In vitro reconstitution of the catabolic reactions catalyzed by PcaHG, PcaB, and PcaL: The protocatechuate branch of the β-ketoadipate pathway in Rhodococcus jostii RHA1

Article abstract of DOI:10.1080/09168451.2014.993915

The β-ketoadipate pathway is a major pathway involved in the catabolism of the aromatic compounds in microbes. The recent progress in genome sequencing has led to a rapid accumulation of genes from the β-ketoadipate pathway in the available genetic database, yet the functions of these genes remain uncharacterized. In this study, the protocatechuate branch of the β-ketoadipate pathway of Rhodococcus jostii was reconstituted in vitro. Analysis of the reaction products of PcaHG, PcaB, and PcaL was achieved by high-performance liquid chromatography. These reaction products, β-ketoadipate enol-lactone, 3-carboxy-cis,cis-muconate, γ-carboxymuconolactone, muconolactone, and β-ketoadipate, were further characterized using LC-MS and nuclear magnetic resonance. In addition, the in vitro reaction of PcaL, a bidomain protein consisting of γ-carboxy-muconolactone decarboxylase and β-ketoadipate enol-lactone hydrolase activities, was demonstrated for the first time. This work provides a basis for analyzing the catalytic properties of enzymes involved in the growing number of β-ketoadipate pathways deposited in the genetic database.

Full text of DOI:10.1080/09168451.2014.993915

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In vitro reconstitution of the catabolic reactions
catalyzed by PcaHG, PcaB, and PcaL: the
protocatechuate branch of the β-ketoadipate
pathway in Rhodococcus jostii RHA1
Tomoya Yamanashia, Seung-Young Kima, Hirofumi Harab & Nobutaka Funaa
a Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka,
Shizuoka, Japan
b Department of Environmental Engineering and Green Technology, Malaysia-Japan
International Institute of Technology, Universiti Teknologi Malaysia, Kuala Lumpur,
Malaysia
Published online: 03 Jan 2015.
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To cite this article: Tomoya Yamanashi, Seung-Young Kim, Hirofumi Hara & Nobutaka Funa (2015): In vitro reconstitution
of the catabolic reactions catalyzed by PcaHG, PcaB, and PcaL: the protocatechuate branch of the β-ketoadipate pathway
in Rhodococcus jostii RHA1, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.993915
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Bioscience, Biotechnology, and Biochemistry, 2014
In vitro reconstitution of the catabolic reactions catalyzed by PcaHG, PcaB,
and PcaL: the protocatechuate branch of the β-ketoadipate pathway in
Rhodococcus jostii RHA1
Tomoya Yamanashi1, Seung-Young Kim1, Hirofumi Hara2,* and Nobutaka Funa1,*
1Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan;
2Department of Environmental Engineering and Green Technology, Malaysia-Japan International Institute of
Technology, Universiti Teknologi Malaysia, Kuala Lumpur, Malaysia
Received September 29, 2014; accepted November 25, 2014
http://dx.doi.org/10.1080/09168451.2014.993915
The β-ketoadipate pathway is a major pathway
involved in the catabolism of the aromatic com-
pounds in microbes. The recent progress in genome
sequencing has led to a rapid accumulation of genes
from the β-ketoadipate pathway in the available
genetic database, yet the functions of these genes
remain uncharacterized. In this study, the protoca-
techuate branch of the β-ketoadipate pathway of
Rhodococcus jostii was reconstituted in vitro. Analy-
sis of the reaction products of PcaHG, PcaB, and
PcaL was achieved by high-performance liquid chro-
matography. These reaction products, β-ketoadipate
enol-lactone, 3-carboxy-cis,cis-muconate, γ-carbo-
xymuconolactone, muconolactone, and β-ketoadi-
pate, were further characterized using LC-MS and
