Purification and characterization of two NAD-dependent alcohol dehydrogenases (ADHs) induced in the quinoprotein ADH-deficient mutant of Acetobacter pasteurianus SKU1108

Article abstract of DOI:10.1271/bbb.67.958

High NAD-dependent alcohol dehydrogenase (ADH) activity was found in the cytoplasm when a membrane-bound, quinoprotein, ADH-deficient mutant strain of Acetobacter pasteurianus SKU1108 was grown on ethanol. Two NAD-dependent ADHs were separated and purified from the supernatant fraction of the cells. One (ADH I) is a trimer, consisting of an identical subunit of 42 kDa, while the other (ADH II) is a homodimer, having a subunit of 31 kDa. One of the two ADHs, ADH II, easily lost the activity during the column chromatographies, which could be stabilized by the addition of DTT and MgCl₂ in the column buffer. ADH I but not ADH II contained approximately one zinc atom per subunit. The N-terminal amino acid analysis indicated that ADH I and ADH II have homology to the long-chain and short-chain ADH families, respectively. ADH I showed a preference for primary alcohols, while ADH II had a preference for secondary alcohols. The two ADHs showed clear difference in their kinetics on ethanol, acetaldehyde, NAD, and NADH. The physiological function of both ADH I and ADH II are also discussed.

Full text of DOI:10.1271/bbb.67.958

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Purification and Characterization of Two NAD-
Dependent Alcohol Dehydrogenases (ADHs) Induced
in the Quinoprotein ADH-Deficient Mutant of
Acetobacter pasteurianus SKU1108
Piyawan CHINNAWIROTPISANa, Kazunobu MATSUSHITAb, Hirohide TOYAMAb, Osao ADACHIb,
Savitree LIMTONGa & Gunjana THEERAGOOLa
a Department of Microbiology, Faculty of Science, Kasetsart UniversityBangkok 10900,
Thailand
b Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi
UniversityYamaguchi 753-8515, Japan
Published online: 22 May 2014.
To cite this article: Piyawan CHINNAWIROTPISAN, Kazunobu MATSUSHITA, Hirohide TOYAMA, Osao ADACHI, Savitree
LIMTONG & Gunjana THEERAGOOL (2003) Purification and Characterization of Two NAD-Dependent Alcohol
Dehydrogenases (ADHs) Induced in the Quinoprotein ADH-Deficient Mutant of Acetobacter pasteurianus SKU1108,
Bioscience, Biotechnology, and Biochemistry, 67:5, 958-965, DOI: 10.1271/bbb.67.958
To link to this article: http://dx.doi.org/10.1271/bbb.67.958
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Biosci. Biotechnol. Biochem., 67 (5), 958–965, 2003
Puriˆcation and Characterization of Two NAD-Dependent Alcohol
Dehydrogenases (ADHs) Induced in the Quinoprotein ADH-Deˆcient
Mutant of Acetobacter pasteurianus SKU1108
Piyawan CHINNAWIROTPISAN,1 Kazunobu MATSUSHITA
,
† Hirohide TOYAMA,2 Osao ADACHI
2,
,
2
1
Savitree LIMTONG,1 and Gunjana THEERAGOOL
1Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
2Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515,
Japan
Received September 27, 2002; Accepted December 21, 2002
High NAD-dependent alcohol dehydrogenase (ADH)
activity was found in the cytoplasm when a membrane-
bound, quinoprotein, ADH-deˆcient mutant strain of
Acetobacter pasteurianus SKU1108 was grown on
ethanol. Two NAD-dependent ADHs were separated
and puriˆed from the supernatant fraction of the cells.
One (ADH I) is a trimer, consisting of an identical subu-
nit of 42 kDa, while the other (ADH II) is a homodimer,
having a subunit of 31 kDa. One of the two ADHs,
ADH II, easily lost the activity during the column chro-
matographies, which could be stabilized by the addition
of DTT and MgCl2 in the column buŠer. ADH I but not
ADH II contained approximately one zinc atom per
subunit. The N-terminal amino acid analysis indicated
that ADH I and ADH II have homology to the long-
chain and short-chain ADH families, respectively. ADH
I showed a preference for primary alcohols, while ADH
II had a preference for secondary alcohols. The two
ADHs showed clear diŠerence in their kinetics on
ethanol, acetaldehyde, NAD, and NADH. The physio-
logical function of both ADH I and ADH II are also
discussed.
