Novel reversible indole-3-carboxylate decarboxylase catalyzing nonoxidative decarboxylation

Article abstract of DOI:10.1271/bbb.66.2388

After enrichment culture with indole-3-carboxylate in static culture, a novel reversible decarboxylase, indole-3-carboxylate decarboxylase, was found in Arthrobacter nicotianae FI1612 and several molds. The enzyme reaction was examined in resting-cell reactions with A. nicotianae FI1612. The enzyme activity was induced specifically by indole-3-carboxylate, but not by indole. The indole-3-carboxylate decarboxylase of A. nicotianae FI1612 catalyzed the nonoxidative decarboxylation of indole-3-carboxylate into indole, and efficiently carboxylated indole and 2-methylindole by the reverse reaction. In the presence of 1 mM dithiothreitol, 50 mM Na₂ S₂O ₃, and 20% (v/v) glycerol, indole-3-carboxylate decarboxylase was partially purified from A. nicotianae FI1612. The purified enzyme had a molecular mass of approximately 258 kDa. The enzyme did not need any cofactor for the decarboxylating and carboxylating reactions.

Full text of DOI:10.1271/bbb.66.2388

This article was downloaded by: [Southern Taiwan University of Science and Technology]
On: 30 October 2014, At: 20:56
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbbb20
Novel Reversible Indole-3-carboxylate Decarboxylase
Catalyzing Nonoxidative Decarboxylation
Toyokazu YOSHIDAa, Kohei FUJITAa & Toru NAGASAWAa
a Department of Biomolecular Science, Faculty of Engineering, Gifu University Yanagido
1-1, Gifu 501-1193, Japan
Published online: 22 May 2014.
To cite this article: Toyokazu YOSHIDA, Kohei FUJITA & Toru NAGASAWA (2002) Novel Reversible Indole-3-carboxylate
Decarboxylase Catalyzing Nonoxidative Decarboxylation, Bioscience, Biotechnology, and Biochemistry, 66:11, 2388-2394,
DOI: 10.1271/bbb.66.2388
To link to this article: http://dx.doi.org/10.1271/bbb.66.2388
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Biosci. Biotechnol. Biochem., 66 (11), 2388–2394, 2002
Novel Reversible Indole-3-carboxylate Decarboxylase Catalyzing
Nonoxidative Decarboxylation
†
Toyokazu YOSHIDA, Kohei FUJITA, and Toru NAGASAWA
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1,
Gifu 501-1193, Japan
Received May 21, 2002; Accepted July 15, 2002
After enrichment culture with indole-3-carboxylate in
static culture, a novel reversible decarboxylase, indole-
3-carboxylate decarboxylase, was found in Arthrobac-
ter nicotianae FI1612 and several molds. The enzyme
reaction was examined in resting-cell reactions with A.
nicotianae FI1612. The enzyme activity was induced
speciˆcally by indole-3-carboxylate, but not by indole.
The indole-3-carboxylate decarboxylase of A. nicotia-
nae FI1612 catalyzed the nonoxidative decarboxylation
of indole-3-carboxylate into indole, and e‹ciently car-
boxylated indole and 2-methylindole by the reverse reac-
we used the carboxylation reaction for the synthesis
of pyrrole-2-carboxylic acid.13) The reverse reaction
of pyrrole-2-carboxylate decarboxylase occurs when
there is reaction equilibrium, and equilibrium was
shifted towards pyrrole-2-carboxylic acid by optimi-
zation of the reaction conditions. 4-Hydroxybenzo-
ate decarboxylase and 3,4-dihydroxybenzoate decar-
boxylase, which are involved in the anaerobic
metabolism of phenolic compounds, also are reversi-
ble decarboxylases catalyzing nonoxidative decar-
8,14)
boxylation in vitro
.
Although the carboxylation
tion. In the presence of 1 m
M
dithiothreitol, 50 m
M
Na2
activities of puriˆed 4-hydroxybenzoate decarboxy-
lase and 3,4-dihydroxybenzoate decarboxylase are
weak, their physiological functions are the carboxy-
lation of phenol and catechol, respectively, under
anaerobic conditions.8,14) Zhang and Young investi-
gated the anaerobic biodegradation of naphthalene
and phenanthrene, and showed that the initial key
reaction for the use of both polycyclic aromatic
hydrocarbons in the absence of oxygen is carboxyla-
tion.15) Their results suggest the existence of an en-
zyme group reversibly catalyzing nonoxidative decar-
boxylation. However, there has been no report on
decarboxylases e‹ciently catalyzing reverse carboxy-
lation other than the pyrrole-2-carboxylate decarbox-
ylase of B. megaterium PYR2910. In this study, we
found an indole-3-carboxylate decarboxylase to be a
novel reversible decarboxylase in Arthrobacter
nicotianae FI1612 and several molds isolated by stat-
ic enrichment culture. The enzyme reaction was
characterized with resting cells and the partially puri-
ˆed enzyme of A. nicotianae FI1612.
