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Applied and Environmental Microbiology, August 2005, p. 4602-4609, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4602-4609.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of an Inducible Phenylserine Aldolase from Pseudomonas putida 24-1

Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan

Received 25 October 2004/ Accepted 6 March 2005

	ABSTRACT

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An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5' phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (K_m, 1.3 mM), L-erythro-3-phenylserine (K_m, 4.6 mM), L-threonine (K_m, 29 mM), and L-allo-threonine (K_m, 22 mM). In the reverse reaction, threo- and erythro- forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5'-phosphate.

	INTRODUCTION

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Much attention has been paid to 3-hydroxy-2-amino acids as components of antibiotics and immunosuppressants (5-7, 9, 18, 19, 32-34) and as a drug for Parkinson's disease therapy (21), and enzymatic syntheses of 3-hydroxy-2-amino acids with L- and D-threonine aldolases have been done extensively (5, 6, 9, 18, 19, 32-34).

Phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26) catalyzes the reversible conversion of L-threo- and L-erythro-3-phenylserine to benzaldehyde and glycine. Although the occurrence of phenylserine aldolase in animals has been reported previously (2), the enzyme has not been purified and characterized. Since serine hydroxymethyltransferase (SHMT) (27, 28, 30) and threonine aldolase (13-16) show the phenylserine aldolase activity, it has been thought that the phenylserine aldolase activity is due to SHMT or threonine aldolase. During the course of a study on microbial metabolism of DL-threo-3-phenylserine, we found a phenylserine aldolase activity in a soil bacterium identified as Pseudomonas putida 24-1. To use the enzyme for the synthesis of 3-hydroxy-2-amino acids, we purified the enzyme to homogeneity from the bacterium and obtained evidence that the enzyme is an inducible phenylserine aldolase but not SHMT and threonine aldolase.

This paper presents the first identification of phenylserine aldolase from P. putida 24-1 and its enzymologic characteristics as well as cloning and overexpression of its gene in Escherichia coli.

	MATERIALS AND METHODS

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Materials.
DL-threo-3-Phenylserine, DL-threo-3-hydroxynorvaline, L-threonine, D-threonine, L-allo-threonine, D-allo-threonine, L-serine, D-serine, and DL-3-hydroxyphenylethylamine were obtained from Sigma, St. Louis, MO. DEAE-cellulose was supplied by Serva, Heidelberg, Germany. A Mono Q HR10/10 column was obtained from Pharmacia, Uppsala, Sweden, and a YMC-Pack C₄ column was purchased from YMC, Kyoto, Japan. DL-erythro-3-Phenylserine was synthesized according to the method of Greenstein and Winitz (4). D-threo-3-Phenylserine was prepared from its DL isomer with phenylserine dehydratase (26). Other chemicals used were of analytical grade.

Medium and culture conditions.
The bacterium was cultured with a medium (pH 7.2) containing 1.0% peptone, 0.2% DL-threo-3-phenylserine, 0.2% K₂HPO₄, 0.2% KH₂PO₄, 0.2% NaCl, 0.01% MgSO₄ · 7H₂O, and 0.01% yeast extract. Large-scale cultivation was carried out in 2-liter flasks containing 500 ml of the medium at 30°C for 12 h on a reciprocal shaker. The cells were harvested by centrifugation and washed twice with 0.85% NaCl. E. coli clones were grown aerobically at 37°C for 20 h in 100 ml of a Luria-Bertani (LB) medium (1% peptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.2) supplemented with ampicillin (50 µg/ml) and isopropyl-ß-D-thiogalactopyranoside (IPTG; 100 µg/ml).

