JOURNAL OF BACTERIOLOGY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Jan. 1999, p. 55–62Vol. 181, No. 1
Genetic and Biochemical Characterization of a 2-Pyrone-4,6-
Dicarboxylic Acid Hydrolase Involved in the Protocatechuate
4,5-Cleavage Pathway of Sphingomonas paucimobilis SYK-6
EIJI MASAI,1SHOUJI SHINOHARA,1HIROFUMI HARA,1SEIJI NISHIKAWA,2
YOSHIHIRO KATAYAMA,3AND MASAO FUKUDA1*
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188,1New Products and
Technology Laboratory, Cosmo Research Institute, Satte, Saitama 340-0193,2and Graduate School of
Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Fuchu,
Received 31 July 1998/Accepted 19 October 1998
Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl
moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain.
Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and
it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a
10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K.
Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki.
J. Bacteriol. 172:2704–2709, 1990), we found the ligI gene encoding 2-pyrone-4,6-dicarboxylic acid (PDC)
hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and
4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and
consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI
gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be
a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The
optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the Kmvalues
for PDC and CHM are 74 and 49 ?M, respectively. LigI activity was inhibited by the addition of thiol reagents,
suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the
PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557–565, 1983) and Comamonas
testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol.
152:1154–1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete
loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid.
These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not
responsible for the growth of SYK-6 on syringic acid.
Protocatechuate (PCA) is one of the most important inter-
mediate metabolites in the bacterial pathways for various
phenolic compounds, including lignin, which is the most abun-
dant aromatic material in nature. Sphingomonas paucimobilis
SYK-6 is able to degrade a wide variety of dimeric lignin
compounds, including ?-aryl ether, biphenyl, and diarylpro-
pane (20). The resulting lignin degradation enzymes are ex-
pected to be useful tools for the utilization of lignin as biomass.
Dimeric lignin compounds with guaiacyl (4-hydroxy-3-me-
thoxyphenyl) moieties are converted into PCA by the action of
the various lignin degradation enzymes, including ?-etherase
(LigF and LigE) (20, 21), ring cleavage dioxygenase for biphe-
nyl (LigZ) (27), and demethylases for 5,5?-dehydrodivanillic
acid (LigX) (unpublished data) and vanillic acid (LigH) (23),
as well as side chain-cleaving enzymes. Thus, PCA is the key
intermediate metabolite in the lignin degradation pathway in
S. paucimobilis SYK-6, and the PCA metabolic pathway plays
a key role in lignin degradation by this strain. It is generally
known that the aromatic ring of PCA is opened in reactions
catalyzed by three kinds of dioxygenases: PCA 3,4-dioxygenase
(3,4-PCD) (5, 6, 40), PCA 4,5-dioxygenase (4,5-PCD) (24), and
PCA 2,3-dioxygenase (2,3-PCD) (38). Among these dioxygen-
ases, 3,4-PCD is the most commonly characterized enzyme,
and its three-dimensional structure has been elucidated (25).
The ?-ketoadipate pathway genes (pca genes), including that
for 3,4-PCD, have been characterized in detail in Acinetobacter
calcoaceticus, Pseudomonas putida, and Agrobacterium tumefa-
In the case of S. paucimobilis SYK-6, PCA is subjected to
ring cleavage by 4,5-PCD and metabolized through the PCA
4,5-cleavage pathway proposed by Kersten et al. (13). In the
PCA 4,5-cleavage pathway (Fig. 1), 4,5-PCD catalyzes 4,5-
cleavage of PCA to form 4-carboxy-2-hydroxymuconate-6-
semialdehyde (CHMS), which is nonenzymatically converted
into an intramolecular hemiacetal form and then dehydroge-
nated by CHMS dehydrogenase (19). The resulting intermedi-
ate, 2-pyrone-4,6-dicarboxylic acid (PDC) (15), is hydrolyzed
by PDC hydrolase to yield 4-oxalomesaconic acid (OMA) or its
tautomer, 4-carboxy-2-hydroxymuconic acid (CHM) (13, 16).
