Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Article abstract of DOI:10.1271/bbb.60655

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, α-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG-1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM α-ketobutyrate, and 100mM L-glutamate with a molar yield to α-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl₂·4H₂O at 28°C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and α-ketobutyrate was purified from A. simplex AKU 626. Mn²⁺ and pyridoxal 5′-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.

Full text of DOI:10.1271/bbb.60655

Biosci. Biotechnol. Biochem., 71 (7), 1607–1615, 2007
Synthesis of 4-Hydroxyisoleucine by the Aldolase–Transaminase
Coupling Reaction and Basic Characterization of the Aldolase
from Arthrobacter simplex AKU 626
1
1
1
1
1
Jun OGAWA, Hiroyuki YAMANAKA, Junichi MANO, Yuko DOI, Nobuyuki HORINOUCHI,
2
2
3
3
Tomohiro KODERA, Noriki NIO, Sergey V. SMIRNOV, Natalya N. SAMSONOVA,
3
1;y
Yury I. KOZLOV, and Sakayu SHIMIZU
1
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
2
Institute of Life Sciences, Ajinomoto Co., Inc., Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan
Ajinomoto-Genetika Research Institute, 1st Dorozhny pr. 1, Moscow 113545, Russia
3
Received November 21, 2006; Accepted March 29, 2007; Online Publication, July 7, 2007
[doi:10.1271/bbb.60655]
Arthrobacter simplex AKU 626 was found to synthe-
and glucose tolerance.6) Due to this insulinotropic
activity, this isomer may be considered a novel orally-
active drug with potential for the treatment of insulin-
independent diabetes mellitus. Since the extraction of
HIL from fenugreek seeds is done at low yield (about
150 mg HIL from 1 kg of seeds ), the pharmaceutical
industry requires a more efficient method for the syn-
thesis of HIL.
size 4-hydroxyisoleucine from acetaldehyde, ꢀ-ketobu-
tyrate, and L-glutamate in the presence of Escherichia
coli harboring the branched chain amino acid trans-
aminase gene (ilvE) from E. coli K12 substrain MG-
1
)
1
655. By using resting cells of A. simplex AKU 626 and
E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyiso-
leucine was produced from 250 mM acetaldehyde, 75 mM
ꢀ
-ketobutyrate, and 100 mM L-glutamate with a molar
yield to ꢀ-ketobutyrate of 4.3% in 50 mM Tris–HCl
We attempted to synthesize HIL by the aldolase–
transaminase coupling reaction. The coupling reaction
consists of two reactions. In the first reaction, an
aldolase catalyzed the aldol condensation of acetalde-
hyde and ꢁ-ketobutyrate. In the second reaction, a trans-
aminase catalyzed amination of the aldol condensation
product, 4-hydroxy-3-methyl-2-keto-pentanoic acid, to
HIL (Fig. 1). Aldolase is the key enzyme, since it can
generate two of three chiral centers in HIL. The aldolase
catalyzing the first reaction, 4-hydroxy-2-keto-pentanoic
acid aldolase (HKP aldolase), has been found in cell-free
ꢀ
.
buffer (pH 7.5) containing 2 mM MnCl2 4H2O at 28 C
for 2 h. An aldolase that catalyzes the aldol condensation
of acetaldehyde and ꢀ-ketobutyrate was purified from
A. simplex AKU 626. Mn2 and pyridoxal 5 -mono-
phosphate were effective in stabilizing the enzyme. The
native and subunit molecular masses of the purified
aldolase were about 180 and 32 kDa respectively. The N-
terminal amino acid sequence of the purified enzyme
showed no significant homology to known aldolases.
þ
0
7
)
extract of Escherichia coli W3110. The enzyme, how-
ever, catalyzes the condensation of acetaldehyde and ꢁ-
ketobutyrate only under alkaline reaction conditions, in
which the condensation product, 4-hydroxy-3-methyl-2-
keto-pentanoic acid, is easily dehydrated to undesirable
products (our unpublished results). HKP aldolase is
involved in the later stages of many bacterial catabolic
pathways responsible for the degradation of aromatic
Key words: 4-hydroxyisoleucine; aldolase; transaminase;
Arthrobacter simplex; antidiabetics
In 1990, Sharma et al. reported for the first time that
fenugreek seeds lowered serum lipids. Through that
research, fenugreek seeds were found to contain 4-
hydroxyisoleucine (HIL) as a unique major free amino
acid; it has been characterized as one of the active
compounds via meta-cleavage pathways, and is classi-
8–12)
1
–5)
ingredients of these seeds.
covered that the major HIL isomer in fenugreek seeds,
S,3R,4S-HIL, induces glucose-induced insulin secre-
tion through a direct effect on pancreatic ꢀ cells in rats
In 2000, Broca et al. dis-
fied as a class I aldolase.
Hence we screened for
an aldolase-producing microorganism suitable for the
aldolase–transaminase coupling reaction among aromat-
ic compound-assimilating microorganisms and in the
strains of our culture collection in the presence of
Escherichia coli harboring the branched chain amino
2
6)
and humans. Furthermore, in a new rat model of type II
diabetes, 2S,3R,4S-HIL partly corrected hyperglycemia
y
To whom correspondence should be addressed. Fax: +81-75-753-6128; E-mail: sim@kais.kyoto-u.ac.jp

1608
J. OGAWA et al.
Amino acid
α-Keto-acid
O
O
OH
O
OH
NH2
+
COOH
COOH
COOH
Aldolase
Transaminase
Acetaldehyde
α
-Ketobutyrate
4-Hydroxy-3-methyl-
-keto-pentanoic acid
4-Hydroxyisoleucine (HIL)
2
Fig. 1. Synthetic Strategy for HIL by the Aldolase–Transaminase Coupling Reaction.
acid transaminase gene (ilvE) from E. coli K12 substrain
MG1655.
