corresponding to a number of proteins in the 4-oxalocrotonate tautomerase (4-OT) family of enzymes. While these sequences showed identities ranging from 20–25%, the most intriguing identities were those observed between MSAD and the -subunit of CaaD (25%) and 4-OT (24%). The protein sequence of MSAD did not show statistically significant similarities (e.g. <20% sequence identity and no conserved motifs) with any proteins from the other two major families (the 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI) family represented by another bacterial isomerase, CHMI, and the macrophage migration inhibitory factor (MIF) family, represented by the mammalian cytokine MIF with a phenylpyruvate tautomerase activity) in the tautomerase superfamily (6). In addition, the protein sequence did not show any significant similarities with any known decarboxylases or aldolases in the data base.

Several 4-OT homologues have been assigned to the 4-OT family of enzymes on the basis of sequence analysis (15). These homologues are composed of small subunits (typically 61–81 amino acid residues) and have a conserved N-terminal proline. 4-OT and CaaD are the best characterized members of the family with assigned functions, and although some of the other homologues have been studied, their physiological functions are unknown. 4-OT is found in a degradative pathway for aromatic hydrocarbons in the soil bacterium Pseudomonas putida mt-2 (16). 4-OT converts 2-oxo-4-hexenedioate (5, Scheme 2) to 2-oxo-3-hexenedioate (7) through a dienol intermediate, known commonly as 2-hydroxymuconate (6) (17). Pro-1 plays a major role in this reaction, functioning as a general base catalyst. Its ability to function as a base at cellular pH is due to its lowered pK_a value of 6.4 (18). Arg¹¹ has also been identified as a critical residue in the 4-OT-catalyzed reaction (19, 20). It interacts with the C-6 carboxylate group and functions as an electron sink to draw electron density to the C-5 position thereby facilitating protonation at C-5. Likewise, Pro¹ (of the -subunit) and Arg¹¹ (of the -subunit) have been identified as critical residues in the CaaD-catalyzed reaction (4, 5). Pro¹ likely functions as a general acid catalyst (providing a proton at C-2 of 2) while Arg¹¹ may interact with the C-1 carboxylate group.

View larger version (7K): [in this window] [in a new window]   SCHEME 2

View larger version (27K): [in this window] [in a new window]   FIG. 3. Alignment of the amino acid sequence of MSAD with those of YrdN, YodA, 4-OT, and the -subunit of CaaD (CaaD1). Residues conserved throughout all sequences are shown in boldface. Dashes represent residues absent in other sequences. The sequence motif present in the N-terminal region of 4-OT and CaaD1 and in both the N- and C-terminal region of MSAD, is boxed.

Mutagenesis of Pro¹, Arg¹¹, and Arg⁷⁵—In order to investigate the importance of Pro¹, Arg¹¹, and Arg⁷⁵ to the mechanism of MSAD and to confirm further that MSAD is a member of the tautomerase superfamily, three single site-directed mutants were constructed in which Pro-1, Arg-11, and Arg-75 were replaced with an alanine (P1A, R11A, and R75A). DNA sequencing verified that only the intended mutations had been introduced into each mutant gene. The three mutants were produced in E. coli BL21(DE3) and purified to 95% homogeneity (as assessed by SDS-PAGE) as described for wild-type MSAD. The yields (in mg of homogeneous protein per liter of cell culture) of the P1A and R11A mutants were poor (4 mg), while the yield of the R75A mutant (8 mg) was moderate in comparison to wild type (15 mg). Mass spectral analysis of the individual mutants further confirmed the presence of the intended amino acid substitutions and showed that each of the mutants had undergone post-translational processing to remove the initiating methionine. It was further shown by gel filtration chromatography and non-denaturing PAGE (data not shown) that the native molecular mass for each mutant is comparable to that of wild type, indicating that the oligomeric association of the mutants was still intact.

The purified P1A mutant of MSAD showed an activity of 760 mU/mg of protein, which is 1.4% of the activity observed for the wild-type enzyme (Table I). This indicates that Pro¹ is indeed important for the MSAD-catalyzed decarboxylation reaction, although it is not absolutely essential for activity. Mutation of Arg⁷⁵ to alanine results in a mutant, which has 0.17% of the activity observed for wild type, whereas mutation of Arg¹¹ to alanine had no significant influence on activity (Table I). These results clearly indicate that of the two conserved arginines in the decarboxylase, only Arg⁷⁵ is essential for decarboxylase activity. The combination of these observations provides further evidence that MSAD is a member of the tautomerase superfamily and points to important roles for Pro¹ and Arg⁷⁵ in the mechanism of MSAD.