OMA is converted into 4-carboxy-4-hydroxy-2-oxoadipic acid
(CHA) by OMA hydratase (17). Finally, CHA is cleaved by
CHA aldolase to produce pyruvate and oxaloacetate (18, 36).
Each enzyme catalyzing the last four steps in Pseudomonas
ochraceae (15–19) has been purified and characterized. Addi-
tionally, the PDC hydrolase and CHA aldolase of Comamonas
* Corresponding author. Mailing address: Department of Bioengi-
neering, Nagaoka University of Technology, Kamitomioka, Nagaoka,
Niigata 940-2188, Japan. Phone: 81-258-47-9405. Fax: 81-258-47-9450.
testosteroni (13, 36) have been purified. However, little is
known about the genes encoding these four enzymes.
In this paper, we present the structure of the PDC hydrolase
gene and the biochemical properties of the gene product. The
actual role of PDC hydrolase in the degradation of model
lignin compounds by S. paucimobilis SYK-6 is also discussed.
MATERIALS AND METHODS
Strains and plasmids. The strains and plasmids used in this study are listed in
Table 1. S. paucimobilis SYK-6 was grown at 30°C in W minimal salt medium
(27) containing 0.2% model lignin compounds, including vanillic acid and syrin-
gic acid, and in LB medium (Bacto Tryptone, 10 g/liter; yeast extract, 5 g/liter;
NaCl, 5 g/liter).
Preparation of substrates. PDC was prepared from PCA by using cells of P.
putida PpY1100 harboring pVAD4, which conferred transformation activity from
PCA to PDC, but no PDC conversion activity. PCA appeared to be converted
into PDC by 4,5-PCD and CHMS dehydrogenase encoded in pVAD4.
PpY1100(pVAD4) was grown in 2 liters of W medium containing 0.2% succinate
and 25 mg of kanamycin/liter for 20 h at 28°C. Cells were harvested by centrif-
ugation, resuspended in 800 ml of W medium containing 10.4 mmol of PCA, and
incubated for 20 h at 28°C. A culture was acidified to pH 1 and centrifuged to
remove cells. Metabolites were extracted twice with 400 ml of ethyl acetate and
dried in vacuo. The residue was dissolved in water, acidified to pH 1, and kept at
4°C for 3 days. The resultant white crystals were recovered, washed with 2 N HCl,
and air dried. Gas chromatography and mass spectrometry (GC-MS) analysis of
the trimethylsilylated (TMS) derivative of the product was carried out. The gas
chromatogram showed a major peak at the retention time of 30.0 min. The mass
spectrum of this peak had a molecular ion (M) at m/z 328, which corresponded
to the expected molecular mass of the PDC TMS derivative. The product showed
an absorption maximum at 312 nm in 50 mM Tris-HCl buffer (pH 8.5). In 1 N
NaOH, the absorbance at 312 nm decreased and that at 353 nm increased. These
characteristics correspond to the features of PDC reported by Maruyama (15,
16). Thus, it was concluded that the product obtained was PDC. Finally, 8.9
mmol of PDC was obtained from 10.4 mmol of PCA.
The PDC hydrolysate which is the substrate for the PDC synthesis was pre-
pared according to the method of Maruyama (16). One millimole of PDC was
incubated in 0.057 N NaOH at room temperature for 3 h and then neutralized
with 0.5 N HCl. Vanillic acid and other chemicals were purchased from Tokyo
Kasei Kogyo Co. (Tokyo, Japan) or Wako Pure Chemical Industries (Osaka,
DNA manipulations and nucleotide sequencing. DNA manipulations were
carried out essentially as described elsewhere (1, 29). A Kilosequence kit (Takara
Shuzo Co., Ltd., Kyoto, Japan) was used to construct a series of deletion deriv-
atives, whose nucleotide sequences were determined by the dideoxy termination
method with an ALFexpress DNA sequencer (Pharmacia Biotech, Milwaukee,
A Sanger reaction (30) was carried out by using the Thermosequenase fluo-
rescent 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 (IntelliGe-
netics, Inc., Mountain View, Calif.). The GenBank and SwissProt databases were
used for searching homologous proteins. Southern hybridization analyses of
SYK-6 and its PDC hydrolase gene (ligI) insertion mutants were performed with
the DIG System (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) ac-
cording to the procedure recommended by the manufacturer.