(pH 6.0). E. coli BL21(DE3)/pET-15b-ilvE, harboring
the branched chain amino acid transaminase gene from
E. coli K-12 substrain MG1655, was obtained from the
Institute of Life Sciences, Ajinomoto Co., Inc., and
cultivated in Luria Bertrani (LB) medium containing
1.0% (w/v) tryptone, 0.5% (w/v) yeast extract, and
1.0% (w/v) NaCl in tap water (pH 7.2).
In this paper, we describe the screening of an
aldolase-producing bacterium that is suitable for the
aldolase–transaminase coupling reaction and the opti-
mization of HIL synthesis using resting cells of
Arthrobacter simplex AKU 626 and E. coli
BL21(DE3)/pET-15b-ilvE, as well as the basic charac-
terization of the aldolase purified from A. simplex AKU
Screening methods. E. coli BL21(DE3)/pET-15b-
ꢀ
6
26.
ilvE was cultivated at 37 C with shaking (120
strokes/min) in a 2-l shaking flask containing 500 ml
of LB medium until OD555 reached 1.0. Then the trans-
aminase gene was induced by the addition of isopropyl
ꢀ-D-thiogalactoside to the medium at a final concen-
tration of 1 mM, and the culture was subsequently in-
Materials and Methods
Chemicals. An authentic sample of HIL was obtained
from the Institute of Life Sciences., Ajinomoto Co., Inc.
ꢀ
(
Kawasaki, Japan). 3-(2-Hydroxyphenyl)propionic acid
cubated at 37 C. After 4 h, the cells were harvested by
ꢀ
and 3-(4-methylphenyl)propionic acid were purchased
from Lancaster Industrial Chemical Manufacturers
centrifugation (12;000 ꢁ g for 20 min at 4 C, High Mac
CR21; Hitachi, Tokyo), washed twice with physiolog-
ical saline, centrifuged again, and re-suspended in 16 ml
of physiological saline. The microorganisms isolated or
obtained from the type culture collection were inocu-
lated into 5 ml of each medium described above, and
(
Morecambe, England). (S)-2-Aminobutyric acid, cat-
echol, ꢁ-ketobutyrate, acetaldehyde, and L-glutamate
were purchased from Wako Pure Chemical Industries
(Osaka, Japan.). All other chemicals used were of
analytical grade and are commercially available.
ꢀ
then cultivated with shaking (300 strokes/min) at 28 C
for 1–2 d for screening of aldolase activity. After cul-
tivation, the culture broths (5 ml each) were added to the
E. coli BL21(DE3)/pET-15b-ilv cell suspension (200
ml) described above. The combined cell suspension
obtained was centrifuged (2;150 ꢁ g for 10 min at room
temperature, LC-100; Tomy Seiko, Tokyo), and the
cells were resuspended in physiological saline and
centrifuged again. Resting cells consisting of the various
microorganisms for aldolase screening and E. coli
BL21(DE3)/pET-15b-ilvE were examined as biocata-
lysts for the synthesis of HIL. The aldolase–transami-
nase coupling reaction was started by the addition of a
reaction mixture (0.5 ml), comprising a 50 mM potassi-
um phosphate buffer (pH 7.0), 1% (w/v) acetaldehyde
(227 mM), 1% (w/v) ꢁ-ketobutyrate (68 mM), and 1%
(w/v) L-glutamate (68 mM) to the resting cells in a test
tube (12 ꢁ 100 mm). The reaction was carried out at
Microorganisms and culture conditions. All media
described below were adjusted to pH 7.0 unless other-
wise stated. Microorganisms assimilating aromatic com-
pounds were isolated by enrichment culture. The me-
dium (medium A) used was composed of 0.1% (w/v)
.
H O, 0.01% (w/v) yeast extract, 0.2% (w/v) NH Cl,
KH PO , 0.1% (w/v) K HPO , 0.03% (w/v) MgSO
4
2
4
2
4
7
and 0.15% (w/v) carbon source, and was incubated at
2
4
ꢀ
2
8 C. The same medium was used for the cultivation of
aromatic compound-assimilating microorganisms. Cat-
echol, 3-(2-hydroxyphenyl)propionic acid, or 3-(4-meth-
ylphenyl)propionic acid was used as a carbon source.
Microorganisms obtained from the culture collection of
the Faculty of Agriculture of Kyoto University (AKU)
were also examined. The medium for bacteria and yeasts
(
medium B) comprised 1.5% (w/v) peptone, 0.1% (w/v)
ꢀ
yeast extract, 0.3% (w/v) K2HPO4, 1.0% (w/v) glucose,
28 C with shaking (300 strokes/min) for 12–24 h. The
.
0
.2% (w/v) NaCl, and 0.01% (w/v) MgSO4 7H2O. The
acetaldehyde and ꢁ-ketobutyrate solutions were acidic
and were neutralized with 2 N NaOH prior to use.