Enzyme assay.
The standard reaction mixture contained 10 µmol of DL-threo-3-phenylserine, 10 nmol of pyridoxal 5'-phosphate (PLP), 100 µmol of N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) buffer (pH 8.5), and enzyme in a final volume of 0.5 ml. In a blank, the enzyme was replaced with water. Incubation was performed at 30°C for 10 min. The reaction was stopped by the addition of 0.5 ml of 1 M HCl. The benzaldehyde formed was determined by the 2,4-dinitrophenylhydrazine method as follows. To the reaction mixture (0.55 ml), 0.15 ml of 0.1% 2,4-dinitrophenylhydrazine solution in 2 M HCl was added, and the mixture was incubated at 30°C for 20 min. Three milliliters of 99% ethanol and 0.85 ml of 3 M NaOH were then added to the mixture, and the color intensity of 2,4-dinitrophenylhydrazone of benzaldehyde was measured at 475 nm with a Shimadzu UV-1200 spectrophotometer. One unit of the enzyme was defined as the amount that catalyzed the formation of 1 µmol of benzaldehyde per minute in the reaction. Specific activity was expressed as units per milligram of protein. Protein was measured by the method of Lowry et al. (20) with bovine serum albumin as the standard. Concentrations of the purified enzyme were determined from the absorbance at 280 nm. The absorption coefficient (A_{1 cm}^1% cm at 280 nm = 11.9) was estimated by absorption and dry weight determinations.

The threonine aldolase and allo-threonine aldolase activities were measured by estimation of acetaldehyde with yeast alcohol dehydrogenase. The formation of L-3-phenylserine from benzaldehyde and glycine was examined as follows. The reaction mixture (0.5 ml), containing 5 µmol of benzaldehyde, 50 µmol of glycine, 10 nmol of PLP, 100 µmol of Tris-HCl buffer (pH 7.5), and enzyme, was incubated at 30°C for 10 min. After termination of the reaction by the addition of 0.1 ml of 25% trichloroacetic acid, the mixture was centrifuged. The pH of the supernatant was adjusted to 5.0 and filled up to 5.0 ml with a 0.5 mM CuSO₄ solution. A sample (50 µl) was analyzed by high-performance liquid chromatography (HPLC) with a TSK gel Enantio L1 column (0.46 by 25 cm) (24).

Purification of phenylserine aldolase.
All procedures were performed at 0 to 5°C, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer (pH 7.2) containing 0.01% 2-mercaptoethanol, 1 mM EDTA disodium salt (EDTA), and 50 µM PLP was used in the purification procedures unless otherwise stated.

Washed cells (289 g, wet weight) were suspended in 300 ml of 0.1 M buffer (pH 7.2) containing 0.02% 2-mercaptoethanol, 2 mM EDTA, and 50 µM PLP and disrupted by sonication. The supernatant obtained by centrifugation was dialyzed overnight against 10 mM buffer (pH 7.2) and used as the crude extract.

The crude extract was brought to 40% ammonium sulfate saturation. After being kept for 1 h, the precipitate was removed by centrifugation. To the supernatant, solid ammonium sulfate was added to 60% saturation and left for 1 h. The precipitate collected by centrifugation was dissolved in 10 mM buffer (pH 7.2) and dialyzed overnight against the same buffer. The enzyme solution was applied to a DEAE-cellulose column (4.5 by 38 cm) equilibrated with 10 mM buffer (pH 7.2). After the column had been washed thoroughly with the buffer and then with the buffer supplemented with 0.1 M KCl, the enzyme was eluted with the buffer supplemented with 0.15 M KCl. The active fractions were combined and concentrated by ultrafiltration with a Pellicon Labocassete (Nihon Millipore, Tokyo, Japan) equipped with PT filters. The enzyme solution was dialyzed overnight against 10 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol, and 50 µM PLP and applied to a hydroxyapatite column (1.7 by 18 cm) equilibrated with 10 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol and 50 µM PLP. After the column had been washed with the buffer, the enzyme was eluted with 20 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol and 50 µM PLP. The active fractions were concentrated with an Amicon 200 ultrafiltration unit (Amicon, Lexington, MA) equipped with a PM-10 membrane filter and dialyzed against 10 mM TES buffer (pH 7.2). The enzyme solution was applied to a DEAE-cellulose column (1.7 by 19 cm) equilibrated with 10 mM TES buffer (pH 7.2). After the column had been washed thoroughly with the buffer and then with the buffer supplemented with 0.1 M KCl, the enzyme was eluted with the buffer supplemented with 0.15 M KCl. The active fractions were pooled and concentrated by ultrafiltration with the Pellicon Labocassete. The enzyme solution was dialyzed overnight against 10 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol and 50 µM PLP. The enzyme solution was applied to a hydroxyapatite column (1.7 by 18 cm) equilibrated with 10 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol and 50 µM PLP. After the column had been washed with the buffer, the enzyme was eluted with 30 mM potassium phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol and 50 µM PLP. The active fractions were concentrated with the Amicon 200 ultrafiltration unit and dialyzed against 10 mM TES buffer (pH 7.2). The enzyme solution was applied to a Mono Q HR10/10 anion-exchange column (1.0 by 10 cm) equilibrated with 10 mM buffer (pH 7.2). The column was equipped with a Pharmacia fast protein liquid chromatography system and developed at room temperature at a flow rate of 1.0 ml/min with a 50-min linear gradient of KCl (0.2 to 0.35 M) in the same buffer. The active fractions were concentrated with the Amicon 200 ultrafiltration unit, dialyzed against 10 mM buffer (pH 7.2), and stored at –20°C in the presence of 30% glycerol.

