JOURNAL OF BACTERIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Dec. 2000, p. 6651–6658Vol. 182, No. 23
Genetic and Biochemical Characterization of 4-Carboxy-2-
Hydroxymuconate-6-Semialdehyde Dehydrogenase and
Its Role in the Protocatechuate 4,5-Cleavage Pathway
in Sphingomonas paucimobilis SYK-6
EIJI MASAI,1* KIYOTAKA MOMOSE,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, Tokyo 183-8509,3Japan
Received 12 June 2000/Accepted 18 September 2000
Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomo-
nas paucimobilis SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We
characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (ligC). CHMS is
the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found
that ligC was located 295 bp downstream of ligB, which encodes the large subunit of the PCA 4,5-dioxygenase.
The ligC gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590
Da. The deduced amino acid sequence of ligC showed 19 to 20% identity with 3-chlorobenzoate cis-dihydrodiol
dehydrogenase of Alcaligenes sp. strain BR60 and phthalate cis-dihydrodiol dehydrogenases of Pseudomonas
putida NMH102-2 and Burkholderia cepacia DBO1, which are unrelated to group I, II, and III microbial alcohol
dehydrogenases (M. F. Reid and C. A. Fewson, Crit. Rev. Microbiol. 20:13–56, 1994). The ligC gene was
expressed in Escherichia coli and LigC was purified to near homogeneity. Production of PDC from CHMS
catalyzed by LigC was confirmed in the presence of NADP?by electrospray ionization-mass spectrometry and
gas chromatography-mass spectrometry. LigC is a homodimer. The isoelectric point, optimum pH, and
optimum temperature were estimated to be 5.3, 8.0, and 25°C, respectively. The Kmfor NADP?was estimated
to be 24.6 ? 1.5 ?M, which was approximately 10 times lower than that for NAD?(252 ? 3.9 ?M). The Kms
for CHMS in the presence of NADP?and NAD?are 26.0 ? 0.5 and 20.6 ? 1.0 ?M, respectively. Disruption
of ligC in S. paucimobilis SYK-6 prevented growth with vanillate. Only PCA was accumulated during the
incubation of vanillate with the whole cells of the ligC insertion mutant (DLC), indicating a lack of PCA
4,5-dioxygenase activity in DLC. However, the introduction of ligC into DLC restored its ability to grow on
vanillate. PDC was suggested to be an inducer for ligAB gene expression.
Lignin is a major component of the plant cell wall along with
cellulose and hemicellulose, and it is known to be one of the
most abundant aromatic compounds in nature. Utilization of
this abundant biomass has been expected but has not been
established. One of the practical ways to utilize lignin is to
degrade it using the enzyme systems of microorganisms to
produce commercially valuable compounds. Sphingomonas
paucimobilis SYK-6, which has been isolated from pulp efflu-
ent, can degrade various dimeric lignin compounds such as
?-aryl ether, biphenyl, phenylcoumarane, diarylpropane, and
pinoresinol (20). The enzyme genes involved in the degrada-
tion of major components of these compounds, ?-aryl ether
(18, 19, 21, 22) and biphenyl (29, 30), have already been char-
acterized. Vanillate is thought to be a major intermediate
metabolite of these dimeric lignin compounds having guaiacyl
moieties. As illustrated in Fig. 1, protocatechuate (PCA) is
generated following the O demethylation of vanillate (26), and
it is metabolized to pyruvate and oxaloacetate via the PCA
4,5-cleavage pathway in S. paucimobilis SYK-6 (20, 23). Three
different pathways have been reported for PCA metabolism,
the PCA 4,5-cleavage pathway (10, 13–17, 20, 27, 39), the PCA
3,4-cleavage pathway (8), and the PCA 2,3-cleavage pathway
Numerous genetic and biochemical characterizations of the
PCA 3,4-cleavage pathway have been established, while the
PCA 4,5- and PCA 2,3-cleavage pathways are poorly under-
stood. Although the PCA 4,5-cleavage pathway was enzymat-
ically characterized by Dagley and coworkers (10, 39) and
Maruyama and coworkers (13–17) in the 1980s, no genetic
information on the PCA 4,5-cleavage pathway is available ex-
cept for that included in studies of SYK-6 (23, 27). According
to the reports of Kersten et al. (10), PCA is first converted into
4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) by the
PCA 4,5-dioxygenase in the PCA 4,5-cleavage pathway (Fig.
1). The three-dimensional structure of PCA 4,5-dioxygenase
has been elucidated previously (38). The resultant CHMS is in
equilibrium between the open form and the cyclic hemiacetal
form. The hemiacetal form of CHMS is thought to be oxidized
by CHMS dehydrogenase to produce 2-pyrone-4,6-dicarboxy-
late (PDC). PDC hydrolase transforms PDC to 4-carboxy-2-
hydroxymuconate (CHM) or its tautomer, 4-oxalomesaconate
(OMA), and the resultant product is converted into 4-carboxy-
* Corresponding author. Mailing address: Department of Bioengi-
neering, Nagaoka University of Technology, Kamitomioka, Nagaoka,
Niigata 940-2188, Japan. Phone: 81-258-47-9428. Fax: 81-258-47-9450.
4-hydroxy-2-oxoadipate (CHA) by a hydratase. Finally, an al-
dolase cleaves CHA to generate pyruvate and oxaloacetate.