roloquinoline quinone (PQQ) as the prosthetic
group, and classiˆed into type III quinoprotein
ADH, which is unique in acetic acid bacteria. Recent-
ly, a thermotolerant strain, Acetobacter pasteurianus
SKU1108 (re-identiˆed based on 16S rDNA se-
quences), was isolated from pineapple in Thailand
based on its ability to produce a large amount of
acetic acid and to tolerate high concentrations of
acetic acid and ethanol even at higher temperatures.2)
In order to elucidate the role of quinoprotein ADH in
such a thermotolerant strain, we obtained a mutant
designated as CN6-2 that has complete loss in
ethanol-oxidizing ability and acetic acid resistance,
by using
N-methyl-N?-nitro-N-nitrosoguanidine
(NTG) treatment. During the analysis of the mutant,
we found that the mutant strain grows very well in
medium containing ethanol, despite having no ability
to oxidize ethanol, and that two NAD-dependent
ADHs, present in a small amount in the wild-type
strain, are increased dramatically in the cytoplasm
when cultured in the medium containing ethanol.
Thus, the mutant strain seemed to induce two diŠer-
ent NAD-dependent ADHs to survive in a medium
containing ethanol when the quinoprotein ADH is
defective (unpublished data).
NAD(P)-dependent ADHs are widely distributed
in microorganisms, plants, and animals. They can be
divided into three groups according to their struc-
tures.3) Group I is a zinc-dependent, long-chain ADH
family, containing approximately 350 amino acid
residues per subunit, and consisting of dimeric or
tetrameric structures. Group II is a zinc-independent,
short-chain ADH containing approximately 250
residues per subunit. There is no evidence for a metal
requirement in any of this group so far characterized.
Key words: acetic acid bacteria; Acetobacter pasteu-
rianus
;
quinoprotein
alcohol
de-
hydrogenase; NAD-dependent alcohol de-
hydrogenase
Acetobacter species are well known as vinegar
producers, which have been shown to produce acetic
acid from ethanol by two sequential reactions of
membrane-bound alcohol dehydrogenase (ADH) and
aldehyde dehydrogenase (ALDH), localized on the
periplasmic side of the membrane.1) The membrane-
bound ADH is a typical quinoprotein, having pyr-
†
To whom correspondence should be addressed. Tel:
u.ac.jp
81-83-933-5858; Fax: 81-83-933-5859; E-mail: kazunobu agr.yamaguchi-
＋ ＋ ＠
A part of this paper was presented at the annual meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry, held in
Sendai, March 25–27, 2002 (No. 3–5Cp20).

NAD-Dependent Alcohol Dehydrogenases from Acetobacter Strain
959
Group III is an ``iron activated'' ADHs of which the
subunit consists of about 385 amino acid residues.
Many organisms have been reported to contain
multiple NAD-dependent ADHs, the physiological
roles of which have sometimes proven di‹cult to
disentangle.4) Candida guilliermondii has been shown
to have two NAD-dependent ADHs, one of which
appears to be involved in the production of ethanol
from acetaldehyde while the other plays a main role
in the oxidation of ethanol to acetaldehyde.5) In
acetic acid bacteria, ethanol is oxidized extensively by
the membrane-bound quinoprotein ADH and only
low NAD-dependent ADH activity is detected in the
cytoplasm.1) There has been no report on NAD-
dependent ADHs in acetic acid bacteria so far.
Therefore, it is important to know the characteristics
of two NAD-dependent ADHs in the mutant strain
of Acetobacter species.
In this work, we describe the puriˆcation and
characterization of two NAD-dependent ADHs from
the soluble fraction of A. pasteurianus CN6-2.
Characterization of the two ADHs indicated that one
(ADH I) is of the long-chain family and shows a
preference for primary alcohols and another (ADH
II) of the short-chain family shows a preference for
secondary alcohols.
10 min and the supernatant was ultracentrifuged at
×
150,000
g for 90 min. The resulting supernatant
was used for further puriˆcation of enzymes.