Determination of molecular mass.
The molecular mass was estimated by gel filtration on a TSK gel G3000SW column (0.75 by 60 cm) at a flow rate of 0.7 ml/min with 0.1 M potassium phosphate buffer (pH 7.0) containing 0.2 M NaCl. A calibration curve was made with the following proteins (Oriental Yeast Co., Osaka Japan): yeast glutamate dehydrogenase (290 kDa), pig heart lactate dehydrogenase (142 kDa), yeast enolase (67 kDa), yeast adenylate kinase (32 kDa), and horse cytochrome c (12.4 kDa). The molecular mass of the subunit was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12), using the following marker proteins (Nacalai Tesque, Kyoto, Japan): rabbit muscle myosin (200 kDa), ß-galactosidase (116 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa).

Determination of the PLP content.
The PLP content of the enzyme was determined by the phenylhydrazine and cyanohydrin methods. For the phenylhydrazine method, the enzyme (20 nmol) was kept at 37°C for 30 min in the presence of 0.2 N HCl to release the bound cofactor and the amount of free PLP was determined by using the method of Wada and Snell (35). For the cyanohydrin method, the enzyme sample (2 nmol) was hydrolyzed with a 55 mM H₂SO₄ solution at 120°C for 5 h and analyzed by the method of Bonavita (1).

Isolation of peptides obtained by tryptic digestion of the enzyme.
The purified enzyme (1 nmol) was dialyzed against water and lyophilized. The protein was dissolved in 20 µl of 0.4 M ammonium bicarbonate buffer (pH 8.0) containing 8 M urea, incubated at 37°C for 30 min, and then diluted with 60 µl of water. Trypsin was added to the solution in a 1:50 (mol/mol) ratio of protease to substrate. Digestion was performed at 37°C for 24 h. The peptides were separated on a Shimadzu HPLC system equipped with a YMC-Pack C₄ column using a solvent system of 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing 0.07% trifluoroacetic acid (solvent B). A 90-min linear gradient from 5 to 50% solvent B was used to elute peptides at a flow rate of 1.0 ml/min. The absorbance at 210 nm of the effluent was continuously monitored. The peptides were isolated and lyophilized.

Isolation of C-terminal peptide of the enzyme.
The C-terminal peptide (P₆) was obtained with a Shimadzu CTFF-1 automatic C-terminal fragment fractionator after digestion of the enzyme with lysyl endopeptidase (3).

Amino acid sequence analysis.
The amino acid sequence was determined with an Applied Biosystems model 492 protein sequencer linked with a phenylthiohydantoin derivative analyzer.