Enzyme assays. According to the method of Maruyama (16), PDC hydrolysis
and synthesis were spectrophotometrically determined by measuring the de-
crease and increase in the absorbance at 312 nm (ε312? 6,600 M?1cm?1; pH
8.5), respectively, with a DU-7500 spectrophotometer (Beckman, Fullerton, Cal-
if.). The reaction was carried out at 30°C in a cuvette. The 1-ml reaction mixture
for PDC hydrolysis contained 100 ?M PDC and the enzyme in 50 mM Tris-HCl
buffer (pH 8.5). That for PDC synthesis contained 100 ?M CHM and the enzyme
in 50 mM sodium phosphate buffer (pH 7.0). One unit of the enzyme was defined
as the amount that degraded 1 ?mol of substrate per min at 30°C. Specific
activity was expressed as units per milligram of protein. The optimum pHs for
PDC hydrolysis and synthesis were examined in the pH range of 4.0 to 10.0 by
using buffers consisting of 50 mM sodium acetate (pH 4.0 to 5.0), GTA (16.7 mM
[each] 3,3-dimethylglutaric acid, Tris, and 2-amino-2-methyl-1,3-propanediol)
(pH 4.0 to 8.5), sodium phosphate (pH 6.0 to 8.0), Tris-HCl (pH 8.0 to 9.0), or
sodium borate (pH 9.0 to 10.0). Kmand Vmaxvalues were obtained from the
Hanes-Woolf plots and expressed as means from at least three independent
Enzyme purification. Enzyme purification was performed according to the
method described below by using a BioCAD700E apparatus (PerSeptive Biosys-
tems, Framingham, Mass.).
FIG. 1. The proposed degradation pathway of vanillic acid, including the protocatechuate 4,5-cleavage pathway in S. paucimobilis SYK-6. LigA and B, small and
large subunits of 4,5-PCD (24); LigH, O-demethylase for vanillic acid and syringic acid (23); LigI, PDC hydrolase (this study). The PCA 4,5-cleavage pathway is
illustrated according to findings from previous studies (13, 15–18).
56 MASAI ET AL. J. BACTERIOL.
(i) Preparation of cell extract. Cells were grown in 2 liters of LB medium
containing 100 mg of ampicillin/liter. Expression of the ligI gene was induced for
3.5 h by adding isopropyl-?-D-thiogalactopyranoside (final concentration, 1 mM)
when the optical density at 660 nm (OD660) of the culture reached 0.5. Cells were
harvested by centrifugation and sonicated in 50 mM Tris-HCl buffer (pH 8.0)
(buffer A). The cell lysate was centrifuged at 15,000 ? g for 15 min. Streptomycin
(final concentration, 1%) was added to the supernatant, and it was recentrifuged
at 15,000 ? g for 15 min to remove nucleic acids. The supernatant was then
FIG. 2. Deletion analysis of the 10.5-kb EcoRI fragment and lig gene organization. The PDC hydrolase activities of the cells containing each subclone are presented
on the right. The small arrows indicate the direction of transcription from the lac promoter. Large filled arrows, ligI, ligA, and ligB genes. A large partly filled arrow,
the part of the ORF which showed a similarity with the LSD gene (lsdA) (9, 10). E. coli JM109 was used as a host strain except for pVA01 and pVAD4, for which P.
putida PpY1100 was used. E, EcoRI; P, PstI; Sl, SalI; Sm, SmaI; Sp, SphI; St, StuI; X, XhoI; Xb, XbaI.