Enzyme assay. Activities of the two NAD-depen-
dent ADHs were measured, at 259C, spec-
trophotometrically at 340 nm by following the for-
mation or disappearance of NADH. The formation
of NADH follows the oxidation of ethanol to acetal-
dehyde (forward reaction), while the disappearance
of NADH does the reduction of acetaldehyde to
ethanol (reverse reaction). For NAD-dependent
ADH I, the reaction mixture (total 1 ml) for the for-
ward reaction contained 100 m
(pH 8.5), 100 m ethanol, and 0.1 m
the assay mixture for the reverse reaction (1 ml) did
100 m KPB (pH 5.5), 10 m acetaldehyde, and
0.1 m NADH. For NAD-dependent ADH II, the
forward reaction mixture (1 ml) contained 100 m
glycine-NaOH (pH 8.5), 100 m ethanol, and
0.1 m NAD, while that for the reverse reaction
(1 ml) did 100 m 2-morpholinoethanesulfonic acid
(MES) buŠer (pH 6.0), 100 m acetaldehyde, and
0.1 m NADH. The reaction was started by the
M
glycine-NaOH
M
M
NAD, while
M
M
M
M
M
M
M
M
M
addition of enzyme solution. One unit of activity is
deˆned as the amount of enzyme that reduces or
oxidizes 1
min at 25
mmol of NAD or NADH, respectively, per
9
C, where a millimolar extinction coe‹cient
Materials and Methods
of 6.22 was used for the calculation.
Chemicals. NAD and NADH were obtained from
Sigma. All other chemicals were of reagent grade and
obtained from commercial sources.
Puriˆcation of two NAD-dependent ADHs. All
steps were done at 4
9C. KPB (pH 7.5) of various con-
centrations containing 1 m
M
DTT and 1 m
M
MgCl2
Bacterial strains and growth conditions. A. pasteu-
rianus CN6-2, which lacks quinoprotein ADH activi-
ty, was used in this study. The strain was maintained
was used. The resulting supernatant after ultracen-
trifugation was put directly on a DEAE-Toyopearl
column (about 1 ml of bed volume per 3 mg of
on potato medium6) containing 1.5
z
agar. For culti-
vation, bacteria were inoculated into 5 ml of the
potato medium and incubated for 18 h at 30 C with
protein), which had been equilibrated with 50 m
M
KPB (pH 7.5). Under these conditions, most of the
enzyme activity was not adsorbed onto the column.
The combined pass-through fractions having the
9
shaking at 200 rpm. The seed culture was transferred
to 100 ml of the same medium in a 500-ml Erlenmay-
er ‰ask and cultivated for 24 h. Then, the culture was
transferred to 1 liter of a main culture medium (5 g of
yeast extract, 5 g of Polypeptone, 5 g of glycerol, and
enzyme activity was dialyzed with 5 m
M
KPB
(pH 7.5), and put on an alternative column of
DEAE-Toyopearl, which had been equilibrated with
the same buŠer. The column was washed with the
column buŠer and then eluted by a linear gradient of
5 g of glucose in tap water), in which 4
z
ethanol
(v v) was added aseptically, in a 3-liter Erlenmayer
5 m
M
to 50 m KPB (pH 7.5). NAD-dependent ADH
M
W
‰ask, and cultivated for 48 h.
activity was separated into two fractions. One, with
an activity preference for primary alcohols (ADH I),
Preparation of cell-free extract. Bacterial cells
was eluted at 35 m
M
KPB (pH 7.5), while another
×
M
were harvested by centrifugation at 9,000
g
for
potas-
sium phosphate buŠer (KPB), pH 7.5, containing
1 m dithiothreitol (DTT) and 1 m MgCl2. The cell
enzyme activity having a preference for secondary
alcohols (ADH II) was not adsorbed to the column.
The two NAD-dependent ADH activity fractions
were combined separately.
10 min and washed twice with ice-cold 50 m
M
M
paste was suspended in the same buŠer (1 g of wet
weight 4 ml of buŠer), and passed twice through a
The combined active fraction of ADH I was dia-
lyzed with 5 m KPB (pH 7.5). The dialysate was put
M
W
French pressure cell press (American Instruments
Co., Silver Spring, MD., U.S.A.) at 16,000 psi. Cell
on a hydroxyl apatite column (about 3 ml of bed
volume per 3 mg of protein), which had been
equilibrated with the same buŠer. The column was
debris was removed by centrifugation at 9,000
×
g for

960
P. CHINNAWIROTPISAN et al
.