Mechanistic Possibilities for MSAD—Malonate semialdehyde is analogous to a -keto acid, for which decarboxylation mechanisms are well known. Based on a considerable body of literature precedence, three reasonable mechanisms for the decarboxylation of 3 can be formulated (Scheme 3) (23–27). All three involve polarization of the 3-carbonyl group coupled with the destabilization of the 1-carboxylate group. Decarboxylation results in the formation of an enolate species, which ketonizes to form product. In the first two mechanisms, a metal ion (Scheme 3A) or the formation of a Schiff base between the aldehyde and the enzyme (Scheme 3B) facilitates catalysis (23–25, 27). The metal-dependent decarboxylation of 3 is not favored for two reasons: the addition of metals does not stimulate activity and dialysis against buffer containing EDTA does not decrease activity. While these observations do not exclude metal ion catalysis, they argue against it. If a metal ion is involved, it is likely tightly bound to the enzyme. The hallmarks of the Schiff base mechanism are the inactivation of the enzyme in the presence of the substrate and NaBH₄ (or NaCNBH₃) and the incorporation of ¹⁸O isotope into product when the reaction is carried out in H₂¹⁸O (24, 27). Inactivation of the enzyme occurs because the intermediate Schiff base is reduced by the hydride to form a stable amine, which is now covalently attached to an active site residue thereby preventing further reaction. The incorporation of ¹⁸O results from hydrolysis of the Schiff base.

View larger version (14K): [in this window] [in a new window]   SCHEME 3

Irreversible Inhibition of MSAD—In order to determine whether an imine can form between substrate and enzyme, the enzyme was incubated with 3 and 4 (in separate reactions) and the mixture was treated with NaCNBH₃. Reduction of the Schiff base will "fix" the substrate to the enzyme and result in its inactivation (Scheme 4) (27). When MSAD was treated with NaCNBH₃ in the presence of either 3 or 4, enzymatic activity was almost completely lost (Table II). Exhaustive dialysis or gel filtration chromatography did not restore activity, indicative of covalent modification. Treatment of the enzyme with the substrate mixture or NaCNBH₃ alone did not result in the loss of enzymatic activity (Table II). Similarly, when the MSAD was incubated with acetoacetate, acetopyruvate, or succinic semialdehyde, and treated with sodium cyanoborohydride, the enzymatic activity was not affected (Table II). These observations suggest that a Schiff base can form between MSAD and 3 (or 4).

View larger version (10K): [in this window] [in a new window]   SCHEME 4

Identification of the Modified Residue by Mass Spectrometry—The inactivated protein samples were analyzed by ESI-MS in order to determine whether the mass is consistent with the mechanism shown in Scheme 4 and to ascertain whether single or multiple modifications had occurred. Mass spectral analysis of the MSAD control sample showed one major peak corresponding to a mass of 14,106 ± 1 Da. Mass spectral analysis of MSAD inactivated by 3 (in the presence of NaCNBH₃) after a 4-h incubation period showed three major peaks corresponding to masses of 14,134 ± 1 Da, 14,180 ± 1 Da, and 14,259 ± 1 Da. The first two masses (representing differences of + 28 Da and + 74 Da) are in near agreement with those expected (+ 28 Da and + 72 Da, respectively) for the enzyme modified by 4 or 3, (and reduced by NaCNBH₃), respectively (Scheme 4). Modification of MSAD by 4 likely results from decarboxylation of the Schiff base formed between 3 and MSAD or from the reaction between 4 and MSAD as it is generated by the MSAD-catalyzed decarboxylation of 3. This observation is consistent with the mass spectral analysis of the MSAD inactivated by 4 (in the presence of NaCNBH₃), which shows one major peak corresponding to a mass of 14,134 ± 1 Da.

The third species (corresponding to a mass of 14,259 Da) may represent MSAD modified by two molecules of 3. The identity of this signal cannot be assigned with certainty because the expected difference for a double modification is 148 Da compared with the observed difference of 153 Da. However, this signal appears only after prolonged incubation. Incubation of MSAD with 3 for shorter time periods (a 1- or 2-h period) leads to complete inactivation and generates two peaks: the major species has a mass of 14,134 Da (MSAD modified by 4 and reduced by NaCNBH₃) and the minor species has a mass of 14,180 Da (MSAD modified by 3 and reduced by NaCNBH₃).