nuclear magnetic resonance. In addition, the in vitro
reaction of PcaL, a bidomain protein consisting of
enol-lactone (2) (Fig. 1). 2 is then further catabolized to
the tricarboxylic acid cycle intermediates, acetyl-CoA
and succinyl-CoA, by downstream enzymes of the β-ke-
toadipate pathway.2) Since the 1940s, the enzymology
of the β-ketoadipate pathway of Pseudomonas and
Acinetobacter has been extensively studied,4) leading to
the unraveling of this complex reaction pathway and the
chemical structures of the intermediates involved,
including 2, 3-carboxy-cis,cis-muconate (3), γ-carbo-
xymuconolactone (4), and muconolactone (5) (Fig. 1).5)
The protocatechuate branch of the β-ketoadipate path-
way begins with an ortho-cleavage of 1 by PcaHG, a
protocatechuate 3,4-dioxygenase (Fig. 1),6,7) resulting in
the formation of 3. PcaB, a 3-carboxy-cis,cis-muconate
cycloisomerase, catalyzes the lactonization of 3 to form
4.8,9) Decarboxylation of 4 is catalyzed by PcaC, a γ-car-
boxy-muconolactone decarboxylase,10) but this reaction
can also occur spontaneously.11) The resulting decarbox-
ylated product, 2, can be isomerized to form 5, and the
two exist in equilibrium.11) PcaD, β-ketoadipate
enol-lactone hydrolase, also known as 3-oxoadipate-enol
lactonase, hydrolyzes 2 to form β-ketoadipate (6).12,13)
Rhodococcus jostii RHA1 was first isolated from
γ-hexachlorocyclohexane-contaminated soil and initially
characterized for its polychlorinated biphenyl-degrading
properties.14) Genome sequence analyses of R. jostii
RHA1 further indicated the potential ability of R. jostii
RHA1 to degrade a broad range of aromatic com-
pounds.15) For example, Hara et al. demonstrated the
growth of R. jostii RHA1 on both phthalate and tere-
phthalate through the use of pad and tpa genes, which
encode the enzymes for the initial steps of phthalate
and terephthalate degradation, respectively. These gene
clusters were identified on two large linear plasmids of
R. jostii RHA1, namely, pRHL1 and pRHL2.16) By
proteomic analysis, Patrauchan et al. confirmed the
translation of the pad genes, pcaB, pcaL, and pcaF of
R. jostii RHA1 grown on phthalate.17) In addition, they
detected the protocatechuate 3,4-dioxygenase activity
from the cell-free extracts of R. jostii RHA1 grown on
phthalate.17)
γ-carboxy-muconolactone
decarboxylase
and
β-ketoadipate enol-lactone hydrolase activities, was
demonstrated for the first time. This work provides
a basis for analyzing the catalytic properties of
enzymes involved in the growing number of
β-ketoadipate pathways deposited in the genetic
database.
Key words: β-ketoadipate pathway; Rhodococcus
jostii RHA1; protocatechuate
Aromatic compounds, one of the most abundant clas-
ses of naturally occurring compounds, are primarily
found in microbial cells as degradation products of
plant-derived molecules such as lignins.1) Microorgan-
isms have developed the ability to use an impressive
variety of aromatic compounds as carbon and energy
sources.2,3) The β-ketoadipate pathway is one of the
main biodegradation pathways for aromatic compounds
in aerobic soil bacteria and fungi.2) This pathway
includes the protocatechuate and catechol branches,
which catabolize protocatechuate (1) and catechol,
respectively. Both branch converges at β-ketoadipate
*Corresponding authors. Email: hhara@utm.my (H. Hara); funa@u-shizuoka-ken.ac.jp (N. Funa)
© 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
T. Yamanashi et al.
spontaneously
COOH
COOH
O
COOH
O
or
PcaHG
PcaB
PcaL
PcaL
PcaD
O
O
COOH
COOH
COOH
COOH
COOH
PcaC
OH
COOH
OH
O
3-carboxy-
-carboxy-
γ
-ketoadipate
β
-ketoadipate (6)
β
cis,cis-muconate (3) muconolactone (4)
protocatechuate (1)
enol-lactone (2)
O
COOH
O
muconolactone (5)
Fig. 1. The protocatechuate branch of the β-ketoadipate pathway from R. jostii RHA1.
