Role of cysteine residues in 4-oxalomesaconate hydratase from pseudomonas ochraceae NGJ1

Article abstract of DOI:10.1271/bbb.60503

4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wildand mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K_m-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl₂; and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.

Full text of DOI:10.1271/bbb.60503

Biosci. Biotechnol. Biochem., 71 (2), 449–457, 2007
Role of Cysteine Residues in 4-Oxalomesaconate Hydratase
from Pseudomonas ochraceae NGJ1
Suhong LI,1 Maho KIMURA,1 Teruo TAKASHIMA,1 Kunihiko HAYASHI,1 Kazunori INOUE,1
y
1;
Ryou ISHIGURO,1 Hiroyuki SUGISAKI,2 and Kiyofumi MARUYAMA
1Department of Biomolecular Science, Faculty of Engineering, Gifu University,
Yanagido 1-1, Gifu 501-1193, Japan
2Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
Received September 14, 2006; Accepted November 30, 2006; Online Publication, February 7, 2007
[doi:10.1271/bbb.60503]
4-Oxalomesaconate hydratase from Pseudomonas
ochraceae NGJ1 is unstable in the absence of reducing
reagents such as dithiothreitol, and strongly inhibited by
5,50-dithiobis(2-nitrobenzoic acid) (DTNB). To study the
role of cysteine residues in enzyme catalysis, the eight
individual cysteine residues of the enzyme were replaced
with serine residues by site-directed mutagenesis. The
catalytic properties and chemical modification of wild-
and mutant type-enzymes by DTNB showed that (i)
none of eight cysteine residues was essential for enzyme
catalysis; (ii) the inhibition by DTNB was mostly due to
modification of Cys-186; (iii) Cys-96 might be another
residue reacting with DTNB, and its modification caused
an increase in the Km-value for 4-oxalomesaconate; (iv)
the other six cysteine residues were inaccessible to
DTNB, but susceptible to HgCl2; and (v) only replace-
ment of Cys-186 remarkably improved the stability of
the enzyme in the absence of reducing reagent.
PocOMH requires no addition of metal ions for activity,
and shows no absorption peak around 400 nm. Thus,
PocOMH is distinguishable from both metal-activated
hydratases such as Escherichia coli 2-hydroxypentadie-
noic acid hydratase (EC 4.2.1.80) and hydratases
containing the Fe–S cluster, such as E. coli dihydroxy-
acid dehydratase (EC 4.2.1.9).6,7) The catalytic amino
acid residues in PocOMH have not been identified. The
substrate 4-oxalomesaconate is unstable. It easily lacto-
nizes to 2-pyrone-4,6-dicarboxylic acid under acidic pH,
and converts to the enol-form under alkaline pH.8–10)
Therefore, the convenient spectrophotometric assay is
impractical for examination of the activity versus pH
relationship, which is useful in searching for catalytic
amino acid residues. Cysteine residues are of particular
interest, since PocOMH is activated by various thiol
compounds, including dithiothreitol (DTT), cysteine,
GSH, and 2-mercaptoethanol, and is strongly inhibited
by HgCl2 and p-chloromercuribenzoate.2) Thiol com-
pounds are also required for storage of the enzyme,
especially in its highly purified state. However, it is
unclear whether the cysteine residues are essential for
the catalytic function or structural integrity of the
enzyme.
Hara et al. cloned the gene ligJ from lignin-
assimilating bacteria Sphingomonas paucimobilis
SYK-6, and identified its gene product LigJ as OMH
(SpaOMH).11) SpaOMH purified from recombinant
E. coli shows a much higher Km-value for 4-oxalome-
saconate than does PocOMH. SpaOMH also shows no
apparent requirement of thiol compounds for stability,
although it is somewhat activated by cysteine, and
strongly inhibited by HgCl2. Several genes showing a
high degree of homology to ligJ have been found in
other bacterial strains, but their gene products were not
Key words: 5,50-dithiobis(2-nitrobenzoic acid); Ell-
man’s reagent; 4-oxalomesaconate hydra-
tase; Pseudomonas ochraceae
Protocatechuic acid is one of the central intermediates
in the microbial metabolism of aromatic compounds.1)
4-Oxalomesaconate hydratase (2-hydroxy-4-oxobu-
tane-1,2,4-tricarboxylate 2,3-hydro-lyase, EC 4.2.1.83)
(OMH) is found on the bacterial catabolic pathways
responsible for the meta-degradation of protocatechuic
acid.2,3) The enzyme catalyzes the reversible hydration
of 4-oxalomesaconate to l-4-carboxy-4-hydroxy-2-ox-
oadipate, which is degraded to oxaloacetate and py-
ruvate by Mg2þ-dependent aldolase (Fig. 1).4,5) OMH
purified from Pseudomonas ochraceae (PocOMH) is a
dimer with a subunit molecular mass of about 37 kDa.2)
y
To whom correspondence should be addressed. Fax: +81-58-293-3061; E-mail: maruyama@gifu-u.ac.jp
Abbreviations: DTNB, 5,50-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; IPTG, isopropyl ꢀ-thiogalactopyranoside; KE, 20 mM potassium
phosphate, pH 7.0, containing 10% (v/v) ethylene glycol; KED, KE containing 1 mM DTT; OMH, 4-oxalomesaconate hydratase; PAGE,
polyacrylamide gel electrophoresis; PAR, 4-(2-pyridylazo)resorcinol; PocOMH, Pseudomonas ochraceae OMH; RpaOMH, Rhodopseudomonas
palustris OMH; SpaOMH, Sphingomonas paucimobilis OMH

450
S. LI et al.
COO-
-OOC
OH
-OOC
-OOC
H3C
1
2
O
+
O
COO-
-OOC
+
H2O
COO-
-OOC
-OOC
O
O
Fig. 1. Degradation of 4-Oxalomesaconate in the Protocatechuate Meta-Degradation Pathway.