These results show that incubation of MSAD with 3 in the presence of NaCNBH₃ leads to the covalent attachment of species with masses of 28 Da and 74 Da to the enzyme while the incubation of MSAD with 4 (in the presence of NaCNBH₃) leads to the covalent attachment of a species with a mass of 28 Da. In order to identify the site of modification, the two MSAD samples (inactivated by 3 and 4) and the control sample were digested with endoproteinase Glu-C (protease V-8), and the resulting peptide mixtures were analyzed by MALDI-MS. At pH 8.0, in 100 mM ammonium bicarbonate buffer, protease V-8 cleaves peptide bonds at the carboxylate side of glutamate and aspartate residues, with a preference for the former (28). There are seven glutamate and twelve aspartate residues in MSAD.

Mass spectral analysis of the peptide mixtures revealed incomplete digestion of all three V-8 protease-treated MSAD samples with proteolytic cleavage occurring predominantly at Asp⁶, Asp¹³, Asp²¹, Asp²⁹, Asp³⁷, Glu⁴⁹, Glu⁵³, Glu⁷⁷, Glu⁷⁸, Glu¹⁰⁸, and Glu¹²² (Table III). A comparison of the peaks of the MSAD samples (inactivated by 4 and 3 in the presence of NaCNBH₃) to those of the unmodified MSAD sample revealed a single modification by a species having a mass of 28 Da on the fragment Pro¹ to Asp¹³ (Table III).⁴ Analysis of the remaining peaks showed no modification of other fragments. Since the overlapping fragments Ile⁷ to Asp¹³ and Ile⁷ to Asp²¹ were not modified (Table III), it can be concluded that the site of modification is localized to the N-terminal fragment from Pro¹ to Asp⁶.

The most likely targets for alkylation within the six residue N-terminal fragment Pro¹ to Asp⁶ (PLLKFD) are Pro¹ and Lys⁴. To identify the amino acid residue modified by a species with a mass of 28 Da, the unmodified MSAD and the two modified peptides were subjected to MALDI-Post-source Decay (PSD) analysis (29). The PSD spectrum of the ion (m/z 1584.9) corresponding to the unlabeled peptide (Pro¹ to Asp¹³) displayed the characteristic immonium ion at m/z 70.1 (Table IV) resulting from Pro¹ and the N-terminal sequence-specific fragment ions a₂ and b₂, which result from the dipeptide, Pro-Leu. PSD analysis of the ions (m/z 1612.9) for the two modified peptides revealed an increase in mass of 28 Da for both the a₂ and b₂ fragment ions (Table IV). Because these fragment ions are generated by the dipeptide Pro-Leu, modification of Lys⁴ is excluded. Thus, only Pro¹ and Leu² remain as potential targets of alkylation. Further evidence implicating Pro¹ as the site of modification was provided by the presence of the immonium ions in the PSD spectra of the two modified peptides, with mass values consistent with the covalent attachment of a single species with a mass of 28 Da to the Pro¹ residue (Table IV).

Mechanistic Implications—The combination of sequence analysis, site-directed mutagenesis, and protein modification studies has identified MSAD as a new member of the tautomerase superfamily, in which Pro¹ and Arg⁷⁵ may play prominent roles in the mechanism. In view of these observations, it is tempting to speculate about possible mechanistic roles for these two residues. At first glance, the results of the protein modification studies and the apparent non-metal-dependent activity seem to point to a Schiff base mechanism. In such a mechanism, the positively charged imine facilitates decarboxylation (Scheme 3B). Subsequent hydrolysis of the Schiff base releases the enzyme and product. In order to be sufficiently nucleophilic at cellular pH to react with the C-3 carbonyl group (and eventually form the Schiff base), Pro¹ is likely to have a lowered pK_a value. If the pK_a is comparable to that observed for Pro¹ of 4-OT (6.4), then under the conditions of the NaCNBH₃ trapping experiments (pH 9), it can be estimated that 99.7% of the enzyme is in the correct protonation state to react with the carbonyl group.