TABLE 1. Strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s)
P. putida PpY1100
E. coli JM109
Wild type; NalrSmr
Mutant derivative of SYK-6; Kmrgene insertion mutant of ligI; NalrSmrKmr
recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ?(lac-proAB) F?[traD36 proAB?
pUC18 and pUC19
pBluescript II KS(?)
pSS32F, pSS14F, pSS55F, pSS73F, pSS32R,
pSS14R, pSS55R, pSS73R
Cloning vectors; Apr
Cloning vector; Apr
oriT sacB Kmr
Broad-host-range vector; Kmr
pUC18 with a 10.5-kb EcoRI fragment of SYK-6 carrying ligAB and ligI
pUC18 carrying the same fragment as pHN139F in the opposite direction
KS(?) with a 5.0-kb SmaI fragment carrying ligI of pHN139
KS(?) carrying the same fragment as pSS50F in the opposite direction
Deletion derivative of pSS50R carrying ligIa
Deletion derivatives of pHN139F and pHN139Ra
pUC19 with a 2.3-kb PstI-SmaI fragment carrying ligI
pUC1923 with insertion of the Kmrgene of pUC4K into a StuI site
pK19mobsacB with a 3.6-kb EcoRI-SmaI fragment of pUC1923K
pKT230 with a 10.5-kb EcoRI fragment carrying ligAB and ligI
Deletion plasmid of pVA01; SalI fragments in the middle of the insert were
aSee Fig. 2.
VOL. 181, 1999HYDROLASE FOR PROTOCATECHUATE 4,5-CLEAVAGE PATHWAY57
centrifuged at 150,000 ? g for 60 min at 4°C, and the crude extract was obtained
after concentration by ultrafiltration using a YM-10 membrane (Amicon, Bev-
(ii) POROS PI anion-exchange chromatography. The crude extract was ap-
plied to a POROS PI (polyethyleneimine) column (4.6 by 100 mm) (PerSeptive
Biosystems) previously equilibrated with buffer A. The enzyme was eluted with
25 ml of a linear gradient of 0 to 0.5 M NaCl. The PDC hydrolase was eluted at
approximately 0.26 M.
(iii) POROS HQ anion-exchange chromatography. The fractions containing
PDC hydrolase activity eluted from a PI column were pooled, desalted, and
concentrated by ultrafiltration using a YM-10 filter. The resulting solution was
applied to a POROS HQ (quaternized polyethyleneimine) 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 PDC hydrolase activity that eluted at approximately 0.2 M were
(iv) POROS HP2 hydrophobic interaction chromatography. The fractions
containing PDC hydrolase activity were pooled, desalted, and concentrated by
ultrafiltration using a YM-10 filter. Ammonium sulfate was added to the enzyme
solution to a final concentration of 2 M. After centrifugation (at 3,000 ? g for 10
min), the supernatant was collected and applied to a POROS HP2 (phenyl)
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
PDC hydrolase activity that eluted at approximately 1.3 M were pooled. Glycerol
was added to a final concentration of 10%, and the purified enzyme was stored
at ?80°C until use.
Analytical methods. The protein concentration was determined by the method
of Bradford (3). The purity of the enzyme preparation was examined by sodium
dodecyl sulfate–12% polyacrylamide gel electrophoresis (SDS–12% PAGE)
(14). The molecular mass of the native enzyme was determined by Superdex200
HR10/30 (Pharmacia Biotech) gel filtration column chromatography using LC
module 1 (Waters Corp., Milford, Mass.). Elution was performed with 50 mM
potassium phosphate buffer (pH 7.0) containing 0.15 M NaCl with a flow rate of
0.25 ml/min. The molecular weight was estimated on the basis of the calibration
curve of reference proteins.
To determine the N-terminal amino acid sequence, the purified enzyme was
subjected to SDS–12% PAGE and electroblotted onto a polyvinylidene difluo-
ride membrane (Bio-Rad, Hercules, Calif.). The enzyme band was cut out and
analyzed on a Procise 492 protein sequencer (Perkin-Elmer, Norwalk, Conn.).
The isoelectric point of LigI was determined by isoelectric focusing with 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 product compounds were identified by using a
GC-MS (model 5971A) with an Ultra-2 capillary column (50 m by 0.2 mm)
(Hewlett-Packard Co., Palo Alto, Calif.). The column temperature was increased
initially from 100 to 150°C, and then from 150 to 300°C, at rates of 20 and 3°C
per min, respectively. Temperatures of injection and detection were 220 and
Identification of the reaction product. PDC was incubated with purified LigI
(0.5 ?g) in 50 mM Tris-HCl buffer (pH 8.5) for 1 h. After the decrease of the
absorbance at 312 nm derived from PDC, the reaction mixture was acidified,
extracted with ethyl acetate, and then trimethylsilylated. GC-MS analysis was
carried out as described above.