Reactions 1 and 2 are catalyzed by OMH and 4-hydroxy-4-methyl-2-oxoglutarate aldolase (EC 4.1.3.17) respectively.
characterized by biochemical methods.12–15) Previously,
we cloned and sequenced the gene cluster responsible
for the meta-degradation of protocatechuate from
P. ochraceae NGJ1 (formerly P. ochraceae) (GenBank
accession nos. AB050935 and AB127969).16,17) In this
study, the gene encoding PocOMH was subcloned into
pKF18k and expressed in E. coli MV1184. To answer
the above question on the role of cysteine residues, the
eight individual cysteine residues in PocOMH were
replaced with serine residues by site-directed muta-
genesis. The wild- and mutant-type enzymes are purified
to investigate their kinetic properties, stability, and
reaction toward Ellman’s reagent [5,50-dithiobis(2-nitro-
benzoic acid), DTNB]. One of the mutant-type enzymes
was found to be more stable than the wild-type enzyme
in the absence of reducing reagent. The results obtained
in this study are discussed based on the X-ray structure
of Rhodopseudomonas palustris CGA009 OMH
(RpaOMH), which reveals that RpaOMH is a homo-
dimer and that it tightly binds one Zn ion per subunit
(Brookhaven Protein Data Bank ID, 2GWG, Forouhar,
F., Abashidze, M., Jayaraman, S., Cunningham, K.,
Ciao, M., Ma, L., Xiao, R., Acton, T. B., Montelione, G.
T., Hunt, J. F., and Tong, L., Northeast Structural
Genomics Consortium (NESG), Crystal structure of 4-
oxalomesaconate hydratase, LigJ, from Rhodopseudo-
monas palustris, Northeast structural genomics target
RPR66, released May 23, 2006).
sequencing kit (PE Applied Biosystems, Warrington,
UK), followed by electrophoresis with an ABI 377 DNA
sequencer (Perkin-Elmer, Norwalk, CT). The sequence
data were analyzed using the DNASIS program (Hitachi
Software Engineering, Yokohama, Japan).
Amino acid sequence. After SDS–polyacrylamide gel
electrophoresis (SDS–PAGE), the protein was electro-
blotted onto a polyvinylidene difluoride membrane
(ATTO, Tokyo), and the amino acid sequence was
determined with a Procise protein sequencer (Perkin-
Elmer). Protein sequence similarities were searched
using the FASTA program and the BLAST pro-
gram.19,20) CLUSTAL W was used for sequence align-
ment.21)
Cloning of the PocOMH gene. Plasmid pBEE7.5,
which was prepared by cloning the EcoRI 7.5-kb DNA
fragment at EcoRI site of pBluescript SK–, harbors the
PocOMH gene.16) The gene is 1,029 bp long, corre-
sponding to a protein of 342 amino acid residues with a
calculated molecular mass of 38,260 Da, and it has an
appropriate ribosome-binding sequence GGAG up-
stream of the initial ATG codon.22) A HincII-digest of
pBEE7.5 gave five major DNA fragments of 3.0, 2.2,
2.0, 1.5, and 0.9 kb on agarose gel electrophoresis.
Among these, the 2.2-kb DNA fragment carrying the
entire PocOMH gene was extracted from the gel,
introduced in the HincII site of pKF18k to obtain
plasmid pKHH2.2, and transformed into E. coli
BMH71-18 mutS for site directed mutagenesis, or into
MV1184 for preparation of wild-type enzyme. An
appropriate transformant, in which the PocOMH gene
was in the same direction as the lac promoter, was
selected by the restriction analysis of plasmid DNA.
Materials and Methods
Materials. DTNB and 4-(2-pyridylazo)resorcinol
(PAR) were obtained from Nacalai Tesque (Kyoto,
Japan) and TCI (Tokyo) respectively. 2-Pyrone-4,6-
dicarboxylic acid was chemically synthesized as de-
scribed previously.8) All other chemicals were commer-
cial products of analytical grade.
Site-directed mutagenesis. Site-directed mutagenesis
of the PocOMH gene was performed by the oligonu-
cleotide-directed dual amber method using a Mutan-
express Km (Takara).23) To replace the TGC/T codon
of cysteine with the AGC or TCT codon of serine at
positions 92, 96, 124, 131, 183, 186, 205, and 260, the
following synthetic oligonucleotides were used as
mutagenic primers (the amino acid changes and their
positions in the amino acid sequences are shown, and the
codon changes are underlined): C92S, 50-pCACAGC-
TCGTTGCTGATGGCGGCCC-30; C96S, 50-pCTGAC-
GCGGAAGCTCAGCTCGTTGC-30; C124S, 50-pAGC-
TCGGGAATGCTGGTTGCAGGGT-30; C131S, 50-pT-
DNA manipulations. DNA was extracted from agar-
ose gel with a GeneClean (Qbiogene, Carlsbad, CA).
Plasmid DNA was prepared from recombinant E. coli
cells with a MiniPrep (Qiagen, Hilden, Germany).
Synthetic oligonucleotides, restriction enzymes, a liga-
tion kit, and competent cells of E. coli BMH71-18 mutS
and MV1184 were obtained from Takara (Kusatsu,
Japan). DNA digestion, ligation, and transformation
were carried out as described by Sambrook et al.18) DNA
sequencing was performed on denatured double-strand-
ed DNA templates with a BigDye terminator cycle

Role of Cysteine Residues in 4-Oxalomesaconate Hydratase
451
ACTGCTCCACAGACTTGACCAGCT-30; C183S, 50-
pAAGCATGAATTGCTGCTGGTGGAGA-30; C186S,
50-pGTGGTGTGAAAGCTTGAATTGCAGC-30; C205S,
50-pTCCGAAGTCAGGCTCTGCATGAAGG-30; and
C260S, 50-pTGGTGGTAGACGCTGGTGTCGAAAT-30.