Note: PcaL, instead of PcaC and PcaD, is responsible of transfer from 4 to 6 via 2 in R. jostii RHA1.
In R. jostii RHA1, the chromosomal pca cluster
consists of genes predicted to encode the enzymes Pca-
JIHGBLF,15,17) which are required to convert 1 to
acetyl-CoA and succinyl-CoA. The genes pcaH and
pcaG encode the two subunits of protocatechuate 3,4-
dioxygenase,15) and pcaB encodes a 3-carboxy-cis,cis-
muconate lactonizing enzyme.15) pcaL is a fused gene
encoding for an enzyme with both γ-carboxy-mucono-
lactone decarboxylase and β-ketoadipate enol-lactone
hydrolase activities (Fig. 1).15,17,18)
In this study, the protocatechuate branch of the β-ke-
toadipate pathway from R. jostii RHA1 was reconsti-
tuted in vitro using recombinant PcaHG, PcaB, and
PcaL. The bifunctional nature of PcaL was character-
ized biochemically for the first time. All compounds of
the protocatechuate branch, including the highly unsta-
ble compound, 3, were characterized by high-perfor-
mance liquid chromatography (HPLC), nuclear
magnetic resonance (NMR), and liquid chromatogra-
phy-electrospray ionization/high-resolution mass spec-
trometry (LC-ESI/HRMS) analyses.
accession numbers of the amino acid sequences of PcaG,
PcaH, PcaB, and PcaL are ABG93160, ABG93159,
ABG93161, and ABG93162, respectively. An NdeI site
was introduced at the start codon of pcaHG by PCR with
primer I: 5′-CGGAATTCCATATGCTGCATCTGCCA-
GCCC-3′ (the EcoRI site is underlined; the NdeI site is
italicized), and primer II: 5′-CGCAAGCTTCGA-
CACCTTTCTGCGTTGTG-3′ (the HindIII site is under-
lined). The amplified fragments were cloned between the
EcoRI and HindIII sites of pUC19, resulting in pUC19-
pcaHG. An NdeI site was introduced at the start codon
of pcaB by PCR with primer III: 5′-CGGAATTCCA-
TATGAACTCTCCGGAGCCTT-3′ (the EcoRI site is
underlined; the NdeI site is italicized), and primer IV: 5′-
CGCAAGCTTGTGTGCGAGTGCGACTG-3′
(the
HindIII site is underlined). The amplified fragments were
cloned between the EcoRI and HindIII sites of pUC19,
resulting in pUC19-pcaB. An NdeI site was introduced
at the start codon of pcaL by PCR with primer V: 5′-
CGTCTAGACATATGACAGTCGCACTCGCA-3′ (the
XbaI site is underlined; the NdeI site is italicized), and
primer VI: 5′-CGCAAGCTTCGTCGGTTCGGTAG-
CGG-3′ (the HindIII site is underlined). The amplified
fragments were cloned between the XbaI and HindIII
sites of pUC19, resulting in pUC19-pcaL. DNA
sequencing of pUC19-pcaHG, pUC19-pcaB, and
pUC19-pcaL confirmed the correct sequence. Each
NdeI-HindIII fragment excised from pUC19-pcaHG,
pUC19-pcaB, and pUC19-pcaL was cloned between the
NdeI and HindIII sites of pColdI, resulting in pColdI-
pcaHG, pColdI-pcaB, and pColdI-pcaL, respectively.
Materials and methods
Chemicals and bacterial strains.
Protocatechuate
was purchased from Wako Pure Chemical Industries,
Ltd. (Osaka, Japan). Luria-Bertani (LB) Lennox med-
ium was purchased from Sigma-Aldrich (St. Louis,
MO, USA). Escherichia coli HST08, the pColdI plas-
mid, restriction enzymes, and other DNA-modifying
enzymes used for DNA manipulation were purchased
from Takara Bio Inc (Shiga, Japan). E. coli BL21 was
purchased from the National BioResource Project
(National Institute of Genetics of Japan). R. jostii RHA1
was obtained from Prof. Masao Fukuda. All other
reagents were purchased from Wako Pure Chemicals.