After incubation, the reaction mixture was centrifuged
(2;150 ꢁ g for 10 min at room temperature, LC-100),
and the supernatant was diluted 1:500 with MilliQ-
filtered de-ionized water, and 20 ml of the diluted sol-
medium for actinomycetes comprised 0.4% (w/v) sol-
uble starch, 0.4% (w/v) yeast extract, and 1.0% (w/v)
malt extract. The medium for molds and basidiomycetes
comprised 0.5% (w/v) peptone, 0.3% (w/v) yeast ex-
tract, 1.0% (w/v) glucose, and 0.3% (w/v) malt extract

Enzymatic Synthesis of 4-Hydroxyisoleucine
1609
ꢀ
ution obtained was reacted with AccQ-Tag fluorescence
derivatization reagent (Waters, Milford, MA) to deriva-
tize HIL to AccQ-Tag-derivated HIL, and 10 ml of the
reaction mixture was subjected to amino acid analysis.
626. All procedures were carried out at 0–5 C, and a 20
mM Tris–HCl buffer (pH 7.4) containing 0.1 mM dithio-
0
.
threitol, 1 mM MnCl 4H O, 0.05 mM pyridoxal 5 -
phosphate (PLP), and 5% ethylene glycol (buffer A)
was used as the standard buffer.
2
2
Analytical methods. Quantitative analysis of AccQ-
Tag-derivatized HIL was performed with an amino acid
analyzer (Waters 2695 Separation Module System;
Waters) equipped with an AccQ-Tag column (3:9 ꢁ
Step 1: Preparation of a cell-free extract. A. simplex
AKU 626 was cultivated in 10 liters of Medium B at
ꢀ
28 C for 48 h with shaking, and the cells were collected
ꢀ
by centrifugation at 12;000 ꢁ g for 20 min at 4 C (High
1
50 mm; Waters). The AccQ-Tag-derivatized HIL was
Mac CR21). The cells obtained (about 100 g wet-
weight) were suspended in 150 ml of buffer A, and then
ultrasonically disrupted for 10 min (19 kHz, Insonator
model 201M; Kubota, Osaka, Japan) with cooling by
water and ice. After centrifugation at 12;000 ꢁ g for
eluted with AccQ-Tag Eluent A (Waters) and acetoni-
trile at a flow rate of 1.0 ml/min. The gradient program
used was as follows: initial 0% acetonitrile (100%
AccQ-Tag Eluent A); linear gradient for 0.5 min to 1%
acetonitrile; linear gradient for 17.5 min to 5% acetoni-
trile; linear gradient for 1 min to 9% acetonitrile; linear
gradient for 10.5 min to 17% acetonitrile; linear gradient
for 3.5 min to 60% acetonitrile; hold for 3 min at 60%
acetonitrile; linear gradient for 6 s to 0% acetonitrile,
and equilibration for 15 min at 0% acetonitrile. The
eluted compound was monitored with a fluorescence
detector at 295 nm with the emission induced by ex-
citation at 250 nm.
ꢀ
20 min at 4 C (High Mac CR21), the supernatant was
used as a cell-free extract.
Step 2: Ammonium sulfate fractionation. The cell-
free extract was fractionated with solid ammonium
sulfate. The precipitate obtained at 70–80% ammonium
sulfate saturation was collected by centrifugation at
16;000 ꢁ g for 20 min (High Mac CR21) and then dis-
solved buffer A (15 ml). The enzyme solution (16 ml)
was dialyzed against 8 liters of buffer A for 12 h.
Step 3: Mono Q column chromatography. The dia-
lyzed enzyme solution was applied to a Mono Q HR 10/
10 column (Amersham Pharmacia Biotech, Uppsala,
Sweden) equilibrated with buffer A. After it was washed
with buffer A, the enzyme was eluted with a linear
gradient of 1–0 M NaCl in 120 ml of buffer A. The active
fractions were combined (5.5 ml).
Step 4: Phenyl-Superose column chromatography.
After the addition of ammonium sulfate to 50% satu-
ration, the enzyme solution was applied to a phenyl-
Superose column, HR 5/5 (Amersham Pharmacia Bio-
tech), equilibrated with buffer A containing ammonium
sulfate (50% saturation) (buffer B). After the column
was washed with buffer B, the enzyme was eluted with a
linear gradient of ammonium sulfate, 50–0% saturation,
in 30 ml of buffer A. The active fractions were combined
Optimization of the reaction conditions for HIL
synthesis using resting cells of A. simplex AKU 626.
ꢀ
A. simplex AKU 626 was cultivated at 28 C with
shaking (120 strokes/min) in a 2-l shaking flask con-
taining 500 ml of medium B for 36–48 h. Five ml of the
culture broth was sampled and used for the reaction. The
reaction was carried out under the conditions described
under ‘‘Screening methods.’’ If necessary, the reaction
was terminated by the addition of 50 ml of 15% (v/v)
perchloric acid, and the solution was neutralized with
4
50 ml of 500 mM potassium phosphate buffer (pH 7.0).
The effects of cultivation time, reaction time, reaction
pH, cofactors, and amino donors on HIL synthesis were
examined under the same conditions, except for the
condition tested.
(1.5 ml).