E. coli MV1184 was used as the host for recombinant
plasmids. The base changes were confirmed by DNA
sequencing with appropriate sequencing primers.
Determination of enzyme activity. 4-Oxalomesaconate
was prepared by alkaline hydrolysis of 2-pyrone-4,6-
dicarboxylic acid. 2-Pyrone-4,6-dicarboxylic acid (20
mg) was incubated at 15 ꢀC for about 1 h in 6 ml of 83
mM NaOH. Then the solution was neutralized with HCl.
To determine the concentration of 4-oxalomesaconate, a
sample (20 ml) of the solution was incubated at 25 ꢀC
in 3 ml of 0.1 M Tris–acetate, pH 8.0, containing 5 mM
potassium phosphate, pH 8.0, 2 mM MgCl2, 3 mg of the
purified PocOMH, 0.14 mM NADH, 15 units of malate
dehydrogenase (Sigma, St. Louis, MO), 15 units of
lactic dehydrogenase (Sigma), and 3 mg of purified 4-
hydroxy-4-methyl-2-oxoglutarate aldolase. The last en-
zyme was prepared from recombinant E. coli as describ-
ed previously.16) The amount of 4-oxalomesaconate was
estimated from the decrease in A340, which indicates the
total amount of oxaloacetate and pyruvate (Fig. 1).
The activity of OMH was determined at 25 ꢀC in the
forward reaction. The standard assay mixture (3 ml)
contained 50 mM Tris–acetate, pH 8.0, 0.08 mM 4-ox-
alomesaconate, and the enzyme. The decrease in A265
Preparation of wild- and mutant-type enzymes. The
wild- and mutant-type enzymes were produced in E. coli
MV1184 cells. Recombinant E. coli was grown at 37 ꢀC
in 800 ml of Luria-Bertani medium containing kanamy-
cin (50 mg/ml).18) When A660 reached about 0.10, 5 mM
isopropyl ꢀ-thiogalactopyranoside (IPTG) was added,
and the culture was continued until late log phase. The
bacterial cells were collected by centrifugation, washed
once with 50 mM potassium phosphate, pH 7.0, and
stored at ꢁ25 ꢀC until needed.
All manipulations for enzyme purification were
performed at 0–4 ꢀC. The bacterial cells were suspended
in 10 ml of 20 mM potassium phosphate, pH 7.0, con-
taining 10% (v/v) ethylene glycol and 1 mM DTT (KED
buffer), and disrupted with a Sonifier (Branson, Dan-
bury, CT). After centrifugation to remove cell debris, the
supernatant (crude extract) was passed through a column
(3 ꢂ 30 cm) of Bio-Gel A-1.5 m (Bio-Rad, Hercules,
CA) equilibrated with KED buffer. The active fractions
were pooled and put on a column (2 ꢂ 10 cm) of
DEAE-cellulose (Serva, Heidelberg, Germany) previ-
ously equilibrated with KED buffer. After washing with
200 ml of KED buffer, the enzyme was eluted with a
linear gradient prepared with 100 ml of KED buffer and
100 ml of KED buffer containing 0.5 M KCl. The active
fractions were pooled, and the enzyme was precipitated
by the addition of solid ammonium sulfate (75% satu-
ration). The precipitate was collected by centrifugation
and dissolved in about 3 ml of 20 mM potassium phos-
phate, pH 7.0, containing 1 mM DTT. The solution was
dialyzed against 200 ml of the same buffer, and then
against 200 ml of 1 mM DTT. It was put on isoelectric
focusing with a 110-ml glass column, as described by
Vesterberg.24) Sucrose was used to maintain the pH
gradient obtained with a 2.5% Pharmalite (pH 4–6.5)
(GE Healthcare, Piscataway, NJ), and 1 mM DTT was
added to prevent enzyme inactivation. Electrophoresis
was done at 900 V for 48 h. The active fractions were
pooled and then dialyzed against 200 ml of KED buffer.
The solution was passed through a Bio-Gel A-1.5 m
column (3 ꢂ 20 cm) equilibrated with KED buffer to
remove Pharmalite. The purified enzyme preparation
was concentrated in a collodion bag apparatus (Sartor-
was meaꢁsu1red. The activity was calculated with an E265
2)
of 6 mM ꢃcmꢁ1
.
One unit of enzyme activity was
defined as mmol of substrate reacted per min under the
assay conditions, and the specific activity was defined as
unit per mg of the enzyme. Protein was determined by
the method of Lowry et al., as modified by Bennett.25,26)
Bovine serum albumin was used as a standard. A subunit
molecular mass of 38 kDa was used for calculation of
the kcat value.
Chemical modification with DTNB. The enzyme
solution (2.7 ml) containing 0.42–0.88 mg of enzyme
in KE buffer was mixed with 0.3 ml of 1 M Tris–HCl,
pH 8.0, and 0.1 ml of 10 mM DTNB dissolved in 50 mM
potassium phosphate, pH 7.0. The mixture was incu-
bated at 25 ꢀC, and the increase in A412 due to the
formation of 2-nitro-5-thiobenzoate dianion was moni-
tored. The number of SH groups was calculated as
described by Riddle et al.27) During incubation, a sample
(2–50 ml) was withdrawn to measure residual enzyme
activity under the standard assay conditions. To deter-
mine the total number of SH groups in the enzyme, 1%
(w=v) SDS was added to the mixture.
Determination of enzyme-bound Zn ion. The purified
enzyme (0.92 mg) in 3 ml of 20 mM Tris–HCl, pH 7.5,
containing 1 mM mercaptoethanol was mixed with
0.15 ml of 1.25 mM PAR (50% ethanolic solution). After
incubation at 25 ꢀC for 10 min, 0.35 ml of 10% (w/v)
SDS was added. Alternatively, the enzyme in 3 ml of
20 mM Tris–HCl, pH 7.5, was mixed with 0.35 ml of
10% (w/v) SDS and boiled for 10 min prior to the
addition of PAR. The increase in A500 was measured
ius, Gottingen, Germany), and stored at ꢁ25 ꢀC.