To understand the whole PCA 4,5-cleavage pathway of S.
paucimobilis SYK-6, we are planning to characterize all the
genes involved in the enzyme reactions and regulation of the
PCA 4,5-cleavage pathway. The genes for the PCA 4,5-dioxy-
genase (ligAB) (27) and PDC hydrolase (ligI) (23) have already
been characterized. In this study, we characterize the CHMS
dehydrogenase gene, which is located next to ligB and involved
in the second step of the PCA 4,5-cleavage pathway. We also
suggest that the reaction product of CHMS dehydrogenase,
PDC, is an inducer of the ligAB gene expression.
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
(29) containing 0.2% (wt/vol) vanillate or syringate or in Luria-Bertani (LB)
medium (Bacto-Tryptone, 10 g/liter; yeast extract, 5 g/liter; NaCl, 5 g/liter).
Preparation of substrates. CHMS was prepared from PCA by using the crude
PCA 4,5-dioxygenase (LigAB) prior to the enzyme assay. PCA (1.5 mM) was
incubated with 100 ?g of crude extract of Escherichia coli JM109 harboring
pAB16 carrying ligAB in 20 mM Tris-HCl buffer (pH 8.0) for 1 h at 30°C. The
complete conversion of PCA into CHMS was confirmed by electrospray ioniza-
tion-mass spectrometry (ESI-MS) and gas chromatography-mass spectrometry
(GC-MS) with the conditions described below. The resultant CHMS solution was
kept at room temperature for 1 h to be equilibrated between the open form and
the cyclic hemiacetal forms. Vanillate and other chemicals were purchased from
Tokyo Kasei Kogyo Co. (Tokyo, Japan) or Wako Pure Chemical Industries
DNA manipulations and nucleotide sequencing. DNA manipulations were
carried out essentially as described elsewhere (1, 32). 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,
Wis.). The Sanger reaction (33) was carried out using the Thermosequenase
fluorescence-labeled primer cycle sequencing kit with 7-deaza dGTP (Amersham
Pharamacia Biotech, Little Chalfont, United Kingdom). Sequence analysis and
homology alignment were done using the programs of GeneWorks (IntelliGe-
netics, Inc., Mountain View, Calif.). The DDBJ databases were employed to
search for homologous proteins. Southern hybridization analysis of SYK-6 and
its CHMS dehydrogenase gene (ligC) insertion mutants was performed with the
DIG system (Boehringer Mannheim Biochemicals, Indianapolis, Ind.).
Enzyme assay. CHMS dehydrogenase activity was spectrophotometrically de-
termined by measuring the increase in the absorbance at 340 nm derived from
the production of NADH (ε340? 6,600 M?1cm?1; pH 8.0) or NADPH (ε340?
5,070 M?1cm?1; pH 8.0) from NAD?or NADP?, respectively, using a DU-7500
spectrophotometer (Beckman, Fullerton, Calif.). Since the reaction product,
PDC, has absorbance at 340 nm (ε340? 2,540 M?1cm?1; pH 8.0) and PDC is
produced in an equimolar amount with NADH or NADPH, the increase of
absorbance at 340 nm represents the sum of the amount of NADH or NADPH
and PDC. We estimated the actual amount of NADH or NADPH by using the
sum of molar extinction coefficients for NADH or NADPH and PDC. The 1-ml
reaction mixture contained 150 ?M CHMS, 200 ?M NAD?or NADP?, and the
enzyme in 20 mM Tris-HCl buffer (pH 8.0). The reaction was carried out at 25°C
in a cuvette. One unit of the enzyme is defined as the amount that converted 1
?mol of NAD?or NADP?to NADH or NADPH, respectively, per min at 25°C.
Specific activity was expressed as units per milligram of protein. The optimum
pHs were examined in the pH range of 6.0 to 9.0 by using buffers consisting of
20 mM potassium phosphate (pH 6.0 to 8.0) and 20 mM Tris-HCl buffer (pH 7.5
to 9.0). The Kmand Vmaxvalues were calculated by the Hanes-Woolf plots
obtained from at least three independent experiments.
Enzyme purification. Enzyme purification was performed according to the
method described below using a BioCAD700E apparatus (PerSeptive Biosys-
tems, Framingham, Mass.).
(i) Preparation of cell extract. The cells were grown in 100 ml of LB medium
containing 100 mg of ampicillin per liter at 37°C. The expression of the ligC gene
was induced for 3.5 h by adding isopropyl-?-D-thiogalactopyranoside (final con-
centration, 1 mM) when the optical density (660 nm) of culture reached 0.5. The
cells were then pelleted and resuspended in 20 mM Tris-HCl buffer (pH 8.0)
(buffer A). Bacterial cells were broken by two passages through a French pres-
sure cell. Crude cell lysate was centrifuged at 15,000 ? g for 15 min. Streptomycin
(final concentration, 1%) was added to the supernatant that was centrifuged at
15,000 ? g for 15 min to remove nucleic acids. The supernatant was recentri-
fuged at 150,000 ? g for 60 min at 4°C and concentrated by ultrafiltration using
a Minicon B15 filter (Amicon, Beverly, Mass.) to obtain the crude extract.
(ii) POROS PI anion-exchange chromatography. The crude extract was ap-
plied to a POROS PI (polyethyleneimine) column (7.5 by 100 mm) (PerSeptive
FIG. 1. Catabolic pathway of vanillate for S. paucimobilis SYK-6 via the PCA 4,5-cleavage pathway. LigA and LigB, small and large subunits of PCA 4,5-dioxygenase
(4,5-PCD) (27, 38); LigC, CHMS dehydrogenase (this study); LigI, PDC hydrolase (23); LigH, an essential gene product for vanillate and syringate O demethylations
6652 MASAI ET AL. J. BACTERIOL.
Biosystems) previously equilibrated with buffer A. The enzyme was eluted with
88 ml of a linear gradient of 0 to 0.5 M NaCl. The CHMS dehydrogenase was
eluted approximately at 0.29 M.