Step 5: Alkyl-Superose column chromatography. Af-
Stereoisomer analysis of HIL by liquid chromato-
graph mass spectrometer (LC–MS). Enzymatically
prepared HIL was reacted with AccQ-Tag fluorescence
derivatization reagent (Waters), and AccQ-Tag-derivat-
ed HIL was separated using a high pressure liquid
chromatograph, Alliance 2695 (Waters) equipped with
column LUNA C18 (150 ꢁ 2 mm, 3 mm; Phenomenex,
ter the addition of ammonium sulfate to 50% saturation,
the enzyme solution was applied to an alkyl-Superose
column, HR 5/5 (Amersham Pharmacia Biotech), equil-
ibrated with buffer B. After it was washed with buffer B,
the enzyme was eluted with a linear gradient of am-
monium sulfate, 50–0% saturation, in 30 ml of buffer A.
The fractions containing the enzyme activity (500 ml)
were concentrated to 50 ml by ultrafiltration with a
Centricon 10 (Amicon, Beverly, MA).
Step 6: Superdex 200 column chromatography. The
enzyme solution was applied to a Superdex 200 HR 10/
30 column (Amersham Pharmacia Biotech) equilibrated
with buffer A containing 200 mM NaCl (buffer C). The
enzyme was eluted with 40 ml of buffer C. The fractions
(0.5 ml) containing the enzyme activity were used for
further characterization.
ꢀ
Torrance, CA) at 26 C. The mobile phase contained
1
0 mM CH COONH at pH 6.8. The flow rate was 0.3
3 4
ml/min. Detection was performed by positive mode
electrospray ionization (ESI) in a Quattro-Micro tandem
quadrupole mass-spectrometer (Waters). The source
conditions were: capillary voltage, 2.0 kV; cone voltage,
ꢀ
2
0 V; source temperature, 119 C; dessolvation temper-
ꢀ
ature, 450 C; cone gas flow, 60 l/h; dessolvation gas
flow, 659 l/h.
Purification of the aldolase from A. simplex AKU

1610
J. OGAWA et al.
Table 1. Microorganisms Producing More Than 1 mM HIL (a) and Showing High Productivity with Good Reproducibility (b)
(a)
Bacteria
176
Yeasts
248
Actinomycetes
78
Molds
177
Basidiomycetes
57
Soil isolates
840
Screened
Showing HIL
production
12
18
0
0
1
10
(> 1 mM)
(b)
HIL concentration
(
AKU number
Strain
Origin
Class
mM)
1
6
9
05
26
95
Alcaligenes denitrificans subsp. denitrificans
Arthrobacter simplex
Bacterium
Bacterium
Bacterium
Yeast
1580
2240
1470
1040
IFO12069
IFO 0149
Ochrobactrum anthropi A37a
Hansenula anomala
4
303
Enzyme assays. Because it was impossible to measure
Results and Discussion
the amount of the aldolase condensation product, 4-
hydroxy-3-methyl-2-keto-pentanoic acid, it was difficult
directly to evaluate the aldolase activity. Hence the ap-
parent aldolase activity was evaluated by the production
of HIL through the aldolase–transaminase coupling re-
action. The standard conditions for HIL-producing ac-
tivity assay were as follows: The reaction mixture, 200
ml, comprised 50 ml of enzyme solution, 30 ml of E. coli
BL21(DE3)/pET-15b-ilvE cell suspension as described
above, 50 mM Tris–HCl buffer (pH 7.4), 0.2 mM PLP,
Screening of the aldolase-producing microorganisms
useful for synthesis of HIL by the aldolase–transaminase
coupling reaction
The aromatic compound-assimilating microorganisms
isolated from soils (840 strains), and the stocked
cultures, including bacteria (176 strains), yeasts (248
strains), actinomycetes (78 strains), molds (177 strains),
and basidiomycetes (57 strains), were examined as
biocatalysts for the synthesis of HIL by the aldolase–
transaminase coupling reaction. The isolated micro-
organisms (10 strains) and the type-cultures (31 strains:
bacteria 12, yeasts 18, and basidomycete 1) were found
to synthesize HIL at more than 1 mM. These 41 strains
were re-examined, and reproducibility for synthesis of
HIL was observed for only four strains (Table 1). The
highest HIL synthesis was observed for Arthrobacter
simplex AKU 626. From 1% (w/v) acetaldehyde (227
mM), 1% (w/v) ꢁ-ketobutyrate (68 mM), and 1% (w/v)
L-glutamate (68 mM), 2.2 mM HIL was synthesized in
12 h in the presence of resting cells of A. simplex AKU
626 and E. coli BL21(DE3)/pET-15b-ilvE under the
screening conditions. A. simplex AKU 626 was there-
fore selected and used for further investigation.
2
mM L-glutamate. Each reaction mixture was incubated at
50 mM acetaldehyde, 75 mM ꢁ-ketobutyrate, and 100
ꢀ
2
8 C for 12 h. Under the conditions employed, the rate
of 4-hydroxy-3-methyl-2-keto-pentanoic acid amination
was assumed to be proportionate to the rate of 4-hy-
droxy-3-methyl-2-keto-pentanoic acid synthesis. Thus
the specific activity was indirectly estimated by the rate
of HIL formation. After incubation, the reaction mixture
ꢀ
was centrifuged at 14;000 ꢁ g for 10 min at 4 C (MX-
1
50; Tomy Seiko, Tokyo), and then the supernatant was
analyzed as described above. One unit of aldolase ac-
tivity corresponded to the formation of 1 mmol of HIL/
min.