Cloning and sequencing of the gene coding for phenylserine aldolase.
The chromosomal DNA of P. putida 24-1 was prepared by the method of Saito and Miura (29). Sense (S) and antisense (A) primers were designed on the basis of the amino acid sequences of the peptide P₁, which was obtained from the tryptic digest of the enzyme, and the C-terminal peptide (P₆), respectively. Sequences were 5'-GGGAATTCAGGCGGGCCCGTATGGCACCGACGA-3' (primer S containing an EcoRI site [underlined]) and 5'-GGGGATCCCCAGCGGGTCGTGATAGAAGCCGAA-3' (primer A containing a BamHI site [underlined]). PCR was done with AmpliTaq DNA polymerase (Perkin-Elmer, Boston, MA). The nucleotides of the amplified DNA fragment (880 bp) were sequenced with an Applied Biosystems 373 DNA sequencer and a DNA sequencing kit (ABI Prism dye terminator cycle sequencing ready reaction kit; Perkin Elmer). From the sequence obtained, oligonucleotide probes U (5'-TGATGACCGTCGACGGCCCG-3') and C (5'-CGGCCTTCAGCAGCGCATCGA-3') were prepared and labeled with ³²P with a DNA labeling kit (MEGALABEL; Takara Shuzo), T4 polynucleotide kinase, and [-³²P]ATP. The chromosomal DNA of P. putida 24-1 was digested with various restriction enzymes at 37°C overnight and electrophoresed on a 0.7% agarose gel. The DNA fragments were transferred onto a nylon membrane (GeneScreen Plus; DuPont Company, Wilmington, DE) by blotting with a Pharmacia VacGene XL instrument. The blotted DNAs were hybridized with radioactive probes U and C. Hybridization was done at 50°C and monitored by autoradiography of the blotted DNA.

Cloning of the phenylserine aldolase gene.
The SphI fragments (6.7 kb), which hybridized with the radioactive probes U and C, were collected from the 0.7% agarose gel by centrifugation in a SUPREC-01 tube and ligated into the SphI site of pUC19. The ligated DNA was inserted into E. coli JM109 cells. Transformants were selected on LB agar plates containing ampicillin (50 µg/ml), IPTG (120 µg/ml), and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal; 100 µg/ml). A positive clone was selected from the transformants by colony hybridization with the radioactive probe U. A plasmid isolated from the positive clone was named pPSA. The sequence of the gene for the enzyme in the plasmid pPSA was analyzed in both directions.

To obtain a high expression strain, the structural gene for the enzyme was amplified by PCR with Ex Taq DNA polymerase and with a sense primer containing a EcoRI site (underlined) (5'-GGGAATTCGACCATCAGGCGAGCGTCAA-3') and an antisense primer containing a HindIII site (underlined) (5'-GGAAGCTTCCAGAGCGAGCACAGCCGCCAC-3'). The amplified DNA fragment was ligated into the EcoRI-HindIII site of pKK223-3. The constructed plasmid was named pKPSA.

Purification of recombinant enzyme from E. coli cells.
To purify the recombinant enzyme from E. coli cells, all procedures were performed at 0 to 5°C, and the buffer containing 0.01% 2-mercaptoethanol, 1 mM EDTA, and 50 µM PLP was used, unless otherwise stated.

E. coli JM109 cells carrying pKPSA were cultured aerobically at 37°C for 24 h in LB medium containing ampicillin (50 µg/ml) and IPTG (120 µg/ml). The cells (8 g, wet weight) were suspended in 30 ml of 0.1 M TES buffer (pH 7.2) containing 0.02% 2-mercaptoethanol, 2 mM EDTA, and 50 µM PLP and disrupted by sonication for 10 min. The supernatant obtained by centrifugation was dialyzed overnight against 10 mM TES buffer (pH 7.2) and used as the crude extract. To the crude extract, solid ammonium sulfate was added to 40% saturation with stirring. After 1 h, the precipitate was collected by centrifugation, dissolved in 10 mM TES buffer (pH 7.2), and dialyzed overnight against the same buffer. The enzyme solution was loaded onto a DEAE-Toyopearl column (3 by 15 cm) equilibrated with 10 mM TES buffer (pH 7.2). After the column had been washed with 10 mM TES buffer (pH 7.2) containing 0.1 M KCl, the enzyme was eluted with the buffer containing 0.125 M KCl. The active fractions were collected, concentrated with an Amicon PM-10 membrane, and dialyzed overnight against 10 mM potassium phosphate butter (pH 7.2). The enzyme solution was applied to a hydroxyapatite column (2 by 10 cm) equilibrated with 10 mM potassium phosphate butter (pH 7.2). After the column had been washed with 50 mM potassium phosphate buffer (pH 7.2), the enzyme was eluted with 0.1 M potassium phosphate buffer (pH 7.2). The active fractions were collected, concentrated with an Amicon ultrafiltration unit with a PM-10 membrane, and stored at –20°C in the presence of 50% glycerol until use.