(iii) POROS HQ anion-exchange chromatography. The fractions containing
CHMS dehydrogenase activity eluted from a PI column were pooled, desalted,
and concentrated by ultrafiltration using a Minicon B15 filter. The resulting
solution was applied to a POROS HQ (quaternized PI) 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 CHMS dehydrogenase activity eluting at approximately 0.23 M were
(iv) POROS PE hydrophobic-interaction chromatography. The fractions con-
taining CHMS dehydrogenase activity were pooled, desalted, and concentrated
as described above. Ammonium sulfate was added to the enzyme solution to a
final concentration of 2 M. After the centrifugation at 15,000 ? g for 10 min, the
supernatant was applied to a POROS PE (phenylether) 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 CHMS dehydrogenase
activity eluting at approximately 1.62 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.
Analytical methods. The protein concentration was determined by the method
of Bradford (4). The homogeneity of the enzyme preparation was examined by
sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis (SDS-PAGE)
(12). The molecular mass of the native enzyme was determined by Superdex200
HR10/30 (Pharmacia Biotech) gel filtration column chromatography using a
BioCAD700E apparatus. The elution was performed using 50 mM potassium
phosphate buffer (pH 7.0) containing 0.15 M NaCl with a flow rate of 0.5 ml/min.
The molecular weight was estimated based on the calibration curve of reference
To determine the N-terminal amino acid sequence, the purified enzyme was
subjected to SDS-PAGE (12% polyacrylamide gel) and electroblotted onto a
polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.). The enzyme
band was cut out and analyzed on a PPSQ-21 protein sequencer (Shimadzu,
Kyoto, Japan). The isoelectric point of LigC 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).
Identification of the substrate and the reaction products was carried out using
a GC-MS (model 5971A; Hewlett-Packard Co., Palo Alto, Calif.) with an Ultra-2
capillary column (50 m by 0.2 mm; Hewlett-Packard Co.) and an ESI-MS
(HP1100 series LC-MSD; Hewlett-Packard Co.). For GC-MS analysis, the sub-
strate and the reaction products in the buffer were acidified and extracted with
ethylacetate, and then the extract was dried in vacuo and trimethylsilylated
(TMS). The analytical conditions for GC-MS were the same as described in the
previous study (23). In the analysis by ESI-MS, mass spectra were obtained by
negative-mode ESI, with a needle voltage of ?3.5 kV and a source temperature
of 350°C. The sample was diluted 10-fold with 10 mM Tris-acetate buffer (pH
8.0) and injected into the flow system; the water/methanol ratio was 90:10
(vol/vol), and the flow rate was 0.2 ml/min.
Disruption of the ligC gene. The 2.5-kb XbaI-EcoRI fragment carrying ligAB
and part of ligC was cloned into pBluescript II SK(?) to generate pABC25. The
BsmI fragment in the ligC gene of pABC25 was replaced by the 1.2-kb PstI
fragment carrying the kanamycin resistance gene from pUC4K. The XbaI-EcoRI
fragment of the resultant plasmid pABC25K containing the inactivated ligC gene
was cloned into pK19mobsacB (34) to generate pLCD1.
The ligC-disrupted plasmid, pLCD1, was introduced into the SYK-6 cells by
electroporation with a Genepulser (Bio-Rad). The colonies resistant to both
kanamycin and sucrose were selected as described earlier (23). Southern hybrid-
ization analysis of the PstI digests of total DNAs prepared from the candidates
for mutants was carried out with the 1.0-kb XhoI-EcoRI and 1.2-kb PstI fragment
probes containing ligC and kanamycin resistance genes, respectively.
The metabolites of vanillate by the ligC insertion mutant (DLC) were ana-
lyzed. DLC cells grown in 10 ml of LB medium were washed with 10 mM
Tris-acetate buffer (pH 8.0). The cells were resuspended in the same buffer and
incubated with 12 mM vanillate for 12 h at 30°C. After centrifugation, the
supernatant was diluted 20-fold with the buffer and analyzed by ESI-MS as
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. AB035122 and M34835.