Expression and purification of recombinant PcaHG,
PcaB, and PcaL.
E. coli BL21 harboring pColdI-
pcaHG, pColdI-pcaB, or pColdI-pcaL was grown at
37 °C in 50 mL of LB broth containing 100 μg mL−1
of ampicillin. When an optical density at 600 nm
reached 0.5, cells were incubated for 30 min at 15 °C
and then protein expression was induced by the addi-
tion of 0.1 mM isopropyl β-D-thiogalactoside. Cells
were then cultured for an additional 24 h at 15 °C.
Cells were harvested by centrifugation and resuspended
in 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM
Construction of pColdI-pcaHG, pColdI-pcaB, and
pColdI-pcaL. Media, growth conditions, and general
recombinant DNA techniques of E. coli were described
by Sambrook et al.19) The chromosomal DNA of R. jostii
RHA1 was used as a template for PCR experiments. The

In vitro reconstitution of the β-ketoadipate pathway
3
imidazole, and 10% glycerol. The resuspended cells
were collected by centrifugation, and a crude cell lysate
was prepared by sonication and cell debris was
removed by centrifugation at 20,000 × g for 20 min.
The cleared lysate was applied to Ni-nitrilotriacetic acid
columns (Qiagen, Hilden, Germany), washed twice
with 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl,
20 mM imidazole, and 10% glycerol. The purified his-
tidine-tagged protein was eluted with a buffer contain-
ing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl,
250 mM imidazole, and 10% glycerol and was dialyzed
two times against 2 L of 10 mM Tris–HCl (pH 7.5)
containing 10% glycerol. The protein concentration
was measured by Bradford assay with a Bio-Rad
Protein Assay Kit (Bio-Rad Laboratories, Hercules,
CA, USA) using bovine serum albumin as a standard.
The purity of N-terminal His-tagged (MNHKVHHH-
HHHIEGRH) PcaHG, PcaB, and PcaL was verified by
SDS analysis (Supplemental Fig. S1). The proteins
were stored at −80 °C before use.
COSMOSIL 5C18-PAQ column (2.0 × 150 mm; Nacalai
Tesque Inc., Kyoto, Japan) was used, and analytes were
eluted under the isocratic condition of 3% acetonitrile in
water (both containing 0.1% formic acid) at a flow rate
of 0.2 mL min−1 at 40 °C.
NMR and MS data. β-ketoadipate enol-lactone (2).
1H NMR (400 MHz, D2O): δ 3.36 (d, 2H, J = 2.0 Hz,
C2H2), 3.49 (s, 2H, C3H), 5.58 (s, 1H, C5H2). Nega-
tive mode LC-ESI/HRMS, [M − H]− = 141.01835 (cal-
culated for C6H5OÀ4 , 141.01933).
1
3-carboxy-cis,cis-muconate (3). H NMR (400 MHz,
CD3OD): δ 6.01(dd, 1H, J = 0.9, 12.0 Hz, C5H), 6.50
(m, 1H, C2H), 6.77 (dd, 1H, J = 1.7, 12.0 Hz, C4H).
Negative mode LC-ESI/HRMS, [M − H]− = 185.00800
(calculated for C7H5OÀ6 , 185.00916).
γ-carboxymuconolactone (4). Negative mode LC-
ESI/HRMS, [M − H]− = 185.00841 (calculated for
C7H5OÀ6 , 185.00916).
muconolactone (5). 1H NMR (400 MHz, D2O): δ
2.71 (dd, 1H, J = 8.2, 16.6 Hz, C1′Ha), 2.95 (dd, 1H,
J = 4.8, 16.6 Hz, C1′Hb), 5.57 (dddd, 1H, J = 1.4, 1.9,
4.8, 8.2 Hz, C5H), 6.24 (dd, 1H, J = 1.9, 5.8 Hz,
C3H), 7.81 (dd, 1H, J = 1.4, 5.8 Hz, C4H). Negative
mode LC-ESI/HRMS, [M − H]− = 141.01831 (calcu-
lated for C6H5OÀ4 , 141.01933).