Site-directed mutagenesis.
Mutant enzymes, K213Q and K238Q, were prepared according to the method of Kunkel et al. (11). The substitutions were confirmed by DNA sequencing. The mutant enzymes were produced in E. coli JM109 cells and purified by the same procedure as that used for the recombinant enzyme.

Nucleotide sequence accession number.
The nucleotide sequence data have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession number AB191192.

	RESULTS

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Isolation of a bacterium having an inducible phenylserine aldolase.
We isolated 22 strains of soil bacteria that could use L-threo-3-phenylserine as the sole carbon and nitrogen source. Crude extracts of 8 strains showed phenylserine aldolase activity. Among these bacteria, strain 24-1 had the highest activity. Strain 24-1 was a gram-negative, aerobic, motile, and nonsporing bacterium. It was a rod-shaped cell with polar flagella. The degradation of protocatechuate was of the ortho type. The 16S ribosomal DNA sequence of the strain showed similarity of 99.4% to that of Pseudomonas putida. Therefore, we named this strain P. putida 24-1. We investigated the culture conditions for aldolase production using P. putida 24-1. The enzyme production was induced by the addition of 0.5% DL-threo-3-phenylserine into a 1% peptone medium. However, L-threonine, D-threonine, L-serine, D-serine, and glycine did not affect enzyme production. The highest specific and total activities were obtained by cultivating the cells in a 1% peptone medium containing 0.5% DL-threo-3-phenylserine at 30°C for 12 h on a reciprocal shaker.

Purification and molecular mass of phenylserine aldolase.
A typical summary of the purification procedure is shown in Table 1. The enzyme was purified 440-fold with a 10% yield from the crude extract. The purified enzyme showed a single band on SDS-PAGE (Fig. 1A). The molecular mass of the enzyme was estimated to be about 210 kDa by gel filtration on a TSK gel G3000SW column. The molecular mass of the subunit was calculated to be 38 kDa by SDS-PAGE. These results suggest that the enzyme is a hexamer composed of identical subunits.

View larger version (80K): [in this window] [in a new window]   FIG. 1. SDS-PAGE of the enzyme purified from P. putida 24-1 (A) and the recombinant enzyme (B). (A) Lane M, molecular marker proteins; lane 1, purified enzyme (5 µg of protein). (B) Lane M, molecular marker proteins; lane 2, cell extract of E. coli JM109 (10 µg); lane 3, cell extract of the E. coli JM109/pKPSA clone (10 µg); lane 4, enzyme purified from E. coli JM109/pKPSA clone cells (1 µg).

Substrate specificity.
The enzyme was specific for the L form of threo- and erythro-3-phenylserine, and the D enantiomers were not substrates. When DL-threo-3-phenylserine or DL-erythro-3-phenylserine was used, analysis of the reaction mixture by HPLC with a TSK gel Enantio L1 column proved that only the L form was a substrate. The addition of D-threo-3-phenylserine (10 µmol) to the standard reaction mixture did not affect the enzyme activity, showing that the D form of 3-phenylserine did not inhibit the reaction. L-Threonine and L-allo-threonine served as substrates, though only slightly (Table 2). The enzyme did not act on L-serine, D-serine, D-threonine, D-allo-threonine, DL-3-hydroxyphenylethylamine, DL-2-amino-3-phenyl-n-butanoate, L-3-phenyllactate, DL-homoserine, D-glucosaminate, D-mannosamine, D-glucosamine, or D-galactosamine. No SHMT activity of the enzyme was detected.