S2O3, and 20 (v v) glycerol, indole-3-carboxylate
%
W
decarboxylase was partially puriˆed from A. nicotianae
FI1612. The puriˆed enzyme had a molecular mass of
approximately 258 kDa. The enzyme did not need any
cofactor for the decarboxylating and carboxylating
reactions.
Key words: decarboxylase; nonoxidative decarboxyla-
tion; carboxylation; indole-3-carboxylate;
indole
In the microbial degradation of various carboxylic
acids, enzymatic decarboxylation reactions are com-
mon. The decarboxylation activities of the decarbox-
ylases involved in the metabolism of amino acids and
2-oxo-acids depend on pyridoxal 5?-phosphate and
thiamine pyrophosphate, respectively, and their
characterization, structures, and reaction mechan-
isms have been reported.1–3) In decarboxylases such as
ferulic acid decarboxylase,4,5)
p-coumaric acid decar-
boxylase,5) gallic acid decarboxylase,6,7) 4-hydroxy-
benzoate decarboxylase,8) 2,3-dihydroxybenzoic acid
Materials and Methods
decarboxylase,9) and orotidine-5
?-monophosphate
decarboxylase,10) no prothetic group is needed for the
catalytic reaction.
Materials. Indole-3-carboxylate and indole were
obtained from Wako Pure Chemical Industiries
(Osaka, Japan) and Tokyo Kasei Kogyo Co. (Tokyo,
Japan), respectively. DEAE-Sephacel, phenyl-
Sepharose CL-4B, and Sephacryl S-200 HR were
from Amersham Biosciences Co. (Upsala, Sweden).
Butyl-Toyopearl 650M was purchased from Tosoh
(Tokyo), and marker proteins for HPLC from Orien-
We recently puriˆed and characterized a pyrrole-2-
carboxylate decarboxylase from Bacillus megaterium
PYR2910; the enzyme catalyzes the nonoxidative
decarboxylation of pyrrole-2-carboxylate to yield
pyrrole and CO2.11) This enzyme also catalyzes the
carboxylation of pyrrole at an appreciable rate,12) and
†
To whom correspondence should be addressed. Fax: +81-58-293-2647; E-mail: tonagasa
＠
biomol.gifu-u.ac.jp

Reversible Indole-3-carboxylate Decarboxylase
2389
tal Yeast Co. (Tokyo). Unless otherwise stated, all
other chemicals were obtained from commercial
sources and were of reagent grade.
dole formed per minute by cells derived from 1 ml of
culture broth.
Identiˆcation of microorganisms. The identiˆca-
tion of microorganisms was done by the National
Collection of Industrial and Marine Bacteria, Japan
(NCIMB).
Enrichment culture. For the isolation of indole-3-
carboxylate-degrading microorganisms, convention-
al enrichment culture was done by static culture at
28
containing 2 g of indole-3-carboxylic acid, 2 g of
(NH4)2HPO4, 0.5 g of MgSO4 7H2O, and 5 ml of
metal solution in 1 liter of tap water (pH 7.0). The
9
C. Soil samples were put in 40 ml of medium I
Enzyme assay. Unless otherwise stated, the enzyme
activity was evaluated in terms of the decarboxylat-
ing reaction of indole-3-carboxylic acid. A standard
・
・
metal solution consisted of 40 mg of CaCl2 2H2O,
reaction mixture contained 20
boxylate, 100 mol of potassium phosphate buŠer
(pH 7.0), 10 mol of dithiothreitol, and enzyme solu-
mmol of indole-3-car-
・
50 mg of HBO3, 4 mg of CuSO4 5H2O, 10 mg of KI,
m
・
・
・
・
20 mg of FeSO4 7H2O, 40 mg of MnSO4 7H2O,
40 mg of ZnSO4 7H2O, 20 mg of H2MoO4 2H2O,
m
tion in a ˆnal volume of 1 ml. The reaction was start-
ed by the addition of indole-3-carboxylate, continued
and 2 ml of conc. HCl in 1 liter of distilled water.
Microorganisms were isolated on agar plates of the
same medium.
at 30
9C for 10 min, and then stopped with 0.05 ml of
2
N
NaOH. The solution was centrifuged, and the
resultant supernatant was analyzed by HPLC as de-
scribed above. One unit of enzyme was deˆned as the
amount of enzyme catalyzing the formation of
Culture conditions and preparation of resting cells.
9
The ˆrst cultivation was done aerobically at 28 C for
2 d with reciprocal shaking at 115 rpm. For bacteria,
the culture medium contained 5 g of meat extract
(Kyokuto Seiyaku Co., Tokyo), 5 g of Polypepton
(Nihon Pharmacy Co., Tokyo), 0.5 g of yeast extract
(Oriental Yeast Co.) and, 2 g of NaCl in 1 liter of tap
water (pH 7.0). Molds were ˆrst cultivated in medi-
1 mmol of indole per minute.