In vitro reactions with protocatechuate. The enzy-
matic reactions, containing 1 μg each of PcaHG, PcaB,
and/or PcaL, 1 mM of protocatechuate, and 50 mM
Tris–HCl (pH 7.5), were performed in a total volume
of 300 μL. The boiled enzymes were used as negative
controls. The reaction mixtures were incubated at
30 °C for 5 min and were stopped by filtration with
Amicon Ultra-0.5 Centrifugal Filters (Merck Millipore,
Darmstadt, Germany). Aliquots (5 μL) of the filtrate
were directly analyzed via HPLC (JASCO International
Co., Tokyo, Japan). Reverse-phase HPLC analysis was
carried out using a COSMOSIL 5C18-PAQ column
(4.6 × 150 mm; Nacalai Tesque Inc., Kyoto, Japan),
and the analytes were subjected to an isocratic elution
with 3% acetonitrile in water (both containing 0.1% tri-
fluoroacetic acid) at a flow rate of 1 mL min−1 at
40 °C. UV absorbance was detected at 200 nm.
1
β-ketoadipate (6). H NMR (400 MHz, CD3OD): δ
2.55 (t, 2H, J = 6.5 Hz, C5H), 2.86 (t, 2H, J = 6.5 Hz,
C4H), 3.39 (s, 2H, C2H2). Negative mode LC-ESI/
HRMS, [M − H]− = 159.02898 (calculated for C6H7OÀ5 ,
159.02990).
Results
In vitro catabolism of protocatechuate by PcaHG
The first step of the catabolism of protocatechuate
(1) in the β-ketoadipate pathway is a ring-opening reac-
tion catalyzed by PcaHG.6,7) An SDS PAGE-verified
pure heterodimer of PcaHG (Supplemental Fig. S1)
was incubated with 1 as described in the Methods sec-
tion. HPLC analysis confirmed that 1 was readily con-
verted to the product, 3, which migrated at a retention
time (RT) of 5.0 min. In control experiments with
boiled, inactive PcaHG, 1 was not converted to product
as evidenced by an RT of 10.2 min (Fig. 2(A)). The
structure of the reaction product was confirmed by the
NMR and MS analyses. The compounds, 2, 3, 5,
and 6, used for NMR analyses were prepared from the
in vitro reaction, which was scaled up to 10 mL. For
preparation of 2, 5, and 6, the reaction mixtures were
acidified with 1 mL of 1 M HCl and extracted with ethyl
acetate. The organic layers were evaporated until dry.
The crude materials were dissolved in a small amount
of methanol and purified using a reverse-phase prepara-
tive HPLC equipped with a COSMOSIL 5C18-AR-II
column (20 × 250 mm; Nacalai Tesque Inc., Kyoto,
Japan). The compounds were chromatographed using
10% methanol and 0.1% trifluoroacetic acid in water as
a mobile phase, at a flow rate of 3 mL/min at ambient
temperature. Because 3 was easily transformed into a
geometical isomer under acidic conditions, the reaction
containing 3 was filtrated with Amicon Ultra-0.5
Centrifugal Filters and the combined filtrate was lyophi-
lized. The lyophilizate was used directly for NMR anal-
yses. NMR data were collected on a Bruker AVANCE
III 400 FT-NMR spectrometer (Bruker Corporation,
Billerica, MA, USA). LC-ESI/HRMS analysis was car-
ried out using a Q Exactive™ (Thermo Fisher Scientific
Inc., Waltham, MA, US). For LC-ESI/HRMS analysis, a
1
negative mode LC-ESI/HRMS and H NMR analyses
(Fig. 2(B) and Supplemental Fig. S2). The unstable
nature of the product hindered us from conducting 13C
NMR and two-dimensional NMR analyses. The m/z of
the product, 185.00800, was in good agreement with
the calculated theoretical value, 185.00916, of the
[M − H]− ion of 3. The geometrical isomerism of the
product was characterized from the coupling constants
1
calculated from the H NMR spectrum (Supplemental
Fig. S2). A coupling constant of 12.0 Hz characteristic
of a cis-coupling across a double bond was observed
for C4- and C5-protons (Supplemental Fig. S3), indi-
cating that the geometrical isomerism of the olefin at
C4–C5 position is in a cis-configuration. In addition, a
long-range coupling was observed between the C2- and
C5-protons, since the C5 proton appeared to be a