¨
When removal of DTT was necessary, the enzyme
solution was passed through a column (1:7 ꢂ 25 cm) of
Sephadex G-50 (GE Healthcare), previously equilibrated
with 20 mM potassium phosphate, pH 7.0, containing
10% ethylene glycol (KE buffer).
with the blank mixture without enzyme. Theꢁa1mount of
28)
Zn ions was calculated with an E500 of 66 mM ꢃcmꢁ1
.
Other analytical methods. Disc PAGE was done with

452
S. LI et al.
Fig. 2. Alignment of the Deduced Amino Acid Sequences of OMHs from Various Bacterial Strains.
Sequences: a, P. ochraceae NGJ1 (GenBank accession no. AB050935); b, R. palustris CGA009 (TrEMBL accession no. Q6N0R4); c,
A. keyseri 12B (GenBank accession no. AF331043); d, S. paucimobilis SYK-6 (GenBank accession no. AB073227). The amino acid residues of
each sequence are numbered on the right. Identical residues are shown by plus signs. Cysteine residues replaced by site-directed mutagenesis are
underlined.
7.5% gel at 4 ꢀC by the method of Davis.29) SDS–PAGE
was done with 12% gel by the method of Laemmli.30)
Proteins were stained with Coomassie Brilliant Blue R-
250. The molecular mass of the native enzyme was
determined by gel filtration on Sephadex G-200 (Al-
drich, Milwaukee, WI), previously equilibrated with
KED buffer, as described previously.17) UV and visible
absorption spectra were measured with a UV-300
spectrophotometer (Shimadzu, Kyoto, Japan). The CD
spectrum in the UV region was measured with a cuvette
(light path, 1 mm) at 10 ꢀC using a J-820 CD polarimeter
(Jasco International, Tokyo). The enzyme samples
(0.55–0.74 mg/ml) in KED buffer were scanned 10
times. Each spectrum was corrected by subtracting the
appropriate blank.
nas. sp. LB126.11,13,14) Figure 2 shows a multiple
sequence alignment, in which OMHs from C. testoster-
oni BR6020 and Sphingomonas. sp. LB126 were
omitted, since they are almost identical to PocOMH
and SpaOMH respectively. PocOMH has eight cysteine
residues per subunit. Among these, Cys-92, 183, and
260 appeared to be well conserved in all proteins. All
proteins conserved a motif, HGGG, at residue nos. 223–
226, which constitutes the oxyanion hole in the hydro-
lases belonging to the mammalian hormone sensitive
lipase family.31,32)
Purification and properties of recombinant PocOMH
The PocOMH gene was subcloned from pBEE7.5 to
pKF18k to obtain plasmid pKHH2.2. E. coli MV1184
harboring pKHH2.2 was grown in Luria-Bertani me-
dium containing kanamycin and IPTG. The wild-type
enzyme was purified about 14-fold with a yield of 34%
from the crude extract of E. coli by gel filtration column
chromatography, ion exchange column chromatography,
and isoelectric focusing (Table 1). The purified enzyme
preparation was near homogeneity on SDS–PAGE
(Fig. 3). The molecular mass (72 kDa) and the subunit
molecular mass (38 kDa) were estimated by gel filtration
on Sephadex G-200 and SDS–PAGE respectively. The
N-terminal amino acid sequence (MIIDVHGHYTTA-
Results
Multiple sequence alignment
The deduced amino acid sequences of OMHs from
various bacterial strains were compared. PocOMH
showed a high degree (80–99%) of homology toward
OMHs from Comamonas testosteroni BR6020 and
R. palustris CGA009,12,15) and a medium degree (63–
68%) of homology toward OMHs from Arthrobacter
keyseri 12B, S. paucimobilis SYK-6, and Sphingomo-

Role of Cysteine Residues in 4-Oxalomesaconate Hydratase
10
453
1
2
3
4
5
6
7
8
9
M
(kDa)
200
116
66
45
31
21
Fig. 3. Analysis of Enzyme Preparations by SDS–PAGE.
The crude extract (35 mg, lane 1) and the purified enzyme preparations (1–2 mg, lanes 2–10) were put on SDS–PAGE. Lanes 1 and 2, wild-type
enzyme; lane 3, C92S; lane 4, C96S; lane 5, C124S; lane 6, C131S; lane 7, C183S; lane 8, C186S; lane 9, C205S; lane 10, C260S; lane M,
marker proteins.
Table 1. Purification of PocOMH from E. coli Harboring pKHH2.2
Wild-type
Specific
activity
(unit/mg)
C92S
C96S
Protein
(mg)
Activity
(unit)
Yield
(%)
Step
Crude extract
168
82.0
10.2
4.0
1324
1402
1013
452
7.88
17.1
99.3
100
106
76.5
34.1
C124S
C131S
C183S
C186S
C205S
C260S
Bio-Gel A-1.5 m
DEAE-cellulose
Isoelectric focusing
113
PAALGAWR), determined by Edman degradation,
completely coincided with that deduced from the
nucleotide sequence. The recombinant enzyme showed
properties similar to those of PocOMH purified from
P. ochraceae in respect of pI, absorption spectrum,
optimum pH, and substrate specificity.2)
0
20
40
60
80
100
120
Relative activity (%)
Fig. 4. Stability of Wild- and Mutant-Type Enzymes in Storage at
Low Temperature.
The Km for 4-oxalomesaconate and the Vmax were
determined to be 10.7 mM and 116 mmol/min/mg re-
spectively by double reciprocal plot. The Km-value was
significantly lower than that (138 mM) of SpaOMH.11)
Citrate inhibited the reaction competitively, with a
Ki-value of 0.41 mM, determined by the method of
Dixon.33) When the enzyme was preincubated with
various reagents at 25 ꢀC for 10 min in 50 mM potassium
phosphate, pH 7.0, none of the following reagents (each
1 mM) affected the enzyme activity: EDTA, 1,10-
phenanthroline, 8-hydroxyquinoline, 2,20-dipyridine, tir-
on, Mg2þ, Ca2þ, Fe3þ, Co2þ, and Ni2þ. Cu2þ strongly
inhibited the enzyme, and Zn2þ weakly inhibited it.