Analytical methods. Protein concentration was de-
termined with a Bio-Rad protein assay kit (Bio-Rad,
Hercules, CA). SDS–polyacrylamide gel electrophoresis
Effect of the cultivation time of A. simplex AKU 626
on the aldolase–transaminase coupling reaction
Culture broth (5 ml) was taken during the cultivation
of A. simplex AKU 626 at 6-h intervals, and was used to
examine the effect of cultivation time on the aldolase–
transaminase reaction. High levels of HIL synthesis
were observed when it was cultivated for 24–48 h, which
corresponded to the mid-log and stationary growth
phases. The addition of aromatic compounds such as
catechol, 3-(2-hydroxyphenyl)propionic acid, and 3-(4-
methylphenyl)propionic acid did not increase the
amount of HIL synthesized, indicating that the en-
zyme(s) involved in the aldol condensation of acetalde-
hyde and ꢁ-ketobutyrate are not induced by these
compounds.
(
SDS–PAGE) was performed in a 12.5% polyacryl-
amide gel using the Tris–glycine buffer system. The
native and subunit molecular masses of the enzyme were
determined by comparison of the mobility of the purified
enzyme on gel filtration chromatography on Superdex
2
00 HR 10/30 and SDS–PAGE respectively with those
of the standard protein (MW-Marker, HPLC, for gel
filtration; Oriental Yeast, Osaka, Japan, and M. W.
marker III for SDS–PAGE; Daiichi Kagaku Yakuhin,
Tokyo). The N-terminal amino acid sequence of the
purified enzyme was analyzed by automated Edman
degradation with a 491HT protein sequencer (Applied
Biosystems, Foster City, CA).

Enzymatic Synthesis of 4-Hydroxyisoleucine
1611
A 2
B 1.6
.5
2
1
0
0
.2
.8
.4
0
1
0
.5
1
.5
0
0
100 200 300 400 500 600 700 800
0
100
200
300
400
500
600
Acetaldehyde concentration (mM)
α
-Ketobutyrate concentration (mM)
C 1.6
1
0
0
.2
.8
.4
0
0
100
200
300
400
500
600
L-Glutamate concentration (mM)
Fig. 2. Effects of Substrate Concentrations on HIL Synthesis.
A, acetaldehyde; B, ꢁ-ketobutyrate; C, L-glutamate. Reactions were carried out under standard conditions in a reaction mixture (0.5 ml)
comprising a 50 mM potassium phosphate buffer (pH 7.0), 1% (w/v) acetaldehyde (227 mM), 1% (w/v) ꢁ-ketobutyrate (68 mM), and 1% (w/v) L-
glutamate (68 mM), with the following exceptions: B, 250 mM acetaldehyde was used; C, 250 mM acetaldehyde and 75 mM ꢁ-ketobutyrate were
used. Each point represents the mean ꢂ S.E. (standard error of the mean) for triplicate samples in duplicate experiments.
Optimization of the reaction conditions for HIL
synthesis
2
1.8
Optimum concentrations of the substrates: The opti-
mum concentrations of acetaldehyde, ꢁ-ketobutyrate,
and L-glutamate were found to be 250, 75, and 100 mM,
respectively (Fig. 2). HIL was also synthesized without
the addition of L-glutamate (Fig. 2C), indicating that an
endogenous amino donor probably exists in the resting
cells.
1
1
1
.6
.4
.2
1
0
0
.8
.6
Effect of pH: The highest HIL synthesis was observed
in a 50 mM Tris–HCl buffer, pH 7.5 (Fig. 3). A lower
HIL synthesis was observed at the same pH in a 50 mM
potassium phosphate buffer. HIL synthesis decreased
with increasing concentrations of a potassium phosphate
buffer.
0.4
0.2
0
0
2
4
6
8
10
12
pH
Effects of metal ions: The effects of various metal
ions (2 mM) on HIL synthesis were examined. HIL
Fig. 3. Effect of Reaction pH on HIL Synthesis.
Reactions were carried out under standard conditions, except for
the buffers (50 mM) used: AcONa–HCl (pH 3–3.5); AcOH–AcONa
(pH 3.5–6.0); KPB (pH 6.0–7.5); Tris–HCL (pH 7.5–9.5), and
2þ
2þ
synthesis was enhanced by the addition of Co , Mn ,
2
þ
2þ
Mg , or Fe , and the relative yields were 122, 140,
18, and 117% respectively. The other metal ions tested
Borate (pH 9.0–10.5). Each reaction mixture was incubated at
ꢀ
1
2
8 C for 12 h.
þ þ 2þ 2þ 2þ
(
HIL synthesis.
Na , K , Ca , Zn , and Cu ) were not effective on
Effects of amino donors: Various amino donors were
examined in place of L-glutamate. Among the com-
pounds listed in Table 2, high levels of HIL synthesis
were achieved with aliphatic and aromatic amino acids,
such as valine, leucine, norvaline, phenylalanine, and
phenylglycine. (S)-2-Aminobutyric acid, a by-product of
the aldolase–transaminase coupling reaction (Fig. 6),
also served as an amino donor in this reaction (data not
shown).