To our knowledge, MSAD would be the first example of a decarboxylase that utilizes a secondary amine in a Schiff base. The formation of a Schiff base between the -ammonium group of lysine and substrate is well known in several enzymatic reactions including decarboxylation and aldol reactions (22, 23). Acetoacetate decarboxylase is the prototypical example of a decarboxylase that uses this strategy (24, 27, 30) while fructose-1,6-bisphosphate aldolase is a well-studied aldolase that proceeds through a Schiff base (22, 31). The only other reported example of an enzymatic reaction involving Schiff base formation between Pro¹ and substrate is the one catalyzed by formamidopyrimidine-DNA glycosylase (Fpg), which catalyzes the removal of 8-oxoguanine and formamidopyrimidines from oxidatively damaged DNA (32). Mechanistic and crystallographic studies have shown that the reaction proceeds through a Schiff base intermediate between the enzyme and DNA substrate (33, 34). Interestingly, replacement of Pro¹ in Fpg with a glycine produces a protein, which retains 5.6% of the activity observed for wild type (32). This result is consistent with the observation that the P1A mutant of MSAD retains some activity (1.4% of the wild-type activity). These mutagenesis results might suggest that Pro¹ is not crucial in the mechanism of both reactions. However, mutagenesis of the N-terminal group with any of the common amino acids (including glycine and alanine) produces a mutant that still displays a free amino group. Hence, unless a cyclopentane group is placed at the N-terminal group (35), some degree of activity might still be expected. The reduced activity is likely due to the fact that a less constrained primary amino acid with a different pK_a value now occupies the N-terminal position.

Since 3 is very reactive and may react with exposed amines (e.g. Pro¹), the results do not rule out a second mechanism in which the C-3 keto group of 3 is polarized by hydrogen bonding and/or electrostatic interactions, analogous to the mechanism proposed for methylmalonyl CoA decarboxylase (26). In this mechanism, Pro¹ would be charged and function as a general acid catalyst providing a hydrogen bond or by virtue of its positive charge. The proposed role of Pro¹ in this mechanism might preclude it from being modified under the conditions of the NaCNBH₃ trapping experiments. However, under these conditions (pH 9) 50% of the enzyme would be in the correct protonation state (assuming a pK_a of 9) to react with a carbonyl group. Reaction of Pro¹ with the carbonyl group (and subsequent reduction) will shift the equilibrium and place more of the enzyme in the correct protonation state to react with the carbonyl group. Hence, the results of the trapping experiments do not rule out such a mechanism. The observation that the P1A mutant of MSAD retains some activity does not argue against the mechanism. Again, an N-terminal group is still present.

The sequence analysis and the results of site-directed mutagenesis also implicate Arg⁷⁵ in the mechanism. In both 4-OT and CaaD, Arg¹¹ plays an important role in the mechanism by its interaction with the substrate's carboxylate group. By analogy, Arg⁷⁵ may interact with the carboxylate group of 3. In addition to assisting in substrate binding, this interaction may place the carboxylate group in a favorable orientation for decarboxylation. In order to achieve maximum orbital overlap, which minimizes the transition state energy for bond cleavage and facilitates decarboxylation, it has been proposed that the bond being cleaved should be maintained by the enzyme in a position that is perpendicular to the -orbitals of the imine bond (36). Arginine residues have been assigned this role in other decarboxylases, providing additional support for the proposed role of Arg⁷⁵ (37). The residual activity of the R75A mutant could suggest that another residue is involved in aligning the carboxylate group.

As noted in the beginning of this section, the mechanistic roles assigned to Pro¹ and Arg⁷⁵ are somewhat speculative and therefore tenuous. While the evidence points to their involvement in MSAD activity, it clearly remains possible that these residues function in some other mechanistic capacity. Without a crystal structure, it is also not clear whether the reduced activities of the P1A and R75A mutants are due to structural perturbations in addition to the absence of essential catalytic groups. Mutations of the corresponding residues in 4-OT did not result in major structural perturbations (8, 38). Crystallographic and mechanistic studies are currently underway to delineate the roles of Pro¹ and Arg⁷⁵ in the mechanism as well as to identify other mechanistic residues involved in the activity.

Evolutionary Implications—The common structural unit for the members of the tautomerase superfamily is the -- fold (39). The 4-OT monomer displays a single -- unit, which associates with another monomer to form a stable dimer. Three 4-OT dimers form the 4-OT hexamer, the active enzyme (15). The CHMI and MIF subunits are nearly twice as long as the 4-OT monomer and encode two -- motifs, resembling the 4-OT dimer. The CHMI and MIF subunits assemble into the active trimeric species, which are structurally homologous to the 4-OT hexamer.