4
T. Yamanashi et al.
(A)
(A)
3
3
4
1
1
iii
ii
i
ii
i
0
8
4
12
Time (min)
0
8
4
12
Time (min)
(B)
[M-H]-
185.00800
(B)
magnified
[M-H]-
185.00841
[M-2CO2-H]-
97.02823
[M-CO2-H]-
141.01831
[M-CO2-H]-
141.01820
[M-2CO2-H]-
97.02817
175
190
80
140
200
m/z
80
140
200
m/z
Fig. 3. HPLC and MS analyses of the product from PcaHG and
PcaB reaction.
Fig. 2. HPLC and MS analyses of the product from PcaHG
reaction.
Note: (A) HPLC chromatograms of in vitro reactions of PcaHG
and PcaB. All enzymes were active unless inactivated by boiling
prior to use in assay. Boiled PcaHG and PcaB (i), PcaHG and boiled
PcaB (ii), and PcaHG and PcaB (iii) were used as enzymes. UV
absorption was detected at 200 nm. (B) LC-ESI/HRMS spectrum of
γ-carboxymuconolactone (4). [M − H]−, [M − CO2 − H]−, and
[M − 2CO2 − H]− ions were observed. Decarboxylation of 4 occurred
during ionization resulting in the observation of both [M − CO2 − H]−
and [M − 2CO2 − H]− as major ions.
Note: (A) HPLC chromatograms of in vitro reactions of PcaHG.
Boiled PcaHG (i) and active PcaHG (ii) were used as enzymes. UV
absorption was detected at 200 nm. (B) LC-ESI/HRMS spectrum of
3-carboxy-cis,cis-muconate (3). [M − H]−, [M − CO2 − H]−, and
−
[M − 2CO2 − H] ions were observed.
doublet of doublets (Supplemental Fig. S2). The
geometrical isomerism of the olefin at the C2–C3 posi-
tion was deduced to be in a cis-configuration, since the
coupling constant of the dienylic coupling was calcu-
lated to be 1.7 Hz (Supplemental Fig. S3). Taken
together, molecule 3 was determined to be the product
of the reaction of PcaHG with 1 via an enzyme cata-
lyzed oxidation.
form of 5 is more energetically favorable than 2
(Supplemental Fig. S4). Unfortunately, no NMR data
of 4 were successfully obtained, because 4 proved to
be extremely unstable.
The molecular formula for 5 was determined to be
C6H6O4 since the LC-ESI/HRMS analysis showed that
the m/z of the product of decarboxylation of 4 was
141.01831—a value that was in agreement with the
theoretical value, 141.01933, of the [M − H]− ion of 5
(Supplemental Fig. S5). Similarly, the molecular for-
mula of 2 was also determined as C6H6O4 (Supplemen-
In vitro catabolism of protocatechuate by PcaHG
and PcaB
The second step of the β-ketoadipate pathway is a
cycloisomerization of 3 catalyzed by PcaB, forming
molecule 4.8) When active PcaB and PcaHG were
1
tal Fig. S6). The H NMR data of 5 were assigned as
follows: the doublets at 6.24 and 7.81 ppm were
assigned to the C4 and C3 protons of the dihydrofuran
ring; the dddd at 5.57 ppm was assigned to the C5 pro-
ton; the doublet of doublets at 2.71 and 2.95 ppm were
assigned to the germinal protons at C1′, since the cou-
pling constant between these protons was 16.6 Hz
simultaneously incubated with 1,
a product was
observed at an RT of 3.0 min via HPLC analysis
(Fig. 3(A)). This product was not generated in the reac-
tions when boiled PcaB or PcaHG were used instead of
active enzymes (Fig. 3(A)). The m/z of the product,
185.00841, was in good agreement with the calculated
theoretical value, 185.00916, of the [M − H]− ion of 4
(Fig. 3(B)). A filtrate of the reaction was incubated at
30 °C to examine whether facile decarboxylation of the
product occurred (Supplemental Fig. S4). As a result, a
rapid decrease in the product with a concomitant
increase in 5 (see below for a structural characteriza-
tion) was observed. This suggests that the product of
PcaHG and PcaB reaction is 4, which then undergoes
spontaneous decarboxylation to form 2. It is known
that 2 and 5 exist in equilibrium,11) even though the
1
(Supplemental Fig. S7). In addition, the H NMR data
of 2 were consistent with the structure of β-ketoadipate
enol-lactone (Supplemental Fig. S8).