TABLE 1. Strains and plasmids used in this study
Strain or plasmidRelevant characteristic(s)Source or reference
P. putida PpY1100
Wild type; NalrSmr
Mutant derivative of SYK-6; Kmrgene insertion mutant of ligC; NalrSmrKmr
recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ?(lac-proAB) F?[traD36 proAB?lacIqlacZ?M15]
hsdS gal(?cIts857 ind1 Sam7 nin5 lacUV5-T7 gene 1)
?) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1
Cloning vectors; Apr
Cloning vectors; Apr
Expression vector; AprT7 promoter
Broad-host-range vector; Kmr
Broad-host-range cosmid; KmrTetr
Broad-host-range vector; KmrCmr
oriT sacB Kmr
Source of Kmrcassette; AprKmr
pKT230 with a SYK-6 10.9-kb insert carrying ligABC and ligI
Deletion plasmid of pVA01; a SalI fragment near the 3? terminus of the insert was deleted
Deletion plasmid of pVA01; SalI fragments in the middle of the insert were deleted
pUC18 with a 10.5-kb EcoRI fragment of pVA01 carrying ligAB, part of ligC, and ligI
pUC18 carrying the same fragment as pHN139F in the opposite direction
pET21(?) with a 1.0-kb SmaI-EcoRI fragment carrying part of ligC
pET21(?) with a 1.4-kb SmaI-EcoRI fragment carrying ligC
pBluescript II SK(?) with a 1.6-kb XbaI-XhoI fragment carrying ligAB
pBluescript II SK(?) with a 2.5-kb XbaI-EcoRI fragment carrying ligAB and part of ligC
pABC25 with insertion of the Kmrgene of pUC4K replacing a 0.2-kb BsmI fragment
pK19mobsacB with a 3.5-kb XbaI-EcoRI fragment of pABC25K
pBBR122 with a 1.4-kb SmaI-HindIII fragment carrying ligC of pSM21
pBBRC1220 with an 0.4-kb PvuII fragment carrying the lac promoter of pUC19 in the SmaI site
VOL. 182, 2000DEHYDROGENASE FOR PROTOCATECHUATE 4,5-CLEAVAGE PATHWAY 6653
Isolation and nucleotide sequence of the CHMS dehydroge-
nase gene. CHMS dehydrogenase activity was detected in the
crude extract of Pseudomonas putida PpY1100 harboring
pVA01, which contained the PCA 4,5-dioxygenase (ligAB) and
PDC hydrolase (ligI) genes. Deletion analysis of pVA01, whose
deletions were generated by partial SalI digestion, indicated
that the intact CHMS dehydrogenase gene was included in
pVAD4 but not in pVAD2 (Fig. 2A). These results suggested
that the CHMS dehydrogenase gene was localized downstream
of ligB. However, the E. coli transformant harboring pHN139F
and pHN139R containing the 10.5-kb EcoRI fragment of
pVA01 did not show CHMS dehydrogenase activity (Fig. 2A),
and this suggested that the CHMS dehydrogenase gene was
truncated at the right end of the 10.5-kb EcoRI fragment.
Thus, the 0.4-kb EcoRI fragment located in the right end of
pVA01 seemed to contain a part of the CHMS dehydrogenase
gene. We determined the nucleotide sequences of the 1.0-kb
XhoI fragment and the 0.4-kb EcoRI fragment carrying the
partial CHMS dehydrogenase gene. In the 1,608-bp DNA re-
gion bounded by the PstI and EcoRI sites, a unique open
reading frame (ORF) of 945 bp preceded by a putative ribo-
some binding site (AGGA) (35) was found and seemed to be
the CHMS dehydrogenase gene. This ORF was designated
ligC. The 1.4-kb DNA region bounded by SmaI and EcoRI
sites carrying the entire ligC gene was cloned in pET21(?) to
construct pSM21, and ligC was expressed in E. coli BL21(DE3)
under the control of the T7 promoter. Production of the 35-
kDa protein was observed by SDS-PAGE (data not shown).
The size of this product is close to the molecular mass calcu-
lated from the deduced amino acid sequence of ligC (Mr,
34,590). Incubation of CHMS with the crude cell extract con-
taining the ligC gene product (LigC) revealed the decrease in
the absorbance at 410 nm derived from CHMS and the in-
crease in the absorbance at 340 nm derived from NADH or
NADPH and PDC, which is the reaction product of CHMS
(data not shown). These results indicated that LigC actually
encodes the CHMS dehydrogenase.
A similarity search of the deduced amino acid sequence of
ligC revealed 19 to 20% identity with 3-chlorobenzoate cis-4,5-
dihydrodiol dehydrogenase (CbaC) of Alcaligenes sp. strain
BR60 (25) and phthalate cis-4,5-dihydrodiol dehydrogenases
of P. putida NMH102-2 (Pht4) (28) and Burkholderia cepacia
DBO1 (OphB) (5). These aromatic dihydrodiol dehydroge-
nases, whose substrate is a dihydrodiol compound with a car-
boxyl group, are thought to be a new class of alcohol dehydro-
genase, which has little similarity to the group I, II, and III
microbial alcohol dehydrogenases (24, 31).
Purification of CHMS dehydrogenase. In order to charac-
terize the enzyme properties of CHMS dehydrogenase, LigC
FIG. 2. Deletion analysis of the CHMS dehydrogenase gene and the insertional inactivation of ligC in S. paucimobilis SYK-6. (A) The CHMS dehydrogenase
activities of the cells harboring each subclone are presented. The small arrows indicate the direction of transcription from the lac (Plac) or T7 (PT7) promoters. Large
filled arrows indicate the PCA 4,5-cleavage pathway genes, ligI, ligA, ligB, and ligC. A partly filled large arrow represents part of the ORF of the lignostilbene
?,?-dioxygenase homolog (lsdA). E. coli JM109, E. coli BL21(DE3), and P. putida PpY1100 were used as host strains for pHN139F and pHN139R; pHNC2 and pSM21;
and pVA01, pVAD2, and pVAD4, respectively. E, EcoRI; P, PstI; Sc, SacI; Sl, SalI; Sm, SmaI; Xb, XbaI; Xh, XhoI. (B) Schematic representation of the insertional
inactivation of ligC by the kanamycin resistance gene from pUC4K. Bs, BsmI; E, EcoRI; P, PstI; Sc, SacI; Sl, SalI; Sm, SmaI; Xb, XbaI; Xh, XhoI.
6654MASAI ET AL. J. BACTERIOL.
was purified from the cell extract of E. coli harboring pSM21 by
a series of column chromatography procedures with PI, HQ,
and PE. LigC was purified approximately 14-fold to near ho-
mogeneity with a recovery of 10% (Table 2). N-terminal amino
acid sequencing revealed that the first 20 residues completely
corresponded to the deduced amino acid sequence of ligC.