Insertional inactivation of the ligI gene. The 2.3-kb PstI-SmaI fragment car-
rying ligI was cloned into pUC19 to generate pUC1923. The 1.3-kb PstI fragment
containing the kanamycin resistance gene from pUC4K was inserted into StuI in
the middle of the ligI gene in pUC1923. The resultant plasmid, pUC1923K, was
digested with EcoRI and PstI, and the insert containing the inactivated ligI gene
was cloned into pK19mobsacB (31) to generate pLID1.
When the cell density of SYK-6 cultured in 10 ml of LB reached 0.5 OD660
unit, the cells were harvested, washed twice with 1 ml of ice-cold 0.3 M sucrose,
and resuspended with 300 ?l of ice-cold 0.5 M sucrose. One microgram of pLID1
was mixed with 100 ?l of cells. Electroporation was performed with a Gene
Pulser (Bio-Rad) under the following conditions: 12-kV/cm field strength, 800-?
resistor, and a 25-?F capacitor. After incubation in LB medium for 12 h, kana-
mycin-resistant transformants were selected on an LB agar plate containing 50
mg of kanamycin/liter. They were cultured for 12 h in LB liquid medium con-
taining 10% sucrose. The candidates for mutants were isolated on an LB agar
plate containing 10% sucrose and kanamycin in order to select the cells in which
the sacB-containing vector portion was deleted by a double crossover. Southern
hybridization analyses of the PstI digests of total DNA prepared from the can-
didates for mutants were carried out with the 2.3-kb PstI-SmaI and 1.3-kb PstI
fragment probes containing the ligI and the kanamycin resistance gene, respec-
Nucleotide sequence accession number. The nucleotide sequence reported in
this paper was deposited in the DDBJ, EMBL, and GenBank nucleotide se-
quence databases under accession no. AB015964.
Nucleotide sequence of the PDC hydrolase gene. The ligAB
genes are located near the 3? end of the SYK-6 10.5-kb EcoRI
fragment (24). We examined PDC hydrolase activity on this
10.5-kb fragment, expecting to see evidence of the existence of
the PDC hydrolase gene in it. PDC hydrolase activity was
initially observed in P. putida PpY1100 containing pVA01,
which has a 10.5-kb fragment inserted in a broad-host-range
vector, pKT230. When SalI fragments in the middle of the
10.5-kb insert were deleted from pVA01, no PDC hydrolase
activity was detected or in the resultant plasmid, pVAD4. PDC
hydrolase activity was found in Escherichia coli JM109 harbor-
ing pHN139R, which contained the 10.5-kb EcoRI fragment
(Fig. 2). Among the deletion derivatives, pSS50R, pSS32R,
and pDS15 conferred PDC hydrolase activity. These depended
on the transcription from the lac promoter. The results ob-
tained with the deletion derivatives showed the following fea-
tures of the PDC hydrolase gene: (i) its direction of transcrip-
tion is the same as that indicated for pSS32R in Fig. 2 and
opposite that of the ligAB genes; (ii) it resides in the SphI
fragment of pDS15; and (iii) the 3?-terminal deletion beyond
the SalI site, which was accomplished in pSS14R, eliminated its
function. The first feature suggests that the PDC hydrolase
gene belongs to a transcriptional unit different from that of
Deletion derivatives of pSS50F and pSS50R were generated,
and the nucleotide sequence of the 1.5-kb SphI fragment was
determined. Only one open reading frame (ORF) of 879 bp,
encoding 293 amino acid residues, had the three features men-
tioned above and was therefore designated ligI. A homology
search for the deduced amino acid sequence with the SwissProt
database revealed no similarity with any other proteins. This
suggests that PDC hydrolase may constitute a unique class of
FIG. 3. SDS-PAGE analysis of protein fractions. Proteins were separated on
an SDS–12% polyacrylamide gel and stained with Coomassie brilliant blue.
