Strains and plasmids.
The strains and plasmids used in this study are listed in Table . S. paucimobilis
SYK-6 was grown at 30°C in W minimal salt medium (33
) containing 10 mM vanillate or syringate or in Luria-Bertani (LB) medium (1
Strains and plasmids used in this study
Preparation of substrate.
PDC and OMA were prepared as described earlier (28
). CHA was prepared by incubating 1 mmol of OMA with 500 U of purified OMA hydratase for 5 min (11
). Electrospray-ionization mass spectrometry (ESI-MS) analysis revealed that the m/z
201 showing [M-H]−
of OMA (where M is a molecular ion of OMA) was completely converted into m/z
219, indicating [M-H]−
of CHA by LigJ. Then, the reaction product of OMA catalyzed by purified LigJ was used as a substrate.
DNA manipulations and nucleotide sequencing.
DNA manipulations were carried out essentially as described in references 1
. A Kilosequence kit (Takara Shuzo Co., Ltd., Kyoto, Japan) was used to construct a series of deletion derivatives, whose nucleotide sequences were determined by the dideoxy termination method with an ALFexpress DNA sequencer (Pharmacia Biotech, Milwaukee, Wis.).
A Sanger reaction (39
) was carried out by using the Thermosequenase fluorescence-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Sequence analysis and homology alignment were carried out with the GeneWorks programs (IntelliGenetics, Inc., Mountain View, Calif.). The DDBJ database was used for searching homologous proteins.
According to the method of Maruyama (21
), a coupled assay was used for CHA aldolase. The decrease in the absorbance at 340 nm derived from the oxidation of NADH (340
= 6.6 × 103
; pH 8.0) in a reaction mixture containing 200 μM CHA, 140 μM NADH, coupled enzymes (30 U of lactate dehydrogenase and malate dehydrogenase), 1 mM MgCl2
, and a suitable aliquot of LigK was measured in 0.1 M Tris-acetate buffer (pH 8.0). The enzyme reaction was carried out at 30°C in a cuvette. One unit of enzyme activity is defined as that causing the oxidation of 2 μmol of NADH/min in this assay. Specific activity was expressed as units per milligram of protein. Oxaloacetate decarboxylase activity was determined by measuring the decrease in absorbance at 340 nm derived from the oxidation of NADH. The 1-ml reaction mixture contained 200 μM oxaloacetate, 140 μM NADH, 30 U of lactate dehydrogenase, 1 mM MgCl2
, and LigK enzyme in 0.1 M Tris-acetate buffer (pH 8.0). One unit of enzyme activity is defined as that causing the oxidation of 1 μmol of NADH/min in this assay. Under these conditions, the spontaneous oxaloacetate decarboxylase activity was detected (0.01 U). This spontaneous activity was subtracted from the raw data of oxaloacetate decarboxylase activity of LigK. Specific activity was expressed as units per milligram of protein. The Km
values were obtained from the Hanes-Woolf plots. The inhibition constant (Ki
) for oxaloacetate was determined from the Dixon plot. These kinetic constants were expressed as means from at least three independent experiments.
Enzyme purification was performed according to the method described below by using a BioCAD700E apparatus (PerSeptive Biosystems, Framingham, Mass.).
(i) Preparation of cell extract.
Escherichia coli BL21(DE3) harboring pETK was grown in 100 ml of LB medium containing 100 mg of ampicillin/liter. Expression of ligK was induced for 4 h at 37°C by the addition of isopropyl-β-d-thiogalactopyranoside (final concentration, 1 mM) when the turbidity of the culture at 660 nm reached 0.5. Cells were harvested by centrifugation and resuspended in 20 mM Tris-HCl buffer (pH 8.0) (buffer A). The cells were broken by two passages through a French pressure cell. The cell lysate was centrifuged at 15,000 × g for 15 min. Streptomycin (final concentration, 1% [wt/vol]) was added to the supernatant, which was centrifuged again at 15,000 × g for 15 min to remove nucleic acids. The supernatant was recovered and then centrifuged again at 170,000 × g for 60 min at 4°C. The crude extract was obtained after concentration by ultrafiltration using a minicon B15 (Amicon, Beverly, Mass.).
(ii) POROS PI anion-exchange chromatography.
The crude extract was applied to a POROS polyethyleneimine (PI) column (7.5 by 100 mm) (PerSeptive Biosystems) previously equilibrated with buffer A. The enzyme was eluted with 88 ml of linear gradient of 0 to 0.5 M NaCl. The CHA aldolase was eluted at approximately 0.20 M.