Although diethylpyrocarbonate (5 mM) reacted with
several histidine residues in the enzyme as judged by
the increase in A235, no inhibition was detectable (data
not shown). The purified enzyme gradually lost activity
in the absence of a reducing reagent. At 4 ꢀC, more than
90% of activity was lost for 1 d (Fig. 4). Brief incubation
of the inactivated enzyme with DTT restored about 74%
The wild- and mutant-type enzymes were incubated at 4 ꢀC for 1 d
in the absence (KE buffer, closed bar) or presence (KED buffer) of
1 mM DTT. Then DTT (1 mM) was added to the sample without
DTT, and the mixture was further incubated at 4 ꢀC for 30 min (open
bar). The enzyme activity obtained with incubation with DTT for 1 d
was taken to be 100%.
of the activity. The inactivated enzyme showed mobility
on disc PAGE similar to that of the native enzyme (data
not shown), suggesting that the inactivation involved no
gross change in the molecular form of the enzyme.
When stored at ꢁ25 ꢀC in KED buffer, no appreciable
loss of enzyme activity was observed for at least 2
months.
Measurement of enzyme-bound Zn ion
A recent X-ray crystallographic study demonstrated
that RpaOMH binds one Zn ion per subunit. This
appears to conflict with the observation that the activity

454
S. LI et al.
of PocOMH is hardly affected by various metal
chelators. Hence, the Zn content of PocOMH was
measured with a metallochromic chelator PAR. When
purified wild-type enzyme was incubated at 25 ꢀC for
10 min with PAR in the presence of 1 mM mercaptoe-
thanol, no decrease in enzyme activity was found, and a
small amount of Zn ions, possibly due to non-specific
contaminants, was observed. The molar ratio (r) of Zn
ions to enzyme subunit was estimated to be 0.14.
Incubation at 25 ꢀC in 1% (w/v) SDS prompted the
release of Zn ions (r ¼ 0:43). When the enzyme was
boiled for 10 min in 1% (w/v) SDS prior to the addition
of PAR, the release of Zn ions was further prompted
(r ¼ 1:07). This value is close to 1.00 if non-specific
contaminants are corrected for. These results suggest
that PocOMH possesses one Zn ion per subunit, and that
it strongly binds to the enzyme protein and reacts with
the metal chelators only under denaturing conditions.
A
120
100
80
60
40
20
0
2
1.5
1
0.5
0
1
2
3
4
B
120
100
80
60
40
20
0
2
1.5
1
0.5
0
0
1
2
3
4
Time (min)
Inactivation of wild-type PocOMH by DTNB
Fig. 5. Modification of Wild- and Mutant-Type Enzymes by DTNB.
In A, purified wild-type enzyme (subunit concn., 7.47 mM) was
incubated with 0.32 mM DTNB in the absence (circle) or presence
(triangle) of 10 mM citrate. In B, the purified C186S (4.75 mM) was
similarly incubated with DTNB. The number of SH groups (open
symbols) and the residual activity (closed symbols) were measured
as described in ‘‘Materials and Methods.’’
When wild-type PocOMH (subunit concn., 6.8 mM)
was incubated at 25 ꢀC with an excess amount of DTNB
(0.32 mM) in the presence of 1% (w/v) SDS, about 7.2
SH groups (molar ratio against enzyme subunit) were
modified by DTNB. This is in agreement with the
cysteine content deduced from the nucleotide sequence
of the PocOMH gene. In the absence of SDS, about one
SH group rapidly reacted with DTNB, and then an
additional one SH group reacted at a slower rate
concomitant with a significant loss of enzyme activity
(Fig. 5A). No further reaction of SH groups was
observed during prolonged incubation (30 min). Possi-
bly, the other six SH groups are buried in the enzyme
molecules and are inaccessible to the bulky DTNB
molecules, unless the enzyme is unfolded with denatur-
ing agents. Citrate (10 mM), a weak competitive inhib-
itor, weakly repressed both the increase in A412 and the
loss of enzyme activity.
Table 2. Kinetic Constants of the Wild- and Mutant-Type Enzymes
kcat
(sꢁ1
Km
kcat=Km
(sꢁ1ꢃmM
Enzyme
Wild-type
C92S
ꢁ1
)
(mM)
)
75.4
24.1
10.7
2.90
87.0
7.94
8.38
8.51
9.94
6.18
7.33
41.9
8.04
7.05
8.31
1.83
13.1
8.84
3.98
8.18
9.06
8.29
3.60
9.99
C96S
C124S
159
104
74.1
C131S
C183S
33.9
81.3
56.0
60.8
C186S
C205S
C260S
DTNB-C186S (ꢁDTT)a
151
80.3
Properties of the mutant-type enzymes
DTNB-C186S (+DTT)a
The eight individual cysteine residues in PocOMH,
Cys-92, 96, 124, 131, 183, 186, 205, and 260, were
replaced with serine residues by site-directed muta-
genesis. Each mutant-type enzyme was purified to near
homogeneity (12–71 fold purification with a yield of 25–
34%) (Fig. 3). All the mutant-type enzymes were active,
suggesting that none of the eight cysteine residues is
essential for enzyme catalysis (Table 2). C92S showed
the lowest kcat and Km values, although the kcat=Km value
was slightly higher than that of the wild-type enzyme.