Conversion of CHMS into PDC by purified LigC. The TMS
derivative of CHMS could not be detected by GC-MS, prob-
ably due to its instability, so we employed ESI-MS. Negative-
ion ESI-MS spectra of CHMS yielded a deprotonated molec-
ular ion at m/z 185.0. When 200 ?M CHMS was incubated for
1 h with 0.5 ?g of purified LigC and 200 ?M NADP?, the ion
at m/z 185.0 disappeared completely, and the reaction product
ion at m/z 183.0 appeared, which corresponds to the deproto-
nated molecular ion of PDC. When NADP?was omitted, the
product was not detected. Figure 3 shows the GC and mass
spectrum of the TMS derivative of the reaction product of
LigC from CHMS. A product peak with a retention time of
27.8 min was observed, and the mass spectrum of the product
was identical to that of the authentic PDC. Thus, we concluded
that LigC catalyzed the conversion of CHMS into PDC de-
pending on the presence of NADP?.
Enzyme properties. Gel filtration column chromatography
with a Superdex200 chromatograph demonstrated that the
molecular mass of the native LigC was 67 kDa, indicating its
dimeric structure. The optimum pH and temperature were
estimated to be 8.0 and 25°C, respectively, and the isoelectric
point was determined to be 5.3.
Table 3 shows the kinetic constants of LigC. With setting of
the initial concentration of CHMS to 150 ?M, LigC showed
10-times-higher affinity to NADP?than to NAD?. Kmfor
NAD?was estimated to be 252 ?M. Vmaxfor CHMS oxidation
with NAD?showed a value similar to that with NADP?. With
adjustment of the initial concentration of a cofactor to 200 ?M,
Kmfor CHMS with NAD?agreed with that with NADP?.
However, Vmaxfor CHMS oxidation with NAD?did not agree
with that with NADP?. The former is two times lower than the
latter. These results are probably due to the initial concentra-
tion of NAD?(200 ?M) used being lower than the Kmvalue
for NAD?(252 ?M).
We also examined the influence of sulfhydryl reagents on
LigC. Treatments of 5 ?g of purified LigC with p-chloromer-
curibenzoate (10 ?M), HgCl2(10 ?M), or 5,5?-dithiobis(2-
nitrobenzoate) (100 ?M) for 1 h inhibited 78, 83, or 98% of
LigC activity, respectively. These results suggested that a cys-
teine residue might be involved in the enzyme reaction. Nei-
ther inhibition nor stimulation of enzyme activity was observed
in the presence of 5 mM EDTA.
ligC disruption in S. paucimobilis SYK-6. In order to clarify
the involvement of the ligC gene in the degradation of model
lignin compounds, the ligC gene in SYK-6 was disrupted. The
insertion mutant of ligC was obtained by the introduction of
pLCD1, in which ligC in the 2.5-kb XbaI-EcoRI fragment in-
serted in pK19mobsacB was inactivated by the insertion of the
1.2-kb kanamycin resistance gene. Southern hybridization
analysis of the ligC insertion mutant using the ligC and the
kanamycin resistance gene probes revealed that the ligC gene
was inactivated by homologous recombination through the
double crossover. This mutant strain was designated DLC and
used for the following experiments (Fig. 2B). DLC was not
able to grow on vanillate, but there was no difference in the
growth on syringate between DLC and the wild type, SYK-6.
The metabolites of vanillate accumulated during the incu-
bation of the whole cells of DLC and SYK-6 pregrown in LB
medium were analyzed by ESI-MS. After 12 h of incubation,
FIG. 3. Identification of the reaction product from CHMS catalyzed by LigC.
(A) Gas chromatogram of the TMS derivative of the reaction product from
CHMS catalyzed by purified LigC. CHMS (100 ?M) was incubated with 3 ?g of
purified LigC for 2 min in the presence of 200 ?M NADP?. The reaction product
was extracted by ethylacetate and trimethylsilylated (TMS). (B) Mass spectrum
of the TMS derivative of the product with a retention time of 27.8 min shown in
panel A. (C) Mass spectrum of the authentic TMS-PDC.
TABLE 2. Purification of CHMS dehydrogenase from E. coli
BL21(DE3) harboring pSM21
VOL. 182, 2000DEHYDROGENASE FOR PROTOCATECHUATE 4,5-CLEAVAGE PATHWAY 6655
only the remaining vanillate and the metabolite, PCA, were
detected in the DLC culture, and no CHMS had accumulated
(Fig. 4). In the SYK-6 culture, vanillate disappeared thor-
oughly, and no metabolites were accumulated. When the
broad-host-range vector pBBR122 carrying ligC (pBBRC1221)
was introduced into the DLC cells, pBBRC1221 restored the
ability to grow on vanillate. These results indicated the repres-
sion of PCA 4,5-dioxygenase activity in DLC.