Carboxylation reaction. The carboxylation reac-
tion of indole was done in a tightly closed reaction
vessel. The reaction mixture contained 40 mol of in-
dole, 6 mmol of KHCO3, 200 mol of potassium
phosphate buŠer (pH 6.0), 20 mol of dithiothreitol,
m
m
um I containing 0.2
ture was transferred to 40 ml of medium I containing
0.2 (w v) yeast extract, and cultured by static culti-
vation in a 500 ml- shaking ‰ask at 28
Bacterial cells were harvested by centrifugation at
12,000 for 30 min. Molds mycelia were obtained
by the ˆltration of culture broth. The harvested cells
z
(w v) yeast extract. The cul-
m
W
and resting cells, in a ˆnal volume of 2 ml. The cells
harvested from 20 ml of culture broth were used for
z
W
9
C for 5 d.
the reaction. To increase the solubility of indole, 8
z
(v v) methanol was added to the reaction mixture.
W
×
g
The reaction was started by the addition of indole,
continued at 20
0.1 ml of 2
9C for 10 min, and stopped with
or mycelia were washed once with 0.15
suspended in the same solution.
M
NaCl and
N
NaOH. After centrifugation at 12,000
for 5 min, the supernatant was analyzed by
HPLC.
×
g
Assay of decarboxylation activity of indole-3-car-
boxylate decarboxylase by resting cells. A standard
Enzyme puriˆcation. All puriˆcation steps were at
C and in potassium phosphate buŠer (pH 7.0)
containing 1 m dithiothreitol, 50 m Na2S2O3, and
20 (v v) glycerol was used, unless otherwise speci-
ˆed. Harvested cells from 6.5 liters of culture broth
were suspended in 130 ml of 50 m buŠer and then
reaction mixture contained 10
m
mol of indole-3-car-
4
9
boxylate, 100
m
mol of potassium phosphate buŠer
M
M
(pH 7.0), and resting cells in a ˆnal volume of 2 ml.
Bacterial cells harvested from 20 ml of culture broth
were added to the reaction mixture. Molds mycelia
from 40 ml were used for the decarboxylation reac-
tion, which was started by the addition of indole-3-
carboxylate. After static incubation of the mixture at
z
W
M
disrupted with an ultrasonic oscillator (Insonator
201 M, Kubota, Japan) at 120 W for 30 min. The cell
×
debris was removed by centrifugation at 15,000
g
30
0.2
at 12,000
lyzed by HPLC (Shimadzu LC-6A and SPD 6AV sys-
9
C for 1 h, the reaction was stopped with 0.2 ml of
for 20 min. The supernatant solution was used as a
cell extract. The cell extract was put on a DEAE-
N
NaOH. The resulting mixture was centrifuged
×
×
g
for 5 min, and the supernatant was ana-
Sephacel column (3.5 14.3 cm) equilibrated with
10 m buŠer. After the column was washed with
M
×
M
M
tem) with a Spherisorb ODS2 column (4.6 150 mm,
Waters). A 44:55 (v v) mixture of methanol and
100 m buŠer, the enzyme was eluted with 100 m
buŠer containing 0.1 KCl. The active fractions
were combined and solid ammonium sulfate was
added to 30 saturation. Then the enzyme solution
was placed on a column (1.9 6.2 cm) of phenyl-
Sepharose CL-4B equilibrated with 10 m buŠer
containing ammonium sulfate at 30 saturation.
M
W
0.1
M
potassium phosphate buŠer (pH 4.0) was used
for the elution. The ‰ow rate was 1.0 ml min, and
z
W
×
the absorbance of indole-3-carboxylate and indole
was monitored at 230 nm. For this resting-cell reac-
tion, total activity was deˆned as the amount of in-
M
z

2390
T. YOSHIDA et al.
After the column was washed with 10 m
M
buŠer con-
conditions, indole-3-carboxylate-decarboxylating ac-
tivity remained in all strains isolated under static cul-
tivation. Indole-3-carboxylate decarboxylase activity
of a bacterial strain, FI1612, was conˆrmed by
resting-cell reaction, with the total activity of
taining ammonium sulfate at 5
z
saturation, the en-
buŠer. The active frac-
tions were collected, and solid ammonium sulfate
was added to 30 saturation. The enzyme solution
was put on a butyl-Toyopearl 650 M column (1.9
1.1 cm) equilibrated with 10 m buŠer containing
ammonium sulfate at 30 saturation. The column
was washed with 10 m buŠer containing ammoni-
um sulfate at 20 saturation, and the enzyme activi-
ty was eluted with 10 m buŠer containing ammoni-
um sulfate at 15 saturation. The active fractions
were combined. Solid ammonium sulfate was added
to the enzyme solution to 90 saturation. After cen-
trifugation of the mixture at 15,000 for 30 min,
the precipitate was dissolved in 10 m buŠer contain-
ing 0.2 KCl, and the resulting solution was placed
on a column (1.7 93 cm) of Sephacryl S-200 HR
equilibrated with 10 m buŠer containing 0.2 KCl.