Reversibility of the enzyme reaction.
The enzyme catalyzed the reverse reaction, synthesis of L-3-phenylserine from a mixture of glycine and benzaldehyde. Analysis of the reaction mixture by HPLC with a TSK gel Enantio L1 column revealed that the products were a mixture of L-threo-3-phenylserine and L-erythro-3-phenylserine (2:1) (Fig. 2). The optimum pH for the synthesis of 3-phenylserine isomers from glycine and benzaldehyde was about 7.5. The best result was obtained at the ratio of glycine to benzaldehyde of 10:1 (mol/mol) at pH 7.5, though the K_m values for glycine and benzaldehyde could not be determined. The synthetic reaction rate (4.3 µmol/min/mg of protein), however, was 70-fold less than the cleavage reaction rate of L-threo-3-phenylserine (300 µmol/min/mg of protein).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Formation of L-threo-3-phenylserine and L-erythro-3-phenylserine from glycine and benzaldehyde. The reverse reaction was carried out as described in Materials and Methods. The reaction mixture was analyzed with a TSK gel Enantio L1 column. (A) Standard 3-phenylserine; (B) reaction mixture. Peaks: 1, D-threo-3-phenylserine; 2, D-erythro-3-phenylserine; 3, L-threo-3-phenylserine; 4, L-erythro-3-phenylserine.

View larger version (28K): [in this window] [in a new window]   FIG. 3. Absorption spectrum of the enzyme. Absorption spectra were taken with a Hitachi 220A spectrophotometer. The enzyme (1 mg/ml) in 10 mM TES buffer (pH 7.2) was used. Curve A, native enzyme; curve B, NaBH4-reduced enzyme; curve C, apoenzyme.

View larger version (91K): [in this window] [in a new window]   FIG. 4. Nucleotide sequence of the structural gene encoding the phenylserine aldolase from P. putida 24-1 and the deduced amino acid sequence. The amino acid sequences that were determined by Edman degradation of the peptides isolated from the tryptic digest of the enzyme are indicated by single underlining. The putative ribosome binding sequence is shown by bold underlining, and the stop codon is indicated with an asterisk.

Purification of the recombinant enzyme from E. coli clone cells.
The recombinant enzyme was purified from E. coli cells harboring pKPSA by three steps with a 20% yield as described in Materials and Methods. The purified enzyme showed a single band on SDS-PAGE (Fig. 1B). The enzymologic properties, such as optimum pH (pH 8.5), substrate specificity, and an apparent K_m value (1.3 mM) for L-threo-3-phenylserine, of the recombinant enzyme were almost the same as those of the enzyme purified from P. putida 24-1. The recombinant enzyme and E. coli cloned cells catalyzed the reverse reaction, as did the P. putida enzyme.