The nucleotide sequence of ligC was determined in this
study. The deduced amino acid sequence of the ligC gene
revealed approximately 20% identity with CbaC of Alcaligenes
sp. strain BR60 (25), Pht4 of P. putida NMH102-2 (28), and
OphB of B. cepacia DBO1 (5), which are dehydrogenases for
carboxylic cis-dihydrodiol compounds involved in the degrada-
tion of 3-chlorobenzoate or phthalate. The identities among
these enzymes ranged from 45 to 64%, and they do not have
any apparent relationship with group I (long-chain zinc-depen-
dent enzyme), group II (short-chain zinc-independent en-
zyme), or group III (iron-activated enzyme) microbial
NAD(P)?-dependent alcohol dehydrogenases (31). Among
the enzymes related to CbaC (CbaC family), the amino acid
(24). LigC contains the largest part of this conserved sequence,
spanning amino acid positions 75 to 96 [H-(X)11-K-H-V-Q-V-
E-I-P-L-A]. The CbaC family members also had the consensus
sequence G-X-X-G-X-G at their N terminus, which is thought
to be the dinucleotide-binding motif for NAD(P)?. However,
instead of this motif, LigC had G-X-G-X-X-G. The length of
LigC was 315 amino acids, which is much less than those of
CbaC (397 amino acids), Pht4 (410 amino acids), and OphB
(391 amino acids). Thus, LigC is similar to the CbaC family
enzymes, but it does not have an obvious relationship with
The ligC gene was located 295 bp downstream of the ligB
gene, and the putative ?-independent terminator was found 24
bp downstream of ligC. This suggested that the transcription of
ligC terminates at this terminator. In addition to this sequence,
the 21-bp inverted repeat sequence (nucleotide positions 86 to
106 and 213 to 233 in the sequence of AB035122) was found in
the intergenic region between ligB and ligC. Further research is
needed to address the actual operon structure of ligA, ligB, and
The ligC gene was expressed in E. coli, and its product was
purified to near homogeneity. The reaction product from
CHMS catalyzed by purified LigC was determined as PDC by
both GC-MS and ESI-MS (Fig. 3). These results are consistent
with the reports of Maruyama (13, 14) and Kersten et al. (10).
Maruyama et al. initially proposed that the CHMS dehydro-
genase would catalyze the conversion of CHMS into CHM,
which was not confirmed experimentally (17). Afterwards, Ma-
ruyama identified PDC as the reaction product and proposed
that this enzyme would be involved in the oxidation of the
hemiacetal form to PDC. CHMS seems to be in equilibrium
between the open form and the cyclic hemiacetal form (Fig. 1),
as suggested by Kersten et al. (10). Such an equilibrium be-
tween the open and cyclic hemiacetal forms is also suggested
for the naphthalene metabolism by the NAH7 plasmid-en-
coded enzymes by Eaton and Chapman (7). Based on the
chemical structure of PDC as an ?-pyrone, it would be pro-
duced from the cyclic hemiacetal form of CHMS. Previously,
Maruyama et al. (17) determined the CHMS dehydrogenase
activity by monitoring the decrease in absorbance at 410 nm,
which is the absorption maximum of CHMS (17). In this study,
we monitored the generation of NADH and NADPH from
NAD?and NADP?, respectively, during CHMS oxidation to
examine the CHMS dehydrogenase activity. The absorbance at
FIG. 4. Identification of the metabolite accumulated in DLC culture from vanillate. Vanillate was incubated with the whole cells of DLC for 12 h. The negative-ion
ESI-MS spectrum of a portion of the supernatant of a reaction mixture is shown. The detected ion (II) at m/z 153.0 corresponded to the deprotonated molecular ion
of PCA and was a major product from vanillate (ion I). The ion at m/z 185.0 representing CHMS was not detected.
TABLE 3. Kinetic constants of the CHMS dehydrogenases
150 24.6 ? 1.5 (NADP?) 363 ? 1.4
20.6 ? 1.0 (CHMS)
26.0 ? 0.5 (CHMS)
252 ? 3.9 (NAD?) 449 ? 3.9
175 ? 10
383 ? 3.8
aThe Kms indicated are the values for the compounds shown in parentheses.
bThe results for the P. ochraceae enzyme have been reported in a previous
6656MASAI ET AL. J. BACTERIOL.
410 nm of CHMS generated from PCA by LigAB gradually
decreased; it did not correspond to the amount of CHMS
estimated by ESI-MS. When CHMS was prepared from PCA,
the amount of CHMS estimated by ESI-MS increased as time
went by. However, the absorbance at 410 nm initially increased
and then decreased (unpublished results), which indicates that
the absorbance at 410 nm represents the amount of the open
form of CHMS rather than the total amount of CHMS, and the
absorbance at 410 nm is not a good index of the activity of
CHMS dehydrogenase, whose substrate was suggested to be a
hemiacetal form of CHMS by Kersten et al. (10) and Ma-
The purified LigC showed features in common with CHMS
dehydrogenase of Pseudomonas ochraceae, including the sub-
unit structure, molecular mass, pI, and kinetic parameters (Ta-
ble 3). Both enzymes showed a much higher affinity to NADP?
than to NAD?, which is a remarkable common kinetic feature.
Both were inhibited by the addition of sulfhydryl reagents. This
may suggest that the cysteine residue plays a key role in the
enzyme reaction. One of the possible roles of cysteine residue
is as a ligand to a metal ion. Based on the fact that enzyme
activities of both LigC and P. ochraceae CHMS dehydrogenase
were not affected by the addition of EDTA, it is clear that the
cysteine residue is not involved in the binding of metal ions
such as zinc in the group I dehydrogenases (31).
The ligC insertion mutant, DLC, lost the ability to grow on
vanillate, indicating that the ligC gene is essential for vanillate
catabolism (Fig. 2B). On the other hand, the ligC disruption
did not affect the growth on syringate. These results are con-
sistent with those obtained regarding both the ligB (H.
Aoshima, E. Masai, S. Nishikawa, Y. Katayama, and M.