zyme was eluted with 10 m
M
z
×
5.01 nmol ml min. Strain FI1612 was identiˆed as
W
M
Arthrobacter nicotianae. A fungus with the highest
z
activity of 0.908 nmol ml min was identiˆed as
W
M
Fusarium subglutinans FI31.
z
Distribution of indole-3-carboxylate decarboxylase
in bacteria, molds, and yeasts was examined by static
M
z
cultivation with medium I containing 0.2z (w v)
W
yeast extract. Among the tested strains from the col-
lection in our laboratory, F. oxysporum IAM5009
and Gibberalla fujikuroi IFO 6605 formed a small
amount of indole from indole-3-carboxylate during
cultivation and in the resting-cell reaction. In the fol-
lowing investigations, A. nicotianae FI1612 was used
because of its high activity.
z
×
g
M
M
×
M
M
The rates of sample loading and elution were main-
tained at 30 ml h. Indole-3-carboxylate decarboxy-
Optimization of culture conditions
W
lase activity was eluted with the same buŠer.
To increase the indole-3-carboxylate decarboxylase
activity of A. nicotianae FI1612, culture conditions
of the bacterium were optimized. The decarboxylase
activity was measured with the resting-cell reaction.
The eŠects of carbon and nitrogen sources were ex-
Analytical methods. SDS-PAGE was done in 10
z
polyacrylamide slab gels,16) and the gels were stained
with Coomassie brilliant blue R-250. Protein concen-
trations were measured by the method of Bradford17)
with bovine serum albumin as the standard. The
molecular mass of the native enzyme were estimated
by gel-permeation HPLC with a TSK G-3000 SW
amined with medium I containing 0.05
extract as the base. The following carbon sources
were used at the concentration of 1 (w v): glucose,
z (w v) yeast
W
z
W
fructose, galactose, maltose, sucrose, lactose, sor-
bitol, glycerol, succinic acid, sodium citrate, sodium
×
column (0.75 60 cm, Tosoh) at 0.7 ml min with
W
0.1
M
potassium phosphate (pH 7.0) containing 0.2
M
fumarate, sodium
Maltose was the most eŠective carbon source, and
the optimum concentration was 1 (w v). The fol-
lowing nitrogen sources were tested at the concentra-
tion of 0.2 (w v): Polypepton, meat extract, yeast
L
-glutamate, and Polypepton.
NaCl as the eluent. The molecular mass of the puri-
ˆed enzyme was calculated from line of regression
obtained from the mobilities of glutamate dehydro-
genase (290 kDa), lactate dehydrogenase (142 kDa),
enolase (67 kDa), adenylate kinase (32 kDa) and
z
W
z
W
extract, casamino acids, corn steep liquor (Wako)
NZ amine (Wako), malt extract (Difco, Franklin
Lakes, USA), NH4Cl, NaNO3, and, (NH4)2HPO4.
The addition of meat extract at 0.5 (w v) increased
z
the indole-3-carboxylate decarboxylase activity of A.
nicotianae FI1612.
cytochrome
c (12.4 kDa). Infrared spectroscopy was
done on a Perkim Elmer 1640 FTIR, and NMR spec-
troscopy was done on a JOEL
dimethylsulfoxide-d6 as the solvent.
a
-400 system with
W
Enzyme induction by indole-3-carboxylate and its
analogues was examined (Table 1). The enzyme activ-
ity was strongly induced by the addition of indole-3-
carboxylic acid to the culture medium. No activity
was found without the addition, and the optimum
Results
Screening of indole-3-carboxylate decarboxylase
activity for microorganisms
Through enrichment culture with indole-3-
carboxylate as the sole carbon source, 21 microor-
ganisms that degraded indole-3-carboxylate were iso-
lated. Among them, one bacterial strain and ˆve
molds formed indole by decarboxylation activities in
the culture medium in static culture. In preliminary
experiments on isolation, aerobic cultivation at
concentration was 0.05
z
(w v). 3-Cyanoindole and
W
L
-tryptophan also induced the enzyme activity. Other
compounds tested did not induce enzyme activity.
In screening for indole-3-carboxylate decarboxy-
lase, its weak activity was frequently lost during aero-
bic cultivation. However, after the optimization of
culture conditions of A. nicotianae FI1612, increased
activity of resting cells was found even under aerobic
conditions. The course of the enzyme activity during
aerobic cultivation is shown in Fig. 1. The highest
115 strokes min was inadequate in screening for in-
W
dole-3-carboxylate-decarboxylating microorganisms;
sometimes the activity disappeared during enrich-
ment culture. Although the growth of microorgan-
isms in static cultivation was slower than in aerobic
total activity of about 500 nmol ml min was ob-
W

Reversible Indole-3-carboxylate Decarboxylase
Table 1. Induction of Indole-3-carboxylate Decarboxylase Ac-
2391
tivity
Growth Total activity
Compound
(OD610
)
(nmol ml min)
W
None
7.15
6.79
6.77
7.43
5.96
5.76
6.83
7.76
3.93
5.49
6.27
1.86
8.23
7.00
8.45
0
23.8
0
0
0
0
0
0
0.87
0.35
0
0
0
0
0
Indole-3-carboxylic acid
Indole-2-carboxylic acid
Indole-3-carboxylic acid methyl ester
Indole-3-aldehyde
3-Indoleacetic acid
3-Indoleacetamide
3-Indolepropionic acid
3-Cyanoindole
L
-Tryptophan
-Tryptophan
Fig. 1. Changes with Time in Indole-3-carboxylate Decarboxy-
lase Activity of A. nicotianae FI1612 Cells under Optimized
Aerobic Culture Conditions.