1612
J. OGAWA et al.
Table 2. Effects of Amino Donors on HIL Synthesis from A. simplex AKU 626
Amino donor
Relative activity (%)
Amino donor
Relative activity (%)
Amino donor
Relative activity (%)
L-Glycine
28
94
L-Methionine
L-Cysteine
L-Glutamate
L-Glutamine
L-Aspartate
L-Asparagine
L-Lysine
109
5
DL-Norvaline
L-Kynurenine
L-Ornithine
150
6
L-Alanine
DL-Valine
128
154
109
100
75
100
94
94
26
43
25
12
75
6
L-Leucine
L-Histidinol
6
L-Isoleucine
L-Serine
L-2-Aminoadipic acid
Taurine
9
11
123
56
42
12
L-Threonine
L-Tyrosine
L-Phenylalanine
L-Tryptophan
L-2-Phenylglycine
4-Aminobutyric acid
Ammonium sulfate
None
64
L-Histidine
L-Arginine
ꢀ-Alanine
155
106
Activity with L-glutamate was defined as 100%.
Basic characterization of the aldolase from A. sim-
plex AKU 626
The aldolase activity of the cell-free extract of
A. simplex AKU 626 significantly decreased during
dialysis. Because there have been reports that aldolases
require divalent metal ions, the effects of metal ions
4
3
2
þ
2þ
2þ
were investigated. Mn , Co , and Cd were found to
be effective for maintaining aldolase activity in cell-free
2
1
2
þ
extract. Among these, Mn was selected for further
experiments because it did not produce any color or
precipitate which would disturb purification by column
chromatography or protein analysis on measuring ab-
sorbance. The effects of various cofactors and organic
acids were also investigated. Aldolase activity was
0
0
2
4
6
8
0
maintained by the addition of pyridoxal 5 -monophos-
phate (PLP) to the dialysis buffer. The other cofactors
Reaction time (h)
(
NADH, NADPH, FAD, and thiamine pyrophosphate)
Fig. 4. Time Course of HIL Synthesis by the Aldolase–Transaminase
Coupling Reaction with Resting Cells of A. simplex AKU 626.
The reaction was carried out in 50 mM Tris–HCl buffer (pH 7.5)
containing 250 mM acetaldehyde, 75 mM ꢁ-ketobutyrate, 100 mM L-
and the organic acids (ascorbate, succinate, and citrate)
did not have significant effects. To maintain aldolase
activity, PLP (0.05 mM) and Mn (1 mM) were added to
the buffer during purification.
The aldolase was purified to homogeneity through
five successive steps from cell-free extract of A. simplex
AKU 626. A typical purification is shown in Table 3.
The final preparation gave a single band on SDS–PAGE
2
þ
.
ꢀ
glutamate, and 2 mM MnCl2 4H2O at 28 C. Each point represents
the mean ꢂ S.E. for triplicate samples in duplicate experiments.
Closed circles, reaction with resting cells of A. simplex AKU 626
and E. coli BL21(DE3)/pET-15b-ilvE. Closed square, reaction with
resting cells of A. simplex AKU 626.
(Fig. 5), and showed specific activity of 0.045 U/mg.
Time course of HIL synthesis by the aldolase–trans-
aminase coupling reaction with resting cells of A. sim-
plex AKU 626
The native protein showed a molecular mass of 180 kDa
by gel filtration and of 32 kDa by SDS–PAGE analysis,
indicating that the aldolase was a hexameric protein.
The N-terminal amino acid sequence of the enzyme
was determined to be as follows: PFPVELPDNFAKR-
VTDSXSAQV. This sequence showed no significant
homology to known aldolases on a BLAST database
search. This suggests that the aldolase catalyzing HIL
production was not HKP aldolase but a novel aldolase.
The optimum reaction pH and temperature for the
The time course of HIL synthesis by resting cells of
A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-
ilvE was investigated under optimum reaction condi-
tions, with 250 mM acetaldehyde, 75 mM ꢁ-ketobutyrate,
and 100 mM L-glutamate in 50 mM Tris–HCl buffer
ꢀ
.
(
pH 7.5) containing 2 mM MnCl 4H O at 28 C. HIL
2
2
synthesis was maximum (3.2 mM) at 2 h, and decreased
with longer reaction times (Fig. 4). The amount of HIL
produced corresponding to 470 mg in 1-L of the reaction
mixture is comparable to the amount of extracted HIL
from 3 kg fenugreek seeds (about 150 mg from 1 kg of
fenugreek seeds). Even in the absence of transaminase-
expressing E. coli BL21(DE3)/pET-15b-ilvE cells, HIL
was synthesized, indicating that an endogenous trans-
aminase exists in A. simplex cells.
2
þ
purified enzyme in the presence of PLP and Mn were
pH 7.0 (in a 50 mM potassium phosphate buffer) and
ꢀ
25 C. More than 80% of the initial activity of the
enzyme was retained in a pH range of 6.0–8.0 after
ꢀ
incubation at 28 C for 60 min. The enzyme was stable
ꢀ
below 30 C after incubation at pH 8.0 for 60 min, but
ꢀ
only 20% of the original activity was retained at 35 C
after incubation at pH 8.0 for 60 min.

Enzymatic Synthesis of 4-Hydroxyisoleucine
1613
Table 3. Purification of the Aldolase
Total protein
mg)
Total activity
(ꢁ10ꢃ3 U)
2453
Specific activity
(ꢁ10ꢃ3 U/mg)
0.47
Yield
(%)
(
Cell-free extract
Ammonium surfate
fractionation
5175
100
62
5
89
1518
2.58
Mono Q HR 10/10
Phenyl-Superose HR 5/5
Alkyl-Superose HR 5/5
Superdex 200 HR 10/30
39.4
63.3
21.2
7.95
1.51
1.61
12.2
2.6
1.74
0.054
0.033
0.87
0.32
0.06
148
44.8
Enzyme activity was evaluated by HIL production though aldolase–transaminase coupling reaction, as described in ‘‘Materials and Methods.’’