Fukuda, Abstr. 8th Int. Symp. Microb. Ecol., abstr. 93, 1998)
and ligI (23) disruptions. Although PCA 4,5-dioxygenase has
the ability to convert 3-O-methylgallate, a metabolic interme-
diate of syringate, to PDC (10), these results indicated that
syringate is mainly metabolized through a pathway in SYK-6
other than the PCA 4,5-cleavage pathway encoded by the ligAB,
ligC, and ligI genes.
Interestingly, it was not CHMS but PCA which accumulated
from vanillate during the incubation with the ligC insertion
mutant DLC (Fig. 4). When pBBRC1221 carrying ligC was
introduced into the strain DLC, the resultant transformant
recovered the ability to grow on vanillate. We confirmed that
the PCA 4,5-dioxygenase activity of the cell extract of E. coli
carrying ligAB in response to 100 ?M PCA was not inhibited by
the equivalent molar amount of CHMS (data not shown). This
result suggested that the accumulation of PCA observed in the
DLC culture containing vanillate was not due to the product
inhibition of PCA 4,5-dioxygenase. Thus, PDC seemed to be
an inducing substance for PCA 4,5-dioxygenase encoded by
ligAB. In the absence of PDC, PCA 4,5-dioxygenase is not
induced in DLC and PCA will be accumulated instead of
CHMS. This notion is supported by the results with ligI inser-
tion mutant DLI, which accumulated PDC from vanillate (23).
To address the details of regulation of the PCA 4,5-cleavage
pathway genes, promoter regions and the regulatory genes
governing them should be clarified.
This work was supported in part by a Grant-in Aid for Encourage-
ment of Young Scientists (no. 11760057) from the ministry of Educa-
tion, Science, Sports and Culture, Japan.
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.
Smith, and K. Struhl. 1990. Current protocols in molecular biology. John
Wiley & Sons, Inc., New York, N.Y.
2. Bagdasarian, M., R. Lurz, B. Ru ¨ckert, F. C. H. Franklin, M. M. Bagdasar-
ian, J. Frey, and K. N. Timmis. 1981. Specific purpose plasmid cloning
vectors. II. Broad host range, high copy number, RSF1010 derived vectors,
and a host-vector system for gene cloning in Pseudomonas. Gene 16:237–247.
3. Bolivar, F., and K. Backman. 1979. Plasmids of Escherichia coli as cloning
vectors. Methods Enzymol. 68:245–267.
4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye bind-
ing. Anal. Biochem. 72:248–254.
5. Chang, H.-K., and G. J. Zylstra. 1998. Novel organization of the genes for
phthalate degradation from Burkholderia cepacia DBO1. J. Bacteriol. 180:
6. Crawford, R. L., J. W. Bromley, and P. E. Perkins-Olson. 1979. Catabolism
of protocatechuate by Bacillus macerans. Appl. Environ. Microbiol. 37:614–
7. Eaton, R. W., and P. J. Chapman. 1992. Bacterial metabolism of naphtha-
lene: construction and use of recombinant bacteria to study ring cleavage of
1,2-dihydroxynaphthalene and subsequent reactions. J. Bacteriol. 174:7542–
8. Harwood, C. S., and R. E. Parales. 1996. The ?-ketoadipate pathway and the
biology of self-identity. Annu. Rev. Microbiol. 50:553–590.
9. Katayama, Y., S. Nishikawa, M. Nakamura, K. Yano, M. Yamasaki, N.
Morohoshi, and T. Haraguchi. 1987. Cloning and expression of Pseudomo-
nas paucimobilis SYK-6 genes involved in the degradation of vanillate and
protocatechuate in P. putida. Mokuzai Gakkaishi 33:77–79.
10. Kersten, P. J., S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lips-
comb. 1982. 2-Pyrone-4,6-dicarboxylic acid, a catabolite of gallic acids in
Pseudomonas species. J. Bacteriol. 152:1154–1162.
11. Knauf, V. C., and E. W. Nester. 1982. Wide host range cloning vectors: a
cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid 8:45–54.
12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature (London) 227:680–685.
13. Maruyama, K. 1979. Isolation and identification of the reaction product of
?-hydroxy-?-carboxymuconic-ε-semialdehyde dehydrogenase. J. Biochem.
14. Maruyama, K. 1983. Purification and properties of 2-pyrone-4,6-dicarboxy-
late hydrolase. J. Biochem. 93:557–565.
15. Maruyama, K. 1985. Purification and properties of ?-oxalomesaconate hy-
dratase from Pseudomonas ochraceae grown with phthalate. Biochem. Bio-
phys. Res. Commun. 128:271–277.
16. Maruyama, K. 1990. Purification and properties of 4-hydroxy-4-methyl-2-
oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate.
J. Biochem. 108:327–333.
17. Maruyama, K., N. Ariga, M. Tsuda, and K. Deguchi. 1978. Purification and
properties of ?-hydroxy-?-carboxymuconic-ε-semialdehyde dehydrogenase.
J. Biochem. 83:1125–1134.
18. Masai, E., Y. Katayama, S. Kawai, S. Nishikawa, M. Yamasaki, and N.
Morohoshi. 1991. Cloning and sequencing of the gene for a Pseudomonas
paucimobilis enzyme that cleaves ?-aryl ether. J. Bacteriol. 173:7950–7955.