C124S showed the highest kcat=Km value, whereas C96S
and C183S showed somewhat lower values. The kinetic
constants of C96S were markedly different from those of
the wild-type enzyme. Km increased about 8-fold, and
kcat increased about 2-fold. The increase in the Km-value
suggests that Cys-96 is involved in the binding of 4-
oxalomesaconate. The alteration in the kinetic constants
was not striking with other mutant-type enzymes.
aC186S was treated with DTNB, as described in Fig. 5. After removal of
DTNB by gel filtration on Sephadex G-50, the enzyme was stored in KE
buffer (ꢁDTT) or KED buffer (+DTT).
In the absence of DTT, only C186S fully retained
activity during storage at 4 ꢀC for 1 d, or ꢁ25 ꢀC for 2
months (Fig. 4). Other mutant-type enzymes were less
stable and significantly inactivated, like the wild-type
enzyme. DTT had no effect on the activity of C186S,
while other mutant-type enzymes were reactivated to
various extents. C186S and the wild-type enzyme
showed almost identical heat stability. When heated at
various temperatures for 10 min in KE buffer, both
enzymes lost half of their activity at about 60 ꢀC, and
were completely inactivated at 65 ꢀC (data not shown).
All mutant-type enzymes except for C186S were
strongly inhibited by DTNB, whereas C186S was

Role of Cysteine Residues in 4-Oxalomesaconate Hydratase
455
10
Wild-type
C92S
Wild-type
C186S
C96S
C96S
5
C124S
C131S
C183S
C186S
C205S
C260S
0
- 5
0
20
40
60
80
100
120
- 10
θ
Residual activity (%)
Fig. 6. Effects of DTNB on Activities of Mutant-Type Enzymes.
The purified enzyme was incubated at 25 ꢀC for 5 min with
0.32 mM DTNB, as described in Fig. 5, and the residual enzyme
activity was measured under the standard conditions. The activity
(mmol/min/mg) without the addition of DTNB was taken to be
100%: wild-type enzyme, 111; C92S, 36.0; C96S, 208; C124S, 162;
C131S, 114; C183S, 50.5; C186S, 112; C205S, 86.2; C260S, 93.5.
- 15
200
220
240
260
Wavelength (nm)
Fig. 7. Circular Dichroism Spectra of Wild- and Mutant-Type
Enzymes.
The spectrum of C183S is not shown, since it was almost identical
to that of the wild-type enzyme.
slightly activated, possibly due to the change in kinetic
properties of the enzyme (Fig. 6). The time course of
reaction revealed that DTNB rapidly modified about one
SH group of C186S without a decline in enzyme activity
(Fig. 5B). These results suggest that inhibition by
DTNB was primarily due to modification of Cys-186.
The DTNB-treated C186S remained fully active even in
the absence of DTT, and showed different kinetic
properties from those of C186S (Table 2). Both the
kcat and the Km of C186S markedly increased after the
reaction with DTNB. DTNB-treated C186S was re-
versed to C186S by treatment with 1 mM DTT. All the
mutant-type enzymes completely lost activity with
incubation (25 ꢀC, 10 min) with 1 mM HgCl2.
identified as Cys-186. This is based on the observation
that C186S is not inhibited by DTNB (Fig. 6), and
shows no increase in A412 correlated with the decrease in
enzyme activity (Fig. 5B). Cys-186 might be located
near the active site, since citrate, a weak competitive
inhibitor, weakly restrains modification by DTNB
(Fig. 5A). Although the second SH group reacting at a
very high rate with DTNB could not be specified by
measuring A412, owing to the interference of Cys-186, its
modification appeared not to be inhibitory to the enzyme
activity (Fig. 5B). Modification of the second SH group
causes a significant increase in Km and kcat (Table 2),
suggesting the importance of this SH group in substrate
binding. Among the eight mutant-type enzymes, only
C96S showed a similar increase in the kinetic constants.
Hence, it is inferred that the second SH group is Cys-96.
Probably, Cys-96 and Cys-186 are exposed to the sol-
vent so as to react rapidly with DTNB. The other six
cysteine residues might be buried in the enzyme
molecule, and might react with HgCl2 but not with
DTNB.
The CD spectra in the UV region (200–260 nm) of
C96S, C183S, and C186S were almost identical to that
of the wild-type enzyme, with two negative peaks, at
209 and 218 nm, indicating no gross change in the
secondary structure on amino acid replacement (Fig. 7).
Discussion
Purified recombinant PocOMH showed physical and
catalytic properties similar to those of OMH purified
from P. ochraceae,2) and also required reducing re-
agents such as DTT for stability. Since SpaOMH has
seven cysteine residues per subunit and is stable without
reducing reagents,11) there may be particular cysteine
residues responsible for the instability of PocOMH. Site-
directed mutagenesis showed that none of the eight
cysteine residues in PocOMH are directly involved in
enzyme catalysis. The cysteine residues in fumarase (EC
4.2.1.2) also lie on the outside of the active site, and
their modification leads to enzyme inactivation.34)
DTNB inactivates wild-type PocOMH by modifying
two SH groups per subunit. The first SH group was
In the absence of DTT, only C186S maintained
catalytic activity in storage at 4 ꢀC, while the other
mutant-type enzymes were markedly inactivated
(Fig. 4). Therefore, Cys-186 was largely responsible
for the instability of PocOMH in the absence of a
reducing reagent. The heat stability and CD spectrum in
the UV region of C186S were almost indistinguishable
from those of the wild-type enzyme, reflecting no gross
alteration in protein structure by amino acid replace-
ment. The wild-type enzyme inactivated in the absence
of DTT was fully reactivated by the subsequent
incubation with DTT (Fig. 4). The dimeric structure of
the enzyme remained unaltered after inactivation, as
tested by Disc PAGE. These results suggest that the

456
S. LI et al.
instability of PocOMH was due to the formation of an
S–S bond between Cys-186 and another intramolecular
SH group. This SH group could not be specified by
examining the inactivation and reactivation of the
mutant-type enzymes (Fig. 4). Cys-186 is located on a
flexible loop, as described below. Assuming that Cys-
186 forms an S–S bond with either of the two SH groups
near Cys-186, all mutant-type enzymes except for
C186S are expected to be inactive in the absence of
DTT, as shown in Fig. 4.
ysis. We also found that Cys-186 is mainly responsible
for the inactivation of PocOMH. The SH group of Cys-
186 on the flexible loop easily reacts with DTNB and
other intramolecular cysteine residues in the absence of
a reducing reagent, and consequently may interfere with
the access of 4-oxalomesaconate to the active-site Zn
ion. It is unclear whether the reactivity of Cys-186 is
involved in the cellular physiology. The present study
perhaps gives a clue as to the structure and function of
OMH.