Lanes: 1, molecular weight markers; 2, crude extract of E. coli JM109 harboring
pBluescript II KS(?) (5 ?g of protein); 3, crude extract of E. coli JM109
harboring pDS15 (5 ?g of protein); 4, PI fraction (5 ?g of protein); 5, HQ
fraction (2 ?g of protein); 6, HP2 fraction (2 ?g of protein). Molecular masses
are given on the left.
TABLE 2. Purification of PDC hydrolase from E. coli
58 MASAI ET AL. J. BACTERIOL.
FIG. 4. Identification of the reaction product from PDC catalyzed by LigI. (A) Gas chromatogram of the TMS derivative of the reaction product from PDC
catalyzed by purified LigI. PDC indicates the TMS derivative of PDC. Compounds I and II were the TMS derivatives of the reaction products originating from a
substrate, PDC. (B) Mass spectrum of the TMS derivative of PDC. (C) Mass spectrum of compound I. The mass spectra of compounds I and II are identical.
Compounds I and II were identified as isomeric forms of CHM.
VOL. 181, 1999 HYDROLASE FOR PROTOCATECHUATE 4,5-CLEAVAGE PATHWAY59
Purification of PDC hydrolase. E. coli JM109 harboring
pDS15 was grown, and the ligI gene in pDS15 was expressed.
The ligI gene product (LigI) was purified by a series of column
chromatography with PI, HQ, and HP2. Table 2 and Fig. 3
summarize the results of a typical purification. LigI was puri-
fied approximately 38-fold, with a recovery of 10%. The puri-
fied enzyme was shown to be near homogeneity by SDS-PAGE
(Fig. 3). The molecular mass of a subunit of LigI was estimated
to be 38 kDa, which is close to the value deduced from the
amino acid sequence of the ligI gene product (Mr, 32,737). The
LigI protein in the HP2 fraction was subjected to N-terminal
sequencing. The first six residues, M-T-N-D-E-R, corre-
sponded to the deduced amino acid sequence of the ligI gene
Identification of the reaction product. Figure 4 shows the
gas chromatogram and mass spectra of TMS derivatives of the
reaction product of LigI from PDC. Two product peaks with
retention times of 31.5 (I) and 34.4 (II) min were observed.
The mass spectra of products I and II were identical. We
extracted and analyzed the PDC alkaline hydrolysate accord-
ing to the procedure reported by Maruyama (16). Two kinds of
products yielding the same retention times and mass spectra as
products I and II in Fig. 4 were observed (data not shown). The
two major fragments at m/z 475 and 373 seemed to correspond
to (M-CH3) and (M-COOTMS), respectively (where M is a
molecular ion of the TMS derivative of CHM and COO rep-
resents a carboxyl group). The M-CH3ion is generally found in
mass spectra of TMS derivatives of organic acids. Based on
these results, we concluded that products I and II are the
stereoisomers of CHM.
Enzyme properties. The molecular mass of the native en-
zyme was estimated to be 31.6 kDa on a Superdex 200 gel
filtration column. This suggests that LigI is a monomer. The
isoelectric point of LigI was determined by isoelectric focusing
to be 4.9.
It is known that PDC hydrolase catalyzes both PDC hydro-
lysis (production of CHM from PDC) and PDC synthesis (pro-
duction of PDC from CHM) (13, 16). The pH optima for PDC
hydrolysis and PDC synthesis by LigI were examined. The
enzyme exhibited the highest activity at pH 8.5 for PDC hy-
drolysis and at pH 6.0 to 7.5 for PDC synthesis. These pH
optima were similar to those of the PDC hydrolase of P. ochra-
ceae (16). The ratio of PDC hydrolysis activity to PDC synthe-
sis activity was 2.6 at pH 8.5. LigI showed maximum hydrolase
activity at 50°C. The activity at 50°C, however, was only 1.2
times higher than that at 30°C. Therefore, the following exper-
iments were carried out at 30°C, which is the physiological
temperature for S. paucimobilis SYK-6.