(iii) POROS HQ anion-exchange chromatography.
The fractions containing CHA aldolase activity eluted from a PI column were pooled, desalted, and concentrated by ultrafiltration using a minicon B15. The resulting solution was applied to a POROS quaternized polyethyleneimine (HQ) column (4.6 by 100 mm; PerSeptive Biosystems) previously equilibrated with buffer A. The enzyme was eluted with 33 ml of a linear gradient of 0 to 0.5 M NaCl. The fractions containing CHA aldolase activity that eluted at approximately 0.30 M were pooled.
(iv) POROS PE hydrophobic-interaction chromatography.
The fractions containing CHA aldolase activity eluted from an HQ column were pooled, desalted, and concentrated. Ammonium sulfate was added to the enzyme solution to a final concentration of 2 M. After centrifugation at 15,000 × g for 10 min, the supernatant was recovered and applied to a POROS phenylether (PE) column (4.6 by 100 mm) (PerSeptive Biosystems) equilibrated with buffer B (buffer A containing 2 M ammonium sulfate). The enzyme was eluted with 25 ml of a linear gradient of 2.0 to 0 M ammonium sulfate. The fractions containing CHA aldolase activity that eluted at approximately 1.3 M were pooled, desalted, and concentrated as described above. Glycerol was added to a final concentration of 10%, and the purified enzyme was stored at −80°C until use.
The protein concentration was determined by the method of Bradford (2
). The purity of the enzyme preparation was examined by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-15% PAGE) (17
). The molecular mass of the native enzyme was estimated by Superdex200 HR10/30 (Pharmacia Biotech) gel filtration column chromatography using a BioCAD700E apparatus. Elution was performed with 50 mM potassium phosphate buffer (pH 7.0) containing 0.15 M NaCl at a flow rate of 0.8 ml/min. The molecular weight was estimated on the basis of calibration curve of reference proteins.
To determine the N-terminal amino acid sequence, the cell extract of E. coli BL21(DE3) harboring pETK was subjected to SDS-15% PAGE and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.). The area at 27 kDa was cut out and analyzed on a PPSQ-21 protein sequencer (SHIMADZU, Kyoto, Japan). The isoelectric point of LigK was determined by isoelectric focusing on an Ampholine PAG plate (pH 3.5 to 9.5; Pharmacia Biotech) using a model Multiphor II electrophoresis system (Pharmacia Biotech).
The substrate and the reaction products were detected and identified by gas chromatography (GC)-MS using model 5971A with an Ultra-2 capillary column (50 m by 0.2 mm; Agilent technologies, Palo Alto, Calif.) and ESI-MS using HP1100 series LC-MSD (Agilent technologies). The analytical conditions for GC-MS were the same as described previously (28
). In ESI-MS analysis, mass spectra were obtained by negative-mode ESI, with a needle voltage of −3.5 kV and a source temperature at 350°C. The sample was injected directly into the mass spectrometer; the water/methanol ratio was 90:10 (vol/vol), and the flow rate was 0.2 ml/min.
Identification of the reaction product.
200 μM CHA was incubated with purified LigK (0.5 μg) in 0.1 M Tris-acetate buffer (pH 8.0) containing 1 mM MgCl2 for 1 min or 5 min, the reaction mixture was diluted to 1/10 with 10 mM Tris-acetate buffer (pH 8.0), and the portion of mixture (5 μl) was injected into the ESI-mass spectrometer.
In the case of GC-MS analysis, the reaction product was acidified and extracted with ethylacetate, and then the extract was trimethylsilylated. The resultant trimethylsilylated derivatives were analyzed.
The metabolites of vanillate and syringate by the ligK insertion mutant (DLK) were analyzed. DLK cells grown in 10 ml of LB medium were washed with 0.1 M Tris-acetate buffer (pH 8.0). The cells were resuspended in the same buffer and incubated with 10 mM vanillate and 10 mM syringate for 12 h at 30°C. After centrifugation, the supernatant was diluted 20-fold with 10 mM Tris-acetate buffer (pH 8.0) and analyzed by ESI-MS as described above. On the other hand, the metabolites were extracted by ethylacetate, trimethylsilylated, and analyzed by GC-MS.
Disruption of orf1, ligK, ligR, and orf2.