4
A B
3
2
1
0
A. simplex A. simplex
E. coli
AKU 626
AKU 626 BL21(DE3)/
+
pET-15b-ilvE
E. coli
BL21(DE3)/
pET-15b-ilvE
Fig. 6. HIL Synthesis by the Resting Cells of A. simplex AKU 626,
E. coli BL21(DE3)/pET-15b-ilvE, and A. simplex AKU 626 and
E. coli BL21(DE3)/pET-15b-ilvE.
Fig. 5. SDS–PAGE Analysis of Purified Aldolase from A. simplex
AKU 626.
HIL synthesis was carried out under optimum reaction conditions
with 250 mM acetaldehyde, 75 mM ꢁ-ketobutyrate, 100 mM L-glu-
Lane A, the purified enzyme (approximately 0.5 mg) after Super-
dex 200 column chromatography. Lane B, molecular mass standards
.
ꢀ
tamate, and 2 mM MnCl2 4H2O at 28 C for 1 h with resting cells of
A. simplex AKU 626, E. coli BL21(DE3)/pET-15b-ilvE, or A. sim-
plex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE. Each point
represents the mean ꢂ S.E. for triplicate samples in duplicate
experiments.
(approximately 20 mg); from top: phosphorylase b (97 kDa), bovine
serum albumin (66 kDa), aldolase (42 kDa), carbonic anhydrase (30
kDa), trypsine inhibitor (20 kDa), and lysozyme (14 kDa).
We succeeded in synthesizing HIL by the aldolase–
transaminase coupling reaction, and found that the
aromatic compound-assimilating microorganisms isolat-
ed from soils and certain strains in our culture collection
were useful for this reaction. This is the first report of
HIL synthesis from acetaldehyde, ꢁ-ketobutyrate, and L-
glutamate. After screening, A. simplex AKU 626 was
selected as the most suitable aldolase-producing strain
for HIL synthesis. By using this strain in the aldolase–
transaminase coupling reaction in the presence of E. coli
BL21(DE3)/pET-15b-ilvE harboring the branched chain
amino acid transaminase gene from E. coli K-12 sub-
strain MG1655, 3.2 mM HIL was synthesized under the
optimum reaction conditions (Fig. 4). As shown in
Fig. 6, even A. simplex AKU 626 or E. coli BL21-
that A. simplex AKU 626 and E. coli BL21(DE3)/pET-
15b-ilvE produce their own transaminase and aldolase
respectively. However, the combination of these two
strains resulted in much higher HIL production. Low
transaminase activity in the resting cells of A. simplex
AKU 626 might be a bottleneck in HIL production by
the aldolase–transaminase coupling reaction, and this
was ameliorated by the addition of resting cells of trans-
aminase-expressing E. coli BL21(DE3)/pET-15b-ilvE.
HIL has three chiral centers, which generate eight
stereoisomers. The stereostructure of the ꢁ-carbon of the
HIL synthesized by the aldolase–transaminase coupling
reaction might be of the L-form due to the stereo-
specificity of the transaminase. Therefore, there are four
possible stereoisomers of HIL synthesized by the re-
(
DE3)/pET-15b-ilvE alone produced HIL, indicating

1614
J. OGAWA et al.
2
-Aminobutyric acid
24.2
Unknown
HIL
25.0
25.7
18.9
15.4
Retention time (min)
Fig. 7. Amino Acid Analysis Chromatogram of the Reaction Mixture of the Aldolase–Transaminase Coupling Reaction with Resting Cells of
A. simplex AKU 626.
action. The stereostructure of HIL synthesized by the
aldolase–transaminase coupling reaction was analyzed
by LC–MS after it was reacted with AccQ-Tag fluo-
rescence derivatization reagent. The HIL synthesized
consisted of 2S,3R,4S-HIL and 2S,3R,4R-HIL in the
ratio of 6:1. Only two isomers were found, and the major
isomer was the same as the major isomer contained in
fenugreek seeds.
Three peaks of the reaction products were observed
in the chromatogram when the reaction mixture of the
aldolase–transaminase coupling reaction with resting
cells of A. simplex AKU 626 and E. coli BL21(DE3)/
pET-15b-ilvE was analyzed with an amino acid analyz-
er. Two of these were identified as (S)-2-aminobutyric
acid and HIL, and their retention times were 24.2 and
because the transaminase in the reaction mixture also
requires PLP as a cofactor. Further characterization,
cloning, and expression of the aldolase are in progress.
References
1
)
Narender, T., Puri, A., Shweta, Khaliq, T., Saxena, R.,
Bhatia, G., and Chandra, R., 4-Hydroxyisoleucine an
unusual amino acid as antidyslipidemic and antihyper-
glycemic agent. Bioorg. Med. Chem. Lett., 16, 293–296
(
2006).
2
)
Handa, T., Yamaguchi, K., Sono, Y., and Yazawa, K.,
Effects of fenugreek seed extract in obese mice fed a
high-fat diet. Biosci. Biotechnol. Biochem., 69, 1186–
1188 (2005).