Sequence analysis uncovered numerous 4-OT homologues, which were predicted to form either hexameric or dimeric structures constructed from the -- unit (15). Thus far, crystallographic analysis has confirmed these predictions for the hexameric 4-OT homologue from B. subtilis (designated YwhB) and the dimeric 4-OT homologue from E. coli (designated YdcE) (15). Further oligomerization of the YdcE dimer is precluded by the presence of a helical region.

CaaD represented yet another variation in the tautomerase superfamily using the -- structural unit as well as a new activity (4). CaaD is a heterohexamer, composed of three dimers, where each dimer consists of an - and -subunit. The subunits encode a -- motif. The observation that this motif has been used to make an enzyme that catalyzes a very different reaction, the dehalogenation of haloacrylates by hydration (5), is perhaps more significant as it demonstrates the catalytic versatility of the -- motif.

MSAD is the latest variant using the -- fold where the motif has been assembled into an enzyme that catalyzes a decarboxylation reaction.⁵ MSAD is also the first characterized member of a new enzyme family within the tautomerase superfamily, the MSAD family. The discovery of MSAD further underscores Nature's ability to "stitch together" combinations of the same simple structural unit to create different enzymatic activities. Moreover, the motif has been used to create two different enzymes that catalyze successive reactions in the same pathway. While these observations are intriguing and provocative, there is not yet a sufficient body of knowledge to discern how the active sites of these enzymes evolved to carry out such different reactions. Our ongoing structure function relationship studies of the tautomerase superfamily members will shed additional light on this question and may assist in the determination of physiological functions for the other MSAD family members including YodA and YrdN.

	FOOTNOTES

 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ290446 [GenBank] .

^* This work was supported by United States Public Health Service Grant GM 65324. The costs of publication of this article were defrayed in part by the payment of page charges. This 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. Tel.: 512-471-6198; Fax: 512-232-2606; E-mail: whitman{at}mail.utexas.edu.

¹ The abbreviations used are: CaaD, trans-3-chloroacrylic acid dehalogenase; Ap, ampicillin; CHMI, 5-carboxymethyl-2-hydroxymuconate isomerase; ESI-MS, electrospray ionization mass spectrometry; HPLC, high pressure liquid chromatography; LB, Luria-Bertani; MALDI-PSD, matrix-assisted laser desorption/ionization post-source decay; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MIF, macrophage migration inhibitory factor; MSAD, malonate semialdehyde decarboxylase; dNTPs, deoxynucleotide triphosphates; 4-OT, 4-oxalocrotonate tautomerase; DMSO-d₆, dimethyl-d₆ sulfoxide.

² The nucleotide sequence for the trans-3-chloroacrylate catabolic gene cluster, which includes the orf130 gene, has been deposited in the GenBank^TM database under Accession Number AJ290446 [GenBank] . In light of this work, the orf130 gene has been renamed msaD.

³ Although the estimated native molecular mass of the protein would suggest that it is a pentamer, preliminary crystallographic analysis of MSAD shows that the crystallographic asymmetric unit consists of a trimer (R. Dasgupta, G. J. Poelarends, M. L. Hackert, and C. P. Whitman, unpublished results). This observation suggests that MSAD behaves anomalously on a gel filtration column. Similar observations were made for 4-OT and the 4-OT homologue from E. coli designated YdcE (15).

⁴ In the mass spectrum of the intact protein (inactivated by 3 in the presence of NaCNBH₃), mass shifts of +28 Da and +74 Da are observed. However, the mass spectrum of the digested protein (inactivated by 3 in the presence of NaCNBH₃) shows only a mass shift of +28 Da. This discrepancy is likely due to the decarboxylation of the adduct during the MALDI deposition or in the ionization process.

⁵ MSAD is more accurately referred to as the first biologically relevant decarboxylase found in the tautomerase superfamily. D-Dopachrome tautomerase, which has a decarboxylase activity associated with it, is the first enzyme with decarboxylase activity reported for the tautomerase superfamily (40). However, the physiological relevance of the reaction as well as the substrate, D-Dopachrome, are not yet known.

	ACKNOWLEDGMENTS

 
Electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry was performed by the analytical instrumentation service core supported by Center grant ES 07784. We thank Professor Dick B. Janssen (Department of Biochemistry, University of Groningen, The Netherlands) for the kind gift of the cosmid pPS41 and for stimulating discussions. We also thank Dr. Maria D. Person (Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas, Austin, Texas) for expert assistance in analyzing the mass spectral data. Finally, we thank Steve D. Sorey (Department of Chemistry, The University of Texas) for expert assistance in acquiring the NMR spectra.

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