D
Indole
Pyrrole-2-carboxylic acid
Nicotinic acid
The optimized medium consisted of 0.5 g of indole-3-carbox-
ylic acid, 10 g of maltose, 5 g of meat extract, 0.5 g of yeast ex-
3-Quinolinecarboxylic acid
tract, 1 g of K2HPO4, 0.5 g of MgSO4
solution in 1 liter of tap water, pH 7.0. The cultivation was done
at 28 C in a 500-ml shaking ‰ask containing 40 ml of medium,
・
7H2O, and 5 ml of metal
Each compound was added at the concentration of 0.1
z (w v) in medium
W
9
I containing 0.2 (w v) yeast extract. Decarboxylation activity of cultivat-
z
W
with reciprocal shaking at 115 strokes min. Circles, total activi-
ed cells was evaluated by a resting-cell reaction.
W
ty; triangles, cell growth.
served after 32 h. When the harvested cells were
stored at „20
9
C in 0.15
M
NaCl for 2 weeks, decar-
boxylase activity did not decrease.
Reversibility of catalytic reaction
Throughout the decarboxylation reaction for in-
dole-3-carboxylic acid, a stoichiometric amount of
indole was formed from indole-3-carboxylic acid
(Fig. 2A).
Indole-3-carboxylate
decarboxylase
reached an equilibrium, suggesting that the enzyme
catalyzed the reverse carboxylation reaction. We
used the conditions for the carboxylation reaction of
pyrrole-2-carboxylate decarboxylase12) in optimizing
the reverse reaction of indole-3-carboxylate decar-
Fig. 2. Decarboxylation of Indole-3-carboxylic Acid (A) and
Carboxylation of Indole (B) by Resting Cells of A. nicotianae
FI1612.
The decarboxylation reaction was done under the standard
reaction conditions with resting cells. The reaction conditions
for carboxylation of indole are described in Materials and
boxylase at 20
Resting cells of A. nicotianae FI1612 catalyzed the
carboxylation of indole (Fig. 2B). By 6 h, 6.81 m
indole-3-carboxylic acid had accumulated in the reac-
tion mixture, with a molar conversion yield of 34
(mol mol). The reaction product of the reverse car-
9C in a tightly closed reaction vessel.

$
Methods. , Indole; , indole-3-carboxylic acid.
M
z
ic acid, 2-hydroxynicotinic acid, 6-hydroxynicotinic
acid, 2-chloronicotinic acid, 6-chloronicotinic acid,
2,3-pyridinedicarboxylic acid, 2,5-pyridinedicarbo-
xylic acid, pyrazine-2-carboxylic acid, and 2,3-
pyrazinedicarboxylic acid. Resting cells of A.
nicotianae FI1612 catalyzed the decarboxylation of
2-quinoxalinecarboxylic acid. The formation of
quinoxaline from 2-quinoxalinecarboxylic acid was
detected by HPLC, although the activity was not
measured because only a small amount or quinoxa-
line formed.
Substrate speciˆcity of the carboxylation reaction
for the following compounds was examined: 2-
methylindole, 5-methylindole, 2-hydroxyindole, 4-
hydroxyindole, 5-hydroxyindole, 4-chloroindole, 5-
chloroindole, 6-chloroindole, 5-aminoindole, and
quinoxaline. 2-Methylindole and quinoxaline were
carboxylated by resting cells of A. nicotianae FI1612.
W
boxylation was isolated by a Dowex-1
×
2 anion-ex-
change column, and identiˆed by 1H-NMR, 13C-
NMR, and infrared spectroscopy with the authentic
compound as a reference: NMR dH (Me2SO-
d
6) 7.15
1.5, 6.3 Hz), 8.02 (1H, s),
11.81 (2H, s); NMR dC (Me2SO- 6) 107.4, 112.2,
120.6, 121.0, 122.1, 126.0, 132.3, 136.4, 166.0; IR
(KBr) cm-1: 3305, 3069, 2861, 1651, 1201.