The Kmand Vmaxvalues were estimated for PDC hydrolysis
and PDC synthesis. The Kmfor PDC was 73.8 ?M. For CHM,
it was 48.5 ?M. The Vmaxfor PDC hydrolysis was 506 U/mg.
For PDC synthesis, it was 283 U/mg.
Effects of thiol reagents and metal ions. The influence of
thiol reagents 5,5?-dithiobis(2-nitrobenzoic acid) (Ellman’s re-
agent) and N-ethylmaleimide on the activity for PDC hydroly-
sis and PDC synthesis was examined (Table 3). These thiol
reagents strongly inhibited both PDC hydrolysis and PDC syn-
thesis activity, suggesting that the cysteine residue is involved
in the enzyme reaction. The effects of metal ions on LigI
activity are summarized in Table 3. Zn2?strongly inhibited
both PDC hydrolysis and PDC synthesis. Cu2?inhibited only
PDC synthesis. In the case of the P. ochraceae enzyme, Mn2?
and Co2?inhibited PDC hydrolysis, and inhibition by Zn2?
and Cu2?was similar to that for the SYK-6 enzyme (16). The
SYK-6 PDC hydrolase was therefore shown to be more resis-
tant to metal ions than the P. ochraceae enzyme.
ligI disruption in S. paucimobilis SYK-6. Inactivation of the
ligI gene by the insertion of the kanamycin resistance gene was
performed by using the ligI disruption plasmid pLID1 as de-
scribed in Materials and Methods. The ligI insertion mutant
was confirmed by Southern hybridization analysis (Fig. 5). The
mutant strain DLI, grown on LB medium or syringic acid,
showed no PDC transformation activity and no PDC hydroly-
sis. This strain completely lost the ability to grow on vanillic
acid, even though it grew as well on syringic acid as the wild-
TABLE 3. Effects of thiol reagents and metal ions on PDC
Relative activity (%)
PDC hydrolysis PDC synthesis
aPurified LigI (0.5 ?g) was preincubated with each reagent at 30°C for 10 min,
and the remaining activities were determined.
FIG. 5. Insertional inactivation of the ligI gene in S. paucimobilis SYK-6. (A)
Schematic representation of the insertional inactivation of ligI by the kanamycin
resistance gene from pUC4K. Thick arrows, orientation of transcription of the
ligI and kanamycin resistance genes. E, EcoRI; P, PstI; Sl, SalI; Sm, SmaI; Sp,
SphI; St, StuI; X, XhoI. (B) Southern hybridization analysis of the ligI insertion
mutant (DLI). Lanes 1 and 3, total DNA of SYK-6 digested with PstI; lanes 2 and
4, total DNA of DLI digested with PstI. The 2.3-kb PstI-SmaI fragment carrying
ligI (lanes 1 and 2) and the 1.3-kb PstI fragment of the kanamycin resistance gene
(lanes 3 and 4) were used as probes.
60MASAI ET AL. J. BACTERIOL.
type strain. The metabolite of vanillic acid generated by the
whole cells of DLI grown on LB medium was examined by
GC-MS analysis. After 22 h of incubation, vanillic acid was
transformed completely, and only the accumulation of PDC
This is the first report on the genetic analysis of PDC hy-
drolase, which is one of the protocatechuate 4,5-cleavage path-
way enzymes. The PDC hydrolase gene of S. paucimobilis
SYK-6, designated ligI, encodes a protein of 32,737 Da (293
amino acids). There was no similarity between the LigI amino
acid sequence and those of the proteins in the databases,
including the dienelactone hydrolase (4, 28, 37) and the ?-ke-
toadipate enol-lactone hydrolase (7, 33), which seemed to be
functionally related to LigI.
The ligI gene is located approximately 5.4 kb upstream of
ligA. Interestingly, ligI is transcribed divergently from ligAB.