washed with the column buŠer and then eluted step-
wise with 50 m 100 m and 500 m KPB
(pH 7.5). Almost all of the enzyme activity was elut-
ed with 100 m KPB (pH 7.5). The combined active
found in the supernatant after ultracentrifugation of
the crude extract from A. pasteurianus CN6-2
mutant strain. During the puriˆcation, enzyme activ-
ity against acetaldehyde was measured. NAD-depen-
dent ADH activity was separated into two fractions
on a DEAE Toyopearl column (Fig. 1(A)). One, with
an activity preference for primary alcohols (ADH I),
M
,
M
,
M
M
fraction was concentrated by ultraˆltration with a
UP20 membrane (Advantec Toyo). The concentrated
enzyme (
¿
1.5 ml) was dialyzed with 10 m
M
KPB
(pH 7.5), and put on a DEAE-Toyopearl column,
which had been equilibrated with the same buŠer.
After washing the column with the column buŠer,
the enzyme was eluted with a linear gradient of
was eluted at 35 m
M
KPB (pH 7.5), while another
enzyme activity (ADH II) was passed through the
column. ADH II activity was easily lost during the
column chromatographies, which could be stabilized
by the addition of DTT and MgCl2 in the column
buŠer. The puriˆcation of two NAD-dependent
ADHs are summarized in Table 1. ADH I was
10 m
M
to 40 m
M
KPB (pH 7.5).
The combined active fraction of ADH II in the sec-
ond DEAE-Toyopearl column was directly applied
on a hydroxyl apatite column, which had been
enriched around 35-fold with a 15
z
yield, while
equilibrated with 5 m
column was washed with the same buŠer, the enzyme
was eluted stepwise with 50 m , 100 m , and
500 m KPB (pH 7.5). The whole enzyme activity
was eluted with 100 m KPB (pH 7.5). The com-
M
KPB (pH 7.5). After the
ADH II was enriched around 21-fold with a 7
z
yield.
M
M
M
Purity and molecular mass determination
M
The puriˆed enzyme fractions of ADH I and ADH
II obtained from the ˆnal step, had a single protein
band in SDS-PAGE (Fig. 1(B) and (C)). The relative
molecular masses of ADH I and ADH II were esti-
mated by SDS-PAGE to be 42 kDa and 31 kDa,
respectively. The native molecular sizes measured by
a Superdex S-200 gel ˆltration were 130 kDa and
70 kDa for ADH I and ADH II, respectively (data
not shown), suggesting that the native ADH I is
probably a trimer composed of identical subunits,
while ADH II consists of two identical subunits.
bined active fraction was concentrated by ultraˆltra-
tion with a UP20 membrane (Advantec Toyo). One
and half ml of the concentrated enzyme was put on a
Superdex S-200 column, which had been equilibrated
with 50 m
M
KPB (pH 7.5).
Other analytical procedures. The purity and
molecular mass of the puriˆed enzymes were
analyzed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) as described by
Laemmli.7) Before application, samples were mixed
with 0.33 volume of a sample buŠer and heated at
N-terminal amino acid sequence analysis
The N-terminal amino acid sequence of ADH I
(20 residues) and ADH II (30 residues) were
60
9
on a 10
C for 30 min. Then, the treated samples were put
z
SDS-PAGE slab gel. Standard pre-stained
proteins (BioRad) were used as markers. For N-
terminal sequencing, the separated proteins in SDS-
PAGE were transferred electrophoretically onto a
polyvinylidene di‰uoride microporous membrane.
SGKMKAAVVHEFGKPLTIEE
and
ARVAG-
respec-
KVAIVSGAANGIGKATAQLLAKEGA,
tively. In a database search using the BLAST
algorithm, the ADH I sequence showed the best
matching scores at the amino terminus of prokaryot-
ic long-chain ADHs, especially from Sinorhizobium
meliloti (NC003037), Ralstonia solanacearum
Then, the membrane was stained with 0.1
Coomasie brilliant blue R250 for 1 min, and
destained with 50 methanol. The visible band was
z
z
excised and put into a peptide sequence analyzer
PSQ-2 (Shimadzu). The protein content was meas-
ured by the modiˆed Lowry method.8) The relative
molecular mass of the native enzyme were measured
by gel ˆltration by using a Superdex S-200 column
(AL646073),
and
Pseudomonas
aeruginosa
(AE004955) (Fig. 2(A)). By using the same proce-
dures, ADH II sequence was shown to have identity
with short-chain ADHs, from Agrobacterium
tumefaciens (AE008318), Mycobacterium tuberculo-
sis (AE006970), and Caenorhabitis elegans
(AC006677) (Fig. 2(B)). The data indicated that
ADH I and ADH II belong to a long-chain and a
short-chain ADH family, respectively.