＝
J
(2H, m), 7.46 (2H, dd,
d
[
max
Substrate speciˆcity
The ability to catalyze decarboxylation of the fol-
lowing compounds was examined by resting cell reac-
tions: indole-2-carboxylic acid, indole-5-carboxylic
acid, thiophene-2-carboxylic acid, furan-2-carboxylic
acid, 3-quinolinecarboxylic acid, 2-quinoxalinecar-
boxylic acid, nicotinic acid, isonicotinic acid, picolin-

2392
T. YOSHIDA et al.
Table 2. Partial Puriˆcation of Indole-3-carboxylate Decarboxy-
lase from A. nicotianae FI1612
Table 3. EŠects of Various Compounds on the Decarboxylation
Activity of Indole-3-carboxylate Decarboxylase
Total
Total
Speciˆc
activity
Relative
Relative
Yield
Step
Cell extract
protein activity
Compound
activity Compound activity
(
z)
(mg)
(units) (units mg)
(
z)
(z)
W
2940
155
11.9
4.2
4490
19.4
1.53
100
0.43
0.047
0.037
0.008
None
-Ethylmaleimide
Iodoacetate
-Chloromercuribenzoate
100
3
100
110
65
13
90
96
28
15
119
51
DEAE-Sephacel
0.125
0.176
0.400
0.224
N
ZnSO4
MnSO4
MgSO4
CaCl2
215
215
188
127
106
94
50
48
31
0
Phenyl-Sepharose CL-4B
Butyl-Toyopearl 650M
Sephacryl S-200 HR
2.10
1.68
0.38
p
1.7
Hydroxylamine
Phenylhydrazine
Phenylmethylsulfonyl ‰uoride
EDTA
-Bipyridyl
-Phenanthroline
CoCl2
Fe2(SO4)3
AgNO3
HgCl2
NiCl2
2,2
o
Tiron
?
The activities toward 2-methylindole and quinoxaline
were 37
z and º1z of the activity toward indole,
CuCl2
respectively. The reaction product from 2-methylin-
8-Hydroxyquinoline
NaN3
dole was puriˆed and analyzed by H-NMR, 13C-
1
86
NMR, and infrared spectroscopy. The spectra of
reaction product were analogous to those of indole-3-
carboxylate except the signals for a methyl group at
C-2, and the presence of a carboxyl group at C-3 was
The enzyme reaction was done in the standard reaction mixture contain-
ing the tested compound at 1 m except for -chloromercuribenzoate at
0.1 m
M
p
M
.
suggested: NMR dH (Me2SO-
d
6) 2.63 (3H, s), 7.08
2.9, 5.9 Hz), 7.92 (1H, dd,
3.4, 5.9 Hz), 11.68 (1H, s), 11.84 (1H, s); NMR
6) 13.7, 103.3, 111.0, 120.4, 120.6,
＝
(2H, m) 7.32 (1H, dd,
J
activity were investigated under the standard enzyme
assay conditions (Table 3). -Ethylmaleimide,
phenylhydrazine,
-phenanthroline, and Cu2+ in-
＝
J
N
dC (Me2SO-
d
o
121.4, 127.3, 134.8, 144.3, 166.7; IR
3377, 2910, 1660, 1213.
[
max (KBr) cm„1
:
hibited the activity. The enzyme activity was in-
creased by the addition of several metal ions such as
Zn2+ and Mn2+. However, the addition of these
metal ions to the puriˆcation buŠer did not protect
against loss of enzyme activity. The puriˆed pyrrole-
2-carboxylate decarboxylase from B. megaterium
PYR2910 absolutely required an organic acid as a co-
factor for its decarboxylation and carboxylation ac-
tivities.11) In the absence of an organic acid such as
acetate, propionate, butyrate, or pimelate, the decar-
boxylation of pyrrole-2-carboxylate and the carboxy-
lation of pyrrole were not observed.11) With this in-
dole-3-carboxylate decarboxylase, the decarboxyla-
tion and carboxylation activities were independent of
the addition of organic acids. The Km values for in-
dole-3-carboxylic acid and indole were calculated to
Enzyme characterization
When a cell extract prepared from A. nicotianae
FI1612 was stored without the addition of sulfhydryl-
protecting reagents, 80
lost after storage at 4
was stabilized by the addition of a mixture of 1 m
dithiothreitol, 50 m Na2S2O3, and 20 (v v)
glycerol to the puriˆcation buŠer, with the residual
activity of 80 after storage for 4 d. However, much
z
of the initial activity was
9C for 4 d. The enzyme activity
M
M
z
W
z
loss of activity seemed inevitable during enzyme
puriˆcation (Table 2). Partially puriˆed indole-3-car-
boxylate decarboxylase, which gave three bands on
SDS-PAGE, was obtained from A. nicotianae
FI1612. The molecular mass of the enzyme was esti-
mated to be 258 kDa by gel-permeation HPLC.
The eŠects of temperature and pH on the enzyme
were investigated. When the enzyme had been incu-
be 0.631 and 0.719 m
respectively.