3) Flammang, A. M., Cifone, M. A., Erexson, G. L., and
Stankowski, L. F. Jr., Genotoxicity testing of a fenugreek
extract. Food Chem. Toxicol., 42, 1769–1775 (2004).
2
5.7 min respectively (Fig. 7). (S)-2-Aminobutyric acid
was a by-product synthesized by the amination of ꢁ-
ketobutyrate catalyzed by the transaminase. An un-
known peak was observed at a retention time of 25.0
min. It was probably derived from a compound synthe-
sized from acetaldehyde, ꢁ-ketobutyrate, and/or L-
glutamate. Further identification of this unknown prod-
uct is necessary to elucidate the reaction mechanism in
detail.
Aldolases are divided into two classes: class I
aldolases form a Schiff-base intermediate in the active
site with the donor substrate, and class II aldolases use
divalent metal ions as cofactors. The aldolase produced
by A. simplex AKU626 was inhibited by a potassium
4
)
Fowden, L., Pratt, H. M., and Smith, A., 4-Hydroxyiso-
leucine from seed of Torigonella foenum-graecum.
Phytochemistry, 12, 1707–1711 (1973).
5
)
Haefele, C., Bonfils, C., and Sauvaire, Y., Character-
ization of a dioxgenase from Torigonella foenum-
graecum involved in 4-hydroxyisoleucine biosynthesis.
Phytochemistry, 44, 563–566 (1997).
6
)
Broca, C., Manteghetti, M., Gross, R., Baissac, Y.,
Jacob, M., Petit, P., Sauvaire, Y., and Ribes, G., 4-
Hydroxyisoleucine: effects of synthetic and natural
analogues on insulin secretion. Eur. J. Pharmacol.,
3
90, 339–345 (2000).
7
)
)
Pollard, J. R., Rialland, D., and Bugg, T. D. H., Substrate
selectivity and biochemical properties of 4-hydroxy-2-
keto-pentanoic acid aldolase from Escherichia coli.
Appl. Environ. Microbiol., 64, 4093–4094 (1998).
Arai, H., Yamamoto, T., Ohnishi, T., Shimizu, T.,
Nakata, T., and Kudo, T., Genetic organization and
characteristics of the 3-(3-hydroxyphenyl)propionic acid
degradation pathway of Comamonas testosteroni TA441.
Microbiology, 145, 2813–2820 (1999).
2
þ
2þ
2þ
phosphate buffer, and enhanced by Co , Mn , Mg ,
and Fe . Furthermore, the aldolase was stabilized by
2þ
2
þ
2þ
2þ
Mn , Co , or Cd during purification. These results
suggest that it is a class II aldolase. The Arthrobacter
aldolase also required PLP to maintain its activity during
purification. There have been some reports on PLP-
dependent aldolases. It is not clear that the
Arthrobacter aldolase requires PLP as a coenzyme,
8
1
3–16)
9) Burlingame, R., and Chapman, P. J., Catabolism of

Enzymatic Synthesis of 4-Hydroxyisoleucine
1615
phenylpropionic acid and its hydroxy derivative by
Escherichia coli. J. Bacteriol., 155, 113–121 (1983).
0) Burlingame, R., and Chapman, P. J., Stereospecificity in
meta-fission catabolic pathways. J. Bacteriol., 155, 424–
bacter sp. DK-38. Eur. J. Biochem., 248, 385–393
(1997).
1
1
14) Liu, J.-Q., Dairi, T., Itoh, N., Kataoka, M., Shimizu, S.,
and Yamada, H., A novel metal-activated pyridoxal
enzyme with a unique primary structure, low specificity
D-threonine aldolase from Arthrobacter sp. DK-38. J.
Biol. Chem., 273, 16678–16685 (1998).
15) Kataoka, M., Wada, M., Nishi, K., Yamada, H., and
Shimizu, S., Purification and characterization of L-allo-
threonine aldolase from Arthrobacter jandaei DK-39.
FEMS Microbiol. Lett., 151, 245–248 (1997).
426 (1983).
1) Kim, D., Kim, Y. S., Kim, S. K., Kim, S. W., Zylstra, G.
J., Kim, Y. M., and Kim, E., Monocyclic aromatic
hydrocarbon degradation by Rhodococcus sp. strain
DK17. Appl. Environ. Microbiol., 68, 3270–3278 (2002).
2) Sakai, M., Miyauchi, K., Kato, N., Masai, E., and
Fukuda, M., 2-Hydroxypenta-2,4-dienoate metabolic
pathway genes in a strong polychlorinated biphenyl
degrader, Rhodococcus sp. strain RHA1. Appl. Environ.
Microbiol., 69, 427–433 (2003).
1
16) Liu, J.-Q., Dairi, T., Itoh, N., Kataoka, M., Shimizu, S.,
and Yamada, H., L-allo-Threonine aldolase from Arthro-
bacter jandaei DK-39: gene cloning, nucleotide se-
0
1
3) Kataoka, M., Ikemi, M., Morikawa, T., Miyoshi, T.,
Nishi, K., Wada, M., Yamada, H., and Shimizu, S.,
Isolation and characterization of D-threonine aldolase, a
quencing, and identification of the pyridoxal 5 -phos-
phate-binding lysine residue by site-direct mutagenesis.
J. Bacteriol., 179, 3555–3560 (1997).
0
pyridoxal-5 -phosphate-dependent enzyme from Arthro-