equilibrated with 50 m
M
KPB (pH 7.5) containing
1 m DTT. The standard marker proteins used were
M
obtained from Oriental Yeast, Co., Ltd. The metal
content of the puriˆed enzymes were measured by
induced-coupled plasma atomic emission spectromet-
ry (ICP-AES) using the internal standard method
Kinetic properties of the puriˆed enzymes
＋
with Zn2＋, Mg2＋, and Fe3
.
The apparent Km and Vmax values were measured
for the two substrates, ethanol and acetaldehyde.
Kinetic constants were also measured for the cofac-
tors, NAD and NADH. These kinetic data are sum-
marized in Table 2. Km values of ADH I for ethanol
and acetaldehyde were lower than those of ADH II,
Results
Puriˆcation of two NAD-dependent ADHs
All of the NAD-dependent ADH activity was

NAD-Dependent Alcohol Dehydrogenases from Acetobacter Strain
961
while the
in ADH I than ADH II. From the values of both Km
and max, both enzymes are suggested to operate with
Km values for NAD and NADH were higher
V
a bias toward the reduction of acetaldehyde to
ethanol. Of these kinetics in both enzymes, only
ADH I had substrate inhibition against acetaldehyde
at the higher concentration over 10 m
M
, where the Ki
value was estimated to be 57.3 m (data not shown).
M
Both ADH I and ADH II were NAD-speciˆc and
totally inactive when NADP was used as coenzyme.
Substrate speciˆcity of the puriˆed enzymes
Substrate speciˆcity of crude extract, NAD-depen-
dent ADH I, and ADH II are shown in Table 3. The
crude extracts of A. pasteurianus CN6-2 mutant
strain grown on ethanol contained NAD-dependent
ADHs active on the reduction of a wide range of
aldehydes, and also on the oxidation of primary,
secondary, and cyclic alcohols. The puriˆed ADH I
showed a broad substrate speciˆcity for primary
alcohols (C2 to C10), cyclobutanol, benzylalcohol,
and several aldehydes. Acetaldehyde or propionalde-
hyde, as a substrate for the reverse reaction, was the
most suitable substrate tested. In contrast, the puri-
ˆed ADH II showed a preference for secondary alco-
hols and aldehydes, and butylaldehyde was the best
substrate tested. Cyclic alcohols were also oxidized
with ADH II, but at a lower rate. Of the secondary
alcohols, activity against stereoisomers of 2-butanol,
2-hexanol, and 2-octanol was also examined with
ADH II. As shown in Table 3, the enzyme appears to
Fig. 1. Puriˆcation of Two NAD-Dependent ADHs from A.
pasteurianus CN6-2 Mutant Strain.
(A) Separation of NAD-dependent ADH I and ADH II on
DEAE-Toyopearl column chromatography II. (B) SDS-PAGE
be highly speciˆc for the
shows the enantioselectivity for the
2-butanol, -(–)-2-hexanol, and
R
-(–)-stereoisomers and
isomer, -(–)-
-(–)-2-octanol,
R
R
R
R
respectively, for 2-butanol, 2-hexanol, and 2-oc-
tanol.
of the puriˆed ADH I; Lane 1: Crude extract (10
Lane 2: DEAE-Toyopearl I (10 g), Lane 3: DEAE-Toyopearl
II (10 g), Lane 4: Hydroxyl apatite (10 g), and Lane 5: DEAE-
Toyopearl III (10 g). (C) SDS-PAGE of the puriˆed ADH II;
Lane 1: Crude extract (10 g of protein), Lane 2: DEAE-
Toyopearl I (10 g), Lane 3: DEAE-Toyopearl II (10 g), Lane
4: Hydroxyl apatite (10 g), and Lane 5: Superdex S-200 (10 g).