In vitro catabolism of protocatechuate by PcaHG,
PcaB, and PcaL
PcaL is a fusion protein composed of PcaC and
PcaD that catalyzes the decarboxylation of 4 and the
hydrolysis of 2, by their respective activities. When
PcaL, PcaB, and PcaHG were incubated with 1, a

In vitro reconstitution of the β-ketoadipate pathway
5
product was observed at an RT of 3.3 min via HPLC
2
trace (Fig. 4(A)). This product was not generated in
reactions with boiled, inactive PcaL, PcaB, or PcaHG
(Fig. 4(A)). Similar results were obtained from the
two-step reaction in which PcaL was added to the fil-
trate of the reaction of PcaHG and PcaB, as seen with
HPLC and LC-MS analyses (Supplemental Fig. S9).
The m/z of the product, 159.02892, was in good agree-
ment with the calculated theoretical value, 159.02990,
of the [M − H]− ion of 6 (Fig. 4(B)). In addition, the
1H NMR spectrum of 6 was identical to β-ketoadipate
(Supplemental Fig. S10).20) Furthermore, PcaL exhib-
ited a β-ketoadipate enol-lactone hydrolase activity with
2 in vitro, resulting in the formation of 6. HPLC
analysis revealed that 5 was not utilized as a substrate
in this reaction (Fig. 5). These results indicate that
PcaL is a bifunctional enzyme, functioning as both
γ-carboxy-muconolactone decarboxylase and β-ketoadi-
pate enol-lactone hydrolase.
6
iv
iii
5
ii
i
3
0
6
9
Time (min)
Fig. 5. HPLC chromatograms of in vitro reaction of PcaL.
Note: Reactions of boiled PcaL and 5 (i), active PcaL and 5 (ii),
boiled PcaL and 2 (iii), and active PcaL and 2 (iv).
pathway have not been thoroughly investigated.
Although Ravi et al. reported the proton NMR spec-
trum of 3, they did not observe the long-range cou-
plings of 3.5) On the other hand, Ainsworth et al.
reported the coupling constants of the dienylic
(J = 0.9 Hz) and allylic (J = 2.1 Hz) couplings of 3.21)
They explained that these “abnormal” coupling con-
stants can be attributed to the nonplanarity of 3. How-
ever, we have observed “normal” coupling constants
for the dienylic and allylic couplings as 1.7 and
0.9 Hz, respectively (Supplemental Fig. S4). We sup-
pose that the rapid sample handling allowed us to ana-
lyze the intact 3.
Discussion
Although a complete picture of the β-ketoadipate
pathway was depicted by the Ornston group in the
1970s,4) chromatographic, spectroscopic, and mass
spectrometric studies of the intermediates in this
(A)
6
4
3
In this study, enzymes of the protocatechuate branch
of the β-ketoadipate pathway from R. jostii, whose func-
tions were predicted based on sequence similarity, were
reconstituted in vitro. We accomplished the structural
determination of the reaction products of PcaHG, PcaB,
and PcaL using HPLC, LC-MS, and NMR, leading to a
biochemical characterization of these enzymes. PcaHG
catalyzes the oxidative cleavage of the aromatic ring of
protocatechuate to yield 3. Following cycloisomeriza-
tion by PcaB, the formation of the lactone ring gives 4.