M
from double reciprocal plots,
Discussion
bated at temperatures up to 20
was not decreased. Treatment at 30, 40, 50, and 60
caused 13, 35, 65, and 100 losses of the initial ac-
tivity, respectively. The enzyme was stable on incuba-
tion at 20 C for 1 h in the pH range of 6.0–8.0. When
9
C for 30 min, activity
9
C
In studies of various kinds of decarboxylases, the
interest in nonoxidative decarboxylases is focused on
their reaction mechanisms because they do not use
z
9
any co-factors. With orotidine-5?-monophosphate
the decarboxylation reaction was done for 30 min at
various temperatures and under various buŠer condi-
decarboxylases, reaction mechanisms have been sug-
gested that explain how the enzyme might catalyze
nonoxidative decarboxylation, and the crystal struc-
tures of the enzymes have been analyzed.18,19) On the
other hand, in studies of the microbial anaerobic
degradation of phenolic compounds, two kinds of
reversible decarboxylases catalyzing nonoxidative
tions at the concentration of 75 m
maximal at 50 C and pH 7.0 (potassium phosphate
buŠer). For the carboxylation reaction, temperatures
over 30 C were not appropriate. The activities at 10,
20, and 30 C were about the same. The activity was
maximal at pH 8.0 (Tris-HCl buŠer, 100 m ).
The eŠects of various compounds on the enzyme
M
, the activity was
9
9
9
M
decarboxylation were found in Clostridium hydroxy-
8,14)
benzoicum
.
The primary structure of the 4-

Reversible Indole-3-carboxylate Decarboxylase
2393
hydroxybenzoate decarboxylase of the bacterium has
been published,20) and is similar to many hypothetical
proteins deduced from open reading frames in ge-
nomes from bacteria and archaea; a novel gene fami-
ly has been proposed. However, the catalytic residues
involved in the reversible decarboxylation have not
been veriˆed yet. We also found a reversible pyrrole-
2-carboxylate decarboxylase in Bacillus megaterium
PYR2910,11) and the pyrrole-2-carboxylate decarbox-
ylase e‹ciently catalyzes the carboxylation of pyr-
role.12) In our study, to ˆnd novel reversible decar-
boxylases catalyzing the nonoxidative decarboxyla-
tion of aromatic compounds and then to analyze
their reaction mechanisms, microorganisms with
indole-3-carboxylate decarboxylase activity were
isolated from soils, and the indole-3-carboxylate
decarboxylase of A. nicotianae FI1612 was charac-
terized.
molar conversion ratio of 80
z
(mol mol).12) The car-
W
boxylation of indole into indole-3-carboxylate was
observed by the puriˆed indole-3-carboxylate decar-
boxylase as well as by the resting cells (not shown).
The molar conversion yield of indole into indole-3-
carboxylate was 34
z (mol mol). The low value
W
might derive from the solubility of indole in the reac-
tion mixture. Although the pyrrole-2-carboxylate
decarboxylae activity of B. megaterium PYR2910
was completely dependent on an organic acid such as
acetic acid,11) indole-3-carboxylate decarboxylase did
not require any cofactor for its activity. It is di‹cult
to identify the causes of this diŠerence because of the
scarcity of knowledge on catalytic mechanisms and
primary structures of reversible decarboxylases.
References
1) O'Leary, M. M., Catalytic strategies in enzymic car-
An oxygen-sensitive 4-hydroxybenzoate de-
carboxylase and 3,4-dihydroxybenzoate decarboxy-
lase are induced by 4-hydroxybenzoate and 3,4-di-
hydroxybenzoate, respectively, in the anaerobic
boxylation and decarboxylation. Enzymes
,
20,
235–269 (1992).
2) Nakazawa, H., Sano, K., Kumagai, H., and
Yamada, H., Distribution and formation of aromatic
cultivation of C. hydroxybenzoicum.
8,14) The pyrrole-
L
-amino acid decarboxylase in bacteria. Agric. Biol.
2-carboxylate decarboxylase of B. megaterium
PYR2910 is strongly induced by pyrrole-2-carboxy-
late and its analogues with a carboxyl group, but not
by pyrrole.11) In the presence of dithiothreitol, a pyr-
role-2-carboxylate decarboxylase with high stability
has been puriˆed from B. megaterium PYR2910.11)
We did enrichment culture using indole-3-carboxylic
acid as the sole carbon source in static culture. In-
dole-3-carboxylate decarboxylase activities found in
the isolated bacteria and molds were quickly lost by
aerobic cultivation in all strains. Similarly, some en-
zyme activity was lost during the puriˆcation of in-
dole-3-carboxylate decarboxylase from A. nicotianae
Chem., 41, 2241–2247 (1977).
3) Koga, J., Adachi, T., and Hidaka, H., Puriˆcation
and characterization of indolepyruvate decarboxy-
lase. J. Biol. Chem., 267, 15823–15828 (1992).
4) Huang, Z., Dosta, L., and Rosazza, J. P. N., Puriˆ-
cation and characterization of a ferulic acid decar-
boxylase from Pseudomonas ‰uorescens
teriol., 176, 5912–5918 (1994).
5) Degrassi, G., De Laureto, P. P., and Bruschi, C. V.,
. J. Bac-
Puriˆcation and characterization of ferulate and
coumarate decarboxylase from Bacillus pumilus
Appl. Environ. Microbiol., 61, 326–332 (1995).
p
-
.