The present study also appears to explain the different
properties of SpaOMH and PocOMH. SpaOMH lacks
both Cys-96 and Cys-186 (Fig. 2), and thus shows a
higher Km-value and higher stability in the absence of a
reducing reagent than PocOMH.
Acknowledgments
We thank Dr. T. Suzuki and Dr. H. Suga of the Life
Science Research Center of Gifu University for helpful
advice in DNA sequencing. We also thank Mr. S.
Takemiya and Mr. S. Fukaya for useful discussion.
Forouhar et al. elucidated the X-ray structure of
RpaOMH. The amino acid sequence of RpaOMH,
including the positions of the eight cysteine residues,
is highly homologous to that of PocOMH (Fig. 2).
RpaOMH has one Zn ion per subunit. Three conserved
histidine residues, His-6, 8, and 178, are located at the
bottom of the ꢀ barrel inside the subunit, and coordinate
to the Zn ion. It is appropriate to assume that the active
site is around the Zn ion, since the Zn ion coordinated by
three imidazole groups can promote the heterolysis of
its bound H2O, like human carbonic anhydrase (EC
4.2.1.1).35) Although some biological function is ex-
pected of the HGGG motif (residue nos. 223–226), the
GGG tripeptide moiety is deep in the molecule, and
appears to be independent of enzyme catalysis. The
imidazole group of His-223 is located near the Zn ion,
and may be involved in enzyme catalysis. Unfortunately,
the region between Thr-181 and Thr-190, in which Cys-
183 and Cys-186 are included, is missing in the reported
structure. Computer-aided prediction of protein secon-
dary structure using the DNASIS program suggests that
this missing region forms a loop structure. Probably, the
loop is disordered and invisible in the electron density
map. Since various metal chelators hardly react with the
Zn ion in native PocOMH, this loop may lie just above
the Zn ion so as to protect it from chelators.
In RpaOMH, Cys-260 is located on a turn segment
and a further five cysteine residues, Cys-92, 96, 124,
131, and 205, on ꢁ-helices. The SH groups of these six
residues are at a short distance (0.7–1.9 nm) from the Zn
ion. The SH groups of Cys-92 and 96, which are at a
distance of about 0.7 nm, face the cleft in the enzyme
molecule, and probably affect the substrate binding.
Cys-124, 131, 205, and 260 lie deep in the molecule.
They are also sterically hindered by the surrounding
amino acid residues, and may be ineffective in enzyme
catalysis. These structural features appear to be mostly
compatible with the reactivity of Cys-96 and the
inertness of Cys-124, 131, 205, and 260 in the reaction
of PocOMH toward DTNB.
References
1) Ornston, L. N., Regulation of catabolic pathways in
Pseudomonas. Bacteriol. Rev., 35, 87–116 (1971).
2) Maruyama, K., Purification and properties of ꢂ-oxalo-
mesaconate hydratase from Pseudomonas ochraceae
grown with phthalate. Biochem. Biophys. Res. Commun.,
128, 271–277 (1985).
3) Hara, H., Masai, E., Miyauchi, K., Katayama, Y., and
Fukuda, M., Characterization of the 4-carboxy-4-hy-
droxy-2-oxoadipate aldolase gene and operon structure
of the protocatechuate 4,5-cleavage pathway genes in
Sphingomonas paucimobilis SYK-6. J. Bacteriol., 185,
41–50 (2003).
4) Maruyama, K., Purification and properties of 4-hydroxy-
4-methyl-2-oxoglutarate aldolase from Pseudomonas
ochraceae grown on phthalate. J. Biochem., 108, 327–
333 (1990).
5) Maruyama, K., Activation of Pseudomonas ochraceae 4-
hydroxy-4-methyl-2-oxoglutarate aldolase by inorganic
phosphate. J. Biochem., 108, 334–340 (1990).
6) Pollard, J. R., and Bugg, T. D., Purification, character-
isation and reaction mechanism of monofunctional 2-
hydroxypentadienoic acid hydratase from Escherichia
coli. Eur. J. Biochem., 251, 98–106 (1998).
7) Flint, D. H., Emptage, M. H., Finnegan, M. G., Fu, W.,
and Johnson, M. K., The role and properties of the iron-
sulfur cluster in Escherichia coli dihydroxy-acid dehy-
dratase. J. Biol. Chem., 268, 14732–14742 (1993).
8) Maruyama, K., Isolation and identification of the re-
action product of ꢁ-hydroxy-ꢂ-carboxymuconic "-semi-
aldehyde dehydrogenase. J. Biochem., 86, 1671–1677
(1979).
9) Kersten, P. J., Dagley, S., Whittaker, J. W., Arciero, D.
M., and Lipscomb, J. D., 2-pyrone-4,6-dicarboxylic acid,
a catabolite of gallic acids in Pseudomonas species.
J. Bacteriol., 152, 1154–1162 (1982).
10) Maruyama, K., Purification and properties of 2-pyrone-
4,6-dicarboxylate hydrolase. J. Biochem., 93, 557–565
(1983).