Identification of the active-site lysine residue.
To identify the PLP-binding lysine residue of the enzyme, two mutant enzymes, K213Q and K238Q, were constructed and purified as described in Materials and Methods. The K213Q mutant enzyme showed no detectable activity, and the K238Q mutant enzyme showed about 92% activity of the wild-type enzyme. The K238Q enzyme showed an absorption maximum at 420 nm, but the K213Q enzyme did not show the absorption maximum at 420 nm. These suggest that a Schiff base linkage between PLP and the -amino group of the active-site lysine residue in the wild-type enzyme is not in the K213Q enzyme.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
P. putida 24-1 could utilize L-threo-3-phenylserine as a sole carbon and nitrogen source. The bacterium produces phenylserine aldolase, which catalyzes the cleavage of L-threo-3-phenylserine to yield glycine and benzaldehyde. The enzyme production was induced by L-threo-3-phenylserine but not by L-threonine and L-allo-threonine, suggesting that the enzyme functions in the degradation of L-threo-3-phenylserine. We have purified and characterized phenylserine aldolase from P. putida 24-1 to compare its properties with those of SHMT and threonine aldolases. Phenylserine aldolase from P. putida 24-1 was a hexamer, as was the threonine aldolase from Candida humicola (10). Although L-allo-threonine aldolase (13), the low-specificity L-threonine aldolase (14-17), and SHMT (25) acted on L-threo-3-phenylserine and L-erythro-3-phenylserine, these enzymes were tetramers, except for the E. coli SHMT (a dimer) (31). Good substrates of the phenylserine aldolase from P. putida 24-1 were L-threo-3-phenylserine and L-erythro-3-phenylserine (Table 2). The enzyme did not show SHMT activity and was not inhibited by D-alanine, though SHMTs were inhibited by D-alanine (25, 27, 28, 30). The reactivities (k_cat/K_m) of the phenylserine aldolase from P. putida 24-1 for L-threo-phenylserine and L-erythro-phenylserine were much higher than those of L-allo-threonine aldolase (13) and low-specificity threonine aldolases (14-17). L-allo-Threonine aldolase did not act on L-threonine (13). Threonine aldolase from C. humicola was activated by K⁺ and NH₄⁺ (10). The enzyme from P. putida 24-1, however, was not activated by these ions. These results described above show that the enzyme from P. putida 24-1 is different from L-allo-threonine aldolase (13), L-threonine aldolases (14-17), and SHMTs (27, 28, 30, 31).

The amino acid sequence of the enzyme from P. putida 24-1 was similar to those of other amino acid aldolases (Fig. 5). The percentages of identical amino acids of the enzyme compared with the low-specificity L-threonine aldolases from Pseudomonas sp. strain NCIMB 10558 (16), E. coli (15), Thermotoga maritima (8), Candida albicans (23), and Saccharomyces cerevisiae (17) and with the L-allo-threonine aldolase from Aeromonas jandaei (13) were estimated to be 44, 22, 22, 21, 21, and 21%, respectively. Y40, H94, H139, H178, R183, K213, K238, and R328 were conserved in these enzymes (Fig. 5).

View larger version (109K): [in this window] [in a new window]   FIG. 5. Linear alignment of amino acid sequences of amino acid aldolases. Pp, phenylserine aldolase from P. putida 24-1; Ps, low-specificity L-threonine aldolase from Pseudomonas sp. (16); Ec, low-specificity L-threonine aldolase from E. coli (15); Tm, low-specificity L-threonine aldolase from Thermotoga maritima (8); Ca, low-specificity L-threonine aldolase from Candida albicans (23); Sc, low-specificity L-threonine aldolase from Saccharomyces cerevisiae (17); Aj, L-allo-threonine aldolase from Aeromonas jandaei (13). Gray shading indicates residues identical among the seven sequences. The asterisk shows the lysine residue forming a Schiff base with PLP.

It is assumed that enzymes showing the aldolase activity for 3-hydroxy-2-amino acids, including threonine aldolase (8), possess a general base which will interact with the 3-hydroxy group at the active site. In the phenylserine aldolase, the affinity for the threo form of the substrate amino acid was about three times higher than that for the erythro form, suggesting that L-threo-3-phenylserine is a most suitable substrate for the active-site structure. In addition, the fact that, in the reverse reaction (i.e., the synthesis of 3-phenylserine from glycine and benzaldehyde), the yield of the threo form was about twofold higher than that of the erythro form probably reflects the binding property of the general base to the 3-hydroxy group. The identification and alteration (or modification) of the general base may lead to the improvement of enzymatic 3-phenylserine synthesis. For example, the engineered enzyme and the enzyme-overproducing E. coli cloned cells have potential for the synthesis of useful L-threo-3-phenylserine and its derivatives such as L-threo-3-[4-(methylthio)-phenylserine], which is a key intermediate for the synthesis of the antibiotics florfemicol and thiamphenicol (19). Resolution of the tertiary structure of the useful enzyme and especially detailed analyses of the region interacting with the functional groups on the three positions of the substrate will provide insight into developing strategies for enzyme engineering and application.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan. Phone: 81 88 864 5187. Fax: 81 88 864 5200. E-mail: hmisono{at}cc.kochi-u.ac.jp.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

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