PcaL, a bidomain protein consisting of PcaC and PcaD
activities, catalyzes the sequential decarboxylation and
hydrolysis reactions of 4 that result in the formation 6.
The Pca enzymes of R. jostii share moderate sequence
similarity with the enzymes whose catalytic properties
were characterized biochemically. For example, PcaG is
42% identical in amino acid sequence to the alpha chain
of protocatechuate 3,4-dioxygenase7) of P. aeruginosa.
Similarly, PcaH is 50% identical to the beta chain of
protocatechuate 3,4-dioxygenase7) of P. aeruginosa. In
addition, PcaB is 41% identical to 3-carboxy-cis,cis-
muconate lactonizing enzyme8) of P. putida. While the
N-terminus (residues 1–261) of PcaL is 45% identical
to β-ketoadipate enol-lactone hydrolase11) of P. putida,
the C-terminus (residues 272–400) of PcaL is 49% iden-
tical to γ-carboxy-muconolactone decarboxylase10) of P.
putida. Therefore, it is not surprising that the catalytic
reactions of the Pca enzymes of R. jostii were identical
to those of the homologous enzymes of Pseudomonas
species. To our knowledge, this is the first report
1
iv
iii
ii
i
0
8
4
12
Time (min)
[M-H]-
159.02892
(B)
170
145
m/z
Fig. 4. HPLC and MS analyses of the product from PcaHG, PcaB,
and PcaL reaction.
Note: (A) HPLC chromatograms of in vitro reactions of PcaHG,
PcaB, and PcaL. All enzymes were active unless inactivated by boil-
ing prior to use in assay. Boiled PcaHG, PcaB, and PcaL (i), PcaHG,
boiled PcaB, and PcaL (ii), PcaHG, PcaB, and boiled PcaL (iii),
PcaHG, PcaB, and PcaL (iv) were used as enzymes. UV absorption
was detected at 200 nm. (B) LC-ESI/HRMS spectrum of β-ketoadi-
pate (6). [M − H]− ion was observed.

6
T. Yamanashi et al.
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demonstrating the in vitro reactions of the Pca enzymes
from actinomycetes.
In the course of our study, 4 was found to be an
extremely unstable compound that was readily trans-
formed to 5 via spontaneous decarboxylation and isom-
erization. It is possible that the bifunctional PcaL is
more advantageous than the separate enzymes of PcaC
and PcaD in carbon and energy acquisition due to the
necessity of PcaC to release intermediate 2, allowing it
to equilibrate with 5, and resulting in the loss of the
carbon source from the pathway. We also found that 3
was an unstable compound and was readily trans-
formed into 3-carboxy-trans,trans-muconate (results
not shown). There may be unidentified enzymes that
scavenge these side products and bring them back to
the β-ketoadipate pathway to recover the invested
energy. Recent progress in genome sequencing has
revealed that the β-ketoadipate pathway genes are
distributed widely among microbial taxa. This work
provides a basis for analyzing the catalytic properties
of enzymes involved in the growing number of the
β-ketoadipate pathway genes deposited in the seem-
ingly continuously updated genetic databases.
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β-ketoadipate by Pseudomonas putida. II. Enzymes of the prot-
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Supplemental material
The supplementary material for this paper is available
at http://dx.doi.org/10.1080/09168451.2014.993915.
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Fernandes C, Miyazawa D, Wong W, Lillquist AL, Wang D,
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Schein JE, Shin H, Smailus D, Siddiqui AS, Marra MA, Jones
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Acknowledgment
We are grateful to Prof. M. Fukuda for providing us
R. jostii RHA1.
Funding
This work was, in part, supported by a Grant-in-Aid for
Scientific Research on Innovative Areas from the Ministry of
Education, Culture, Sports, Science and Technology [grant
number 23108521] (to N. F); The Naito Foundation (to N.
F.); The Kato Memorial Bioscience Foundation (to N. F.);
The Uehara Memorial Foundation (to N. F.).
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Eltis LD. Catabolism of benzoate and phthalate in Rhodococcus
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A
1H NMR