11) Hara, H., Masai, E., Katayama, Y., and Fukuda, M., The
4-oxalomesaconate hydratase gene, involved in the
In this study, we investigated the role of cysteine
residues in PocOMH, and found that none of the eight
cysteine residues participates directly in enzyme catal-

Role of Cysteine Residues in 4-Oxalomesaconate Hydratase
457
protocatechuate 4,5-cleavage pathway, is essential to
vanillate and syringate degradation in Sphingomonas
paucimobilis SYK-6. J. Bacteriol., 182, 6950–6957
(2000).
CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weight-
ing, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res., 22, 4673–4680 (1994).
22) Shine, J., and Dalgarno, L., The 30-terminal sequence of
Escherichia coli 16S ribosomal RNA: complementarity
to nonsense triplets and ribosome binding sites. Proc.
Natl. Acad. Sci. USA, 71, 1342–1346 (1974).
23) Hashimoto-Gotoh, T., Mizuno, T., Ogasahara, Y., and
Nakagawa, M., An oligodeoxyribonucleotide-directed
dual amber method for site-directed mutagenesis. Gene,
152, 271–275 (1995).
12) Providenti, M. A., Mampel, J., MacSween, S., Cook, A.
M., and Wyndham, R. C., Comamonas testosteroni
BR6020 possesses a single genetic locus for extradiol
cleavage of protocatechuate. Microbiol., 147, 2157–
2167 (2001).
13) Eaton, R. W., Plasmid-encoded phthalate catabolic
pathway in Arthrobacter keyseri 12B. J. Bacteriol.,
183, 3689–3703 (2001).
14) Wattiau, P., Bastiaens, L., van Herwijnen, R., Daal, L.,
Parsons, J. R., Renard, M.-E., Springael, D., and
Cornelis, G. R., Fluorene degradation by Sphingomonas
sp. LB126 proceeds through protocatechuic acid: a
genetic analysis. Res. Microbiol., 152, 861–872 (2001).
15) Larimer, F. W., Chain, P., Hauser, L., Lamerdin, J.,
Malfatti, S., Do, L., Land, M. L., Pelletier, D. A., Beatty,
J. T., Lang, A. S., Tabita, F. R., Gibson, J. L., Hanson, T.
E., Bobst, C., Torres, J. L. T., Peres, C., Harrison, F. H.,
Gibson, J., and Harwood, C. S., Complete genome
sequence of the metabolically versatile photosynthetic
bacterium Rhodopseudomonas palustris. Nat. Biotech-
nol., 22, 55–61 (2004).
16) Maruyama, K., Miwa, M., Tsujii, N., Nagai, T., Tomita,
N., Harada, T., Sobajima, H., and Sugisaki, H., Cloning,
sequencing, and expression of the gene encoding 4-
hydroxy-4-methy-2-oxoglutarate aldolase from Pseudo-
monas ochraceae NGJ1. Biosci. Biotechnol. Biochem.,
65, 2701–2709 (2001).
17) Maruyama, K., Shibayama, T., Ichikawa, A., Sakou, Y.,
Yamada, S., and Sugisaki, H., Cloning and character-
ization of the genes encoding enzymes for protocatech-
uate meta-degradation pathway of Pseudomonas ochra-
ceae NGJ1. Biosci. Biotechnol. Biochem., 68, 1434–
1441 (2004).
18) Sambrook, J., Fritsch, E. F., and Maniatis, T., ‘‘Molecu-
lar Cloning, a Laboratory Manual’’ 2nd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor (1989).
19) Pearson, W. R., and Lipman, D. J., Improved tools for
biological sequence comparison. Proc. Natl. Acad. Sci.
USA, 85, 2444–2448 (1988).
24) Vesterburg, O., Isoelectric focusing of proteins. Methods
Enzymol., 22, 389–412 (1971).
25) Lowry, O. H., Rosebrough, N. J., Farr, A. L., and
Randall, R. J., Protein measurement with the Folin
phenol reagent. J. Biol. Chem., 193, 265–275 (1951).
26) Bennet, T. P., Membrane filtration for determining
protein in the presence of interfering substances. Nature,
18, 1131–1132 (1967).
27) Riddle, P. W., Blakeley, R. L., and Zerner, B., Elleman’s
reagent: 5,50-dithiobis(2-nitrobenzoic acid): a reexami-
nation. Anal. Biochem., 94, 75–81 (1979).
28) Hunt, J. B., Neece, S. H., and Grinsburg, A., The use of
4-(2-pyridylazo)resorcinol in studies of zinc release from
Escherichia coli aspartate transcarbamoylase. Anal.
Biochem., 146, 150–157 (1985).
29) Davis, B. J., Disc electrophoresis. II. Method and
application to human serum proteins. Ann. NY Acad.
Sci., 121, 404–427 (1964).
30) Laemmli, U. K., Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature
(London), 227, 680–685 (1970).
31) Sussman, J. L., Harel, M., Frolow, F., Oefner, C.,
Goldman, A., Toker, L., and Silman, I., Atomic structure
of acetylcholinesterase from Torpede californica: a
prototypic acetylcholine-binding protein. Science, 253,
872–879 (1991).
32) Laurell, H., Contreras, J. A., Castan, I., Langin, D., and
Holm, C., Analysis of the psychrotolerant property of
hormone-sensitive lipase through site-directed mutagen-
esis. Protein Eng., 13, 711–717 (2000).
33) Dixon, M., The determination of enzyme inhibitor
constants. Biochem. J., 55, 170–171 (1953).
20) Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,
Zhang, Z., Miller, W., and Lipman, D. J., Gapped
BLAST and PSI-BLAST: a new generation of protein
database search programs. Nucleic Acid Res., 25, 3389–
3402 (1997).
34) Hill, R. L., and Teipel, J. W., Fumarase and crotonase.
Enzymes, 5, 539–571 (1971).
35) Sheridan, R. P., and Allen, L. C., The active site
electrostatic potential of human carbonic anhydrase.
J. Am. Chem. Soc., 103, 1544–1550 (1981).
21) Thompson, J. D., Higgins, D. G., and Gibson, T. J.,