6) Yoshida, H., Tani, Y., and Yamada, H., Isolation
and identiˆcation of a pyrogallol producing bacteri-
um from soil. Agric. Biol. Chem., 46, 2539–2546
(1982).
7) Zeida, M., Wieser, M., Yoshida, T., Sugio, T., and
Nagasawa, T., Puriˆcation and characterization of
gallic acid decarboxylase from Pantoea agglomerans
FI1612, and the addition of 50 m
M
Na2S2O3 to the
puriˆcation buŠer stabilized the enzyme activity
somewhat. This result indicates the importance of
Cys residue(s) for the enzyme activity or the protein
structure. A. nicotianae FI1612 grew well in aerobic
culture, and the indole-3-carboxylate decarboxylase
activity of resting cells of the bacterium was fairly
stable. However, the enzyme activity in the cell ex-
tract was sensitive to oxygen. Thus, in the screening
for such reversible decarboxylases, anaerobic or
semianaerobic cultures are probably appropriate for
enrichment. For indole-3-carboxylate decarboxylase
activity of resting cells, after the highest activity was
reached by 32 h of aerobic cultivation of A. nicotia-
nae FI1612, activity was lost rapidly. The disappear-
ance of decarboxylase activity during enrichment
culture under aerobic conditions and the loss of the
activity of isolated strains seem to arise during a long
cultivation.
T71. Appl. Environ. Microbiol.
(1998).
, 64, 4743–4747
8) He, Z., and Wiegel, J., Puriˆcation and characteriza-
tion of an oxygen-sensitive reversible 4-hydroxyben-
zoate decarboxylase from Clostridium hydroxyben-
zoicum. Eur. J. Biochem., 229, 77–82 (1995).
9) Santha, R., Sanvithri, H. S., Rao, N. A., and
Vaidyanathan, C. S., 2,3-Dihydroxybenzoic acid
decarboxylase from Aspergillus niger: a novel decar-
boxylase. Eur. J. Biochem., 230, 104–110 (1995).
10) Cui, W., De Witt, J. G., Miller, S. M., and Wu, W.,
No metal cofactor in orotidine 5?-monophosphate
decarboxylase. Biochem. Biophys. Res. Commun.
259, 133–135 (1999).
,
11) Omura, H., Wieser, M., and Nagasawa, T., Pyrrole-
2-carboxylate decarboxylase from Bacillus megateri-
um PYR2910, an organic-acid-requiring enzyme.
Eur. J. Biochem., 253, 480–484 (1998).
Pyrrole-2-carboxylate decarboxylase of B.
megaterium PYR2910 catalyzed the reverse carboxy-
lation of pyrrole to pyrrole-2-carboxylate, with a

2394
T. YOSHIDA et al.
12) Wieser, M., Fujii, N., Yoshida, T., and Nagasawa,
T., Carbon dioxide ˆxation by reversible pyrrole-2-
carboxylate decarboxylase from Bacillus megaterium
PYR2910. Eur. J. Biochem., 257, 495–499 (1998).
13) Wieser, M., Yoshida, T., and Nagasawa, T.,
Microbial synthesis of pyrrole-2-carboxylate by Bacil-
lus megaterium PYR2910. Tetrahedron Lett., 39,
4309–4310 (1998).
17) Bradford, M. M., A rapid and sensitive method for
the quantitation of microgram of protein utilizing the
principle of protein-dye binding. Anal. Biochem., 72,
248–254 (1976).
18) Appleby, T. C., Kinsland, C., Begley, T. P., and
Ealick, S. T., The crystal structure and mechanism of
orotidine 5?-monophosphate decarboxylase. Proc.
Natl. Acad. Sci. USA, 97, 2005–2010 (2000).
19) Miller, B. G., Hassell, A. M., Wolfendne, R., Mil-
burn, M. V., and Short, S. A., Anatomy of a
14) He, Z., and Wiegel, J., Puriˆcation and characteriza-
tion of an oxygen-sensitive, reversible 3,4-dihydroxy-
benzoate decarboxylase from Clostridium hydroxy-
proˆcient enzyme: the structure of orotidine 5?-
benzoicum
.
J. Bacteriol., 278, 3539–3543 (1996).
phosphate decarboxylase in the presence and absence
of a potential transition state analog. Proc. Natl.
Acad. Sci. USA, 97, 2011–2016 (2000).
15) Zhang, X., and Young, L. Y., Carboxylation as an
initial reaction in the anaerobic metabolism of
naphthalene and phenanthrene by sulˆdogenic con-
sortia. Appl. Environ. Microbiol., 63, 4759–4764
(1997).
20) Huang, J., He, Z., and Wiegel, J., Cloning, charac-
terization, and expression of a novel gene encoding a
reversible 4-hydroxybenzoate decarboxylase from
16) Laemmli, U. K., Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature, 227, 680–685 (1970).
Clostridium hydroxybenzoicum. J. Bacteriol., 181,
5119–5122 (1999).