19. Masai, E., Y. Katayama, S. Kubota, S. Kawai, M. Yamasaki, and N. Moro-
hoshi. 1993. A bacterial enzyme degrading the model lignin compound
?-etherase is a member of the glutathione-S-transferase superfamily. FEBS
20. Masai, E., Y. Katayama, S. Nishikawa, and M. Fukuda. 1999. Characteriza-
tion of Sphingomonas paucimobilis SYK-6 genes involved in degradation of
lignin-related compounds. J. Ind. Microbiol. Biotechnol. 23:364–373.
21. Masai, E., Y. Katayama, S. Nishikawa, M. Yamasaki, N. Morohoshi, and T.
Haraguchi. 1989. Detection and localization of a new enzyme catalyzing the
?-aryl ether cleavage in the soil bacterium (Pseudomonas paucimobilis SYK-
6). FEBS Lett. 249:348–352.
22. Masai, E., S. Kubota, Y. Katayama, S. Kawai, M. Yamasaki, and N. Moro-
hoshi. 1993. Characterization of the C?-dehydrogenase gene involved in the
cleavage of ?-aryl ether by Pseudomonas paucimobilis SYK-6. Biosci. Bio-
technol. Biochem. 57:1655–1659.
23. Masai, E., S. Shinohara, H. Hara, S. Nishikawa, Y. Katayama, and M.
Fukuda. 1999. 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. J. Bacteriol. 181:55–62.
24. Nakatsu, C. H., M. Providenti, and R. C. Wyndham. 1997. The cis-diol
dehydrogenase cbaC gene of Tn5271 is required for growth on 3-chloroben-
zoate but not 3,4-dichlorobenzoate. Gene 196:209–218.
25. Nakatsu, C. H., and R. C. Wyndham. 1993. Cloning and expression of the
transposable chlorobenzoate 3,4-dioxygenase genes of Alcaligenes sp. strain
BR60. Appl. Environ. Microbiol. 59:3625–3633.
26. Nishikawa, S., T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N.
Morohoshi, and Y. Katayama. 1998. Cloning and sequencing of the Sphin-
gomonas (Pseudomonas) paucimobilis gene essential for the O demethylation
of vanillate and syringate. Appl. Environ. Microbiol. 64:836–842.
27. Noda, Y., S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda,
Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. 1990. Mo-
lecular cloning of the protocatechuate 4,5-dioxygenase gene of Pseudomonas
VOL. 182, 2000DEHYDROGENASE FOR PROTOCATECHUATE 4,5-CLEAVAGE PATHWAY 6657
paucimobilis. J. Bacteriol. 172:2704–2709.
28. Nomura, Y., M. Nakagawa, N. Ogawa, S. Harashima, and Y. Oshima. 1992.
Genes in PHT plasmid encoding the initial degradation pathway of phthalate
in Pseudomonas putida. J. Ferment. Bioeng. 74:333–334.
29. Peng, X., T. Egashira, K. Hanashiro, E. Masai, S. Nishikawa, Y. Katayama,
K. Kimbara, and M. Fukuda. 1998. Cloning of a Sphingomonas paucimobilis
SYK-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl
and characterization of the enzyme. Appl. Environ. Microbiol. 64:2520–2527.
30. Peng, X., E. Masai, Y. Katayama, and M. Fukuda. 1999. Characterization of
the meta-cleavage compound hydrolase gene involved in degradation of the
lignin-related biphenyl structure by Sphingomonas paucimobilis SYK-6.
Appl. Environ. Microbiol. 65:2789–2793.
31. Reid, M. F., and C. A. Fewson. 1994. Molecular characterization of microbial
alcohol dehydrogenases. Crit. Rev. Microbiol. 20:13–56.
32. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
33. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.
34. Scha ¨fer, A., A. Tauch, W. Ja ¨ger, J. Kalinowski, G. Thierbach, and A. Pu ¨hler.
1994. Small mobilizable multi-purpose cloning vectors derived from the
Escherichia coli plasmids pK18 and pK19: selection of defined deletions in
the chromosome of Corynebacterium glutamicum. Gene 145:69–73.
35. Shine, J., and L. Dalgarno. 1974. The 3?-terminal sequence of Escherichia
coli 16S ribosomal RNA: complementary to nonsense triplets and ribosome
binding sites. Proc. Natl. Acad. Sci. USA 71:1342–1346.
36. Short, J. M., J. M. Fernandez, J. A. Sorge, and W. Huse. 1988. ?ZAP: a
bacteriophage ? expression vector with in vivo excision properties. Nucleic
Acids Res. 16:7583–7600.
37. Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA
polymerase to direct selective high-level expression of cloned genes. J. Mol.
38. Sugimoto, K., T. Senda, H. Aoshima, E. Masai, M. Fukuda, and Y. Mitsui.
1999. Crystal structure of an aromatic-ring-opening dioxygenase LigAB
which is a protocatechuate 4,5-dioxygenase. Struct. Fold. Des. 15:953–965.
39. Tack, B. F., P. J. Chapman, and S. Dagley. 1972. Purification and properties
40. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived
system for insertion mutagenesis and sequencing with synthetic universal
primers. Gene 19:259–268.
41. Wolgel, S. A., J. E. Dege, P. E. Perkins-Olson, C. H. Juarez-Garcia, R. L.
Crawford, E. Munck, and J. D. Lipscomb. 1993. Purification and character-
ization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new
extradiol catecholic dioxygenase. J. Bacteriol. 175:4414–4426.
42. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage
cloning vectors and host strains: nucleotide sequences of the M13mp18 and
pUC19 vectors. Gene 33:103–119.
6658MASAI ET AL. J. BACTERIOL.