This fact indicated that the PCA 4,5-cleavage pathway was
composed of at least two distinct operons. We could not find
other genes responsible for the PCA 4,5-cleavage pathway
enzymes in the region sequenced. Downstream from ligI, an
incomplete ORF which had the same direction of transcription
as ligI was found. The predicted amino acid sequence of the
product of this ORF showed significant similarity to those of
the lignostilbene-?,?-dioxygenase (LSD) genes of Pseudomo-
nas paucimobilis TMY1009 (9, 10). LSD has been reported to
be a dioxygenase catalyzing the cleavage of the interphenyl
double bond of lignostilbenes. The occurrence of the LSD
gene homolog beside the PCA 4,5-cleavage pathway enzyme
genes is interesting. Further nucleotide sequencing and func-
tional analysis of the 10.5-kb EcoRI fragment may address the
functions of a putative LSD and other enzymes of the PCA
The reaction product from PDC, catalyzed by LigI, was
estimated to be two stereoisomers of CHM. OMA, which is a
tautomer of CHM was not detected. The production of OMA
from PDC was suggested by Maruyama (16). OMA is an
?-keto acid, and its TMS derivative is easily distinguishable in
MS from that of CHM, because a keto group of OMA is not
trimethylsilylated. OMA might have been degraded during the
process of extraction, since ?-keto acid is generally unstable.
The characteristics of the PDC hydrolases of S. paucimobilis
SYK-6, P. ochraceae, and C. testosteroni are summarized in
Table 4. All enzymes are monomeric proteins. The molecular
mass and pH optima of the SYK-6 enzyme are very similar to
those of the P. ochraceae enzyme. A higher affinity with CHM
(or OMA) than with PDC and a higher Vmaxfor PDC hydro-
lysis than for PDC synthesis were common features of the PDC
hydrolases of SYK-6 and P. ochraceae. Thiol reagents, such as
Ellman’s reagent and N-ethylmaleimide, strongly inhibited ac-
tivity, suggesting that the cysteine residue is the catalytic site of
these three enzymes. A similar inhibition was also observed in
the P. ochraceae and C. testosteroni enzymes. In the ?/? hydro-
lase fold enzymes, the catalytic nucleophile is located in a
highly conserved peptide, Gly-X-(Ser/Cys)-X-Gly. The three-
dimensional structure of the dienelactone hydrolase from
Pseudomonas sp. strain B13 was solved, and its catalytic nu-
cleophile, cysteine residue 123, was shown to constitute part of
a catalytic triad of residues (26). Recently, Schrag and Cygler
reported that a consensus sequence of these enzymes might
better be described as Sm (small amino acid)-X-Nuc (nucleo-
phile)-X-Sm, since the glycine residues at Nuc ?2 and Nuc ?2
are sometimes substituted for other small amino acids, includ-
ing alanine and serine (32). Among the three cysteine residues
of LigI, Cys76 may be the catalytic site, since the sequence
Ala74-Ser75-Cys76-His77-Gly78 corresponds to the consensus
The ligI gene insertion mutant, DLI, showed no PDC hy-
drolase activity when the DLI cells were grown on LB medium
and on syringic acid. DLI was not able to grow on vanillic acid,
and the cells of DLI grown on LB medium accumulated PDC
from vanillic acid. These results indicated that the ligI gene is
unique in conferring PDC hydrolysis and is essential for growth
of SYK-6 on vanillic acid. According to the proposed meta-
bolic pathway of syringic acid in SYK-6, syringic acid is con-
verted to PDC via 3-O-methylgallic acid (11, 23). The produc-
tion of 3-O-methylgallic acid from syringic acid was evident
from the results obtained with the ligH gene. The mutation in
the ligH gene, the product of which is involved in the conver-
sion of syringic acid to 3-O-methylgallic acid, resulted in a
growth defect on syringic acid (23). Kersten et al. reported the
production of PDC from 3-O-methylgallic acid by 4,5-PCD
(13). However, the ligI insertion mutant DLI grew on syringic
acid and showed no PDC transformation activity and no PDC
hydrolysis. These results obviously suggest that syringic acid is
not metabolized via PDC and that neither 4,5-PCD nor LigI is
involved in the metabolism of syringic acid.
The results obtained in this study strongly support the pro-
posed PCA 4,5-cleavage pathway presented in Fig. 1. Further
research is needed to elucidate the correct degradation path-
way of syringic acid in SYK-6.
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