The 4.0-kb Xho
I fragment carrying ligK
was cloned into pBluescript II SK(+) to generate pXS4, and it was digested with Ppu
MI for ligK
disruption or with Sal
I for orf1
disruption. The 1.2-kb Pst
I fragment containing the kanamycin resistance gene from pUC4K (47
) was inserted into the Ppu
MI or Sal
I site of the 4.0-kb Xho
I fragment to construct pXS4K and pXS4K2, respectively. pXS4K and pXS4K2 were digested with Bam
HI and Kpn
I, and their inserts were cloned into pK19mobsacB
) to generate pLKD and pF1D, respectively. The 1.8-kb Cla
I fragment carrying ligR
was cloned into pUC19 to generate pCS18, and it was digested with Eco
47III. The kanamycin resistance gene was inserted into this Eco
47III site. The resultant plasmid, pCS18K, was digested with Kpn
I and Sac
I, and the insert containing the inactivated ligR
gene was cloned into pK19mobSacB
to generate pLRD. The 1.7-kb Pst
I fragment carrying orf2
was cloned into pUC19 to generate pPS17, and it was digested with Sma
I. The kanamycin resistance gene was inserted into the Sma
I site. The resultant plasmid, pPS17K, was digested with Bam
HI and Kpn
I, and the insert containing the inactivated orf2
gene was cloned into pK19mobsacB
to generate pF2D.
Each of plasmids, pLKD, pF1D, pLRD, and pF2D was introduced into SYK-6 cells by electroporation, and the candidates for mutants were isolated as described previously (28
). To examine the disruption of each gene, Southern hybridization analysis was carried out. The total DNA of the candidates for ligK
, and orf2
mutants were digested with Pst
I, and those for orf1
were digested with Sma
I. The 1.2-kb Pst
I fragment carrying the kanamycin resistance gene, the 2.3-kb Pst
I fragment carrying ligR
, the 4.0-kb Xho
I fragment carrying orf1
, and the 1.7-kb Pst
I fragment carrying orf2
were labeled with the DIG system (Roche Diagnostics, Indianapolis, Ind.) and used as probes.
Reverse transcription (RT)-PCR.
Cells of S. paucimobilis SYK-6 were grown in W minimal salt medium containing 10 mM vanillate until they reached the turbidity at 660 nm of 0.5. Total RNA was prepared from 10 ml of culture by using RNeasy Mini columns (Qiagen Inc, Chatsworth, Calif.). To remove any contaminating genomic DNA, the RNA samples were incubated with 1 U of RNase-free DNase (Takara Shuzo Co., Ltd.) in 40 mM Tris-HCl (pH 7.9) containing 1 U of RNase inhibitor (Takara Shuzo Co., Ltd.), 10 mM NaCl, 10 mM CaCl2, and 6 mM MgSO4 for 30 min at 37°C. RT-PCR was carried out with a BcaBEST RNA PCR kit (Takara Shuzo Co., Ltd.). A cDNA library was obtained by an RT reaction using a hexanucleotide random priming mix. The cDNA was used as a template for subsequent PCRs with specific primers, which amplify the boundaries of ligK-orf1-ligI-lsdA and ligR-orf2-ligJ-ligA-ligB-ligC. The forward and reverse primers used were as follows: lsdA-forward (nucleotide positions from 1,363 to 1,383 in the 10.5-kb EcoRI fragment) and ligI-reverse (positions 1,924 to 1,944); ligI-forward (positions 2,528 to 2,548) and orf1-reverse (positions 2,999 to 3,019); orf1-forward (positions 3,489 to 3,509) and ligK-reverse (positions 3,740 to 3,760); internal ligR-forward (positions 4,613 to 4,533) and internal ligR-reverse (positions 5,215 to 5,235); ligR-forward (positions 5,770 to 5,790) and orf2-reverse (positions 6,476 to 6,496); internal orf2-forward (positions 5,843 to 5,863) and orf2-reverse; orf2-forward (positions 6,536 to 6,556) and ligJ-reverse (positions 6,943 to 6,963); ligJ-forward (positions 7,662 to 7,682) and ligA-reverse (positions 8,162 to 8,182); ligA-forward (positions 8,162 to 8,182) and ligB-reverse (positions 8,706 to 8,726); ligB-forward (positions 9,119 to 9,139) and ligC-reverse (positions 9,598 to 9,608); internal ligC-forward (positions 9,609 to 9,629) and internal ligC-reverse (positions 10,098 to 10,118). Control samples in which reverse transcriptase was omitted in RT-PCR and in which genomic DNA was used as a template in PCRs were run in parallel with RT-PCRs.
Nucleotide sequence accession number.
The nucleotide sequence reported in this paper was deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB073227.