mg of protein),
m
m
m
m
Metal content and eŠects of metal ions on the
enzyme activity
Metal content of the NAD-dependent ADHs
m
m
m
m
m
Table 1. Summary of the Puriˆcation of NAD-Dependent ADHs (ADH I and ADH II) from the Cytosolic Fraction of A. pasteurianus
CN6-2 Mutant Strain
Total activity
(U)
Total protein
(mg)
Speciˆc activity
Puriˆcation
(fold)
Yield
Puriˆcation step
(U mg)
(z)
W
Crude extracta
31,100
17,400
1,830
829
16.7
20.9
1
1.2
100
56
DEAE-Toyopearl Ia
ADH Ia
DEAE-Toyopearl IIa
Hydroxyl apatitea
DEAE-Toyopearl IIIa
ADH IIb
12,800
8,150
4,740
282
24.2
7.92
45.3
336
597
2.7
20
35
41
26
15
DEAE-Toyopearl IIb
Hydroxyl apatiteb
Superdex S-200b
9,000
7,820
2,160
207
92.8
6.01
43.6
84.3
361
2.6
5.0
21
29
25
7.0
a
Activity was measured by the reduction of acetaldehyde at the ˆnal concentration of 10 m
Methods.
Activity was measured with 100 m
M
, as described for the reverse reaction for ADH I in Materials and
b
M acetaldehyde as the substrate, as described for the reverse reaction for ADH II in Materials and Methods.

962
P. CHINNAWIROTPISAN et al.
Table 3. Substrate Speciˆcity of NAD-Dependent ADHs Activi-
ties in Crude Extract, ADH I and ADH II of A. pasteurianus
CN6-2 Mutant Strain
Activities for primary, secondary, and cyclic alcohols were
measured by the forward reaction, while those for aldehydes, by
the reverse reaction, as described in Materials and Methods.
Crude extract
ADH I
U mg
ADH II
z
U mg ( )
Substrate
(10 m
M
)
a
a
b
U mg
(
z
)
(
z
)
W
W
W
Primary alcohol
Ethanol
1-propanol
1-Butanol
1-Pentanol
1-Hexanol
1-Heptanol
1-Octanol
1-Nanonol
1-Decanol
0.78 (100) 16.4 (100)
0.20 (
º
º
º
º
1)
0.75 (96) 14.7
0.83 (106) 13.4
(90)
(82)
0.38 (
1.30 (
0.34 (
1)
1)
1)
Fig. 2. Alignment of the N-Terminal Amino Acid Sequence of
NAD-Dependent ADHs from A. pasteurianus CN6-2 Mutant
Strain.
0.07
(9)
2.42 (15)
5.76 (35)
8.55 (52)
1.86 (11)
2.97 (18)
3.16 (19)
0.70 (90)
0.45 (58)
0.22 (28)
0.14 (18)
3.21 (2)
(A) ADH I. (B) ADH II. The consensus motif for NAD-bind-
ing is indicated above the alignment; GXXXGXG, glycine-rich
0.92 (
0.38 (
0.34 (
0
º
º
º
1)
1)
1)
*
consensus; , highly conserved residues.
0.07
(9)
(0)
Secondary alcohol
2-Propanol
2-Butanol
0.91 (117)
1.71 (219)
0.56 (72)
2.66 (341)
2.46 (315)
4.69 (601)
0.42 (54)
4.69 (601)
4.37 (560)
0.10 (13)
3.06 (392)
2.60 (16) 22.9 (12)
1.67 (10) 84.2 (46)
Table 2. Kinetic Constants of NAD-Dependent ADHs from A.
pasteurianus CN6-2 Mutant Strain
S
R
-(
＋
)-2-Butanol
1.30
0.37
(8) 18.4 (10)
(2) 107 (58)
(0) 68.9 (37)
(0) 168 (91)
(0) 9.95 (5)
Kinetics for ethanol were measured with various concentrations
-(–)-2-Butanol
of ethanol and 0.1 m
Methods (the forward reaction), while that for acetaldehyde was
done with various concentrations of acetaldehyde and with 0.1 m
M
NAD, as described in Materials and
2-Pentanol
2-Hexanol
0
0
0
0
0
0
0
M
S )-2-Hexanol
＋
-(
-(–)-2-Hexanol
2-Octanol
-( )-2-Octanol
-(–)-2-Octanol
NADH as described as the reverse reaction for ADH I or for ADH
II in Materials and Methods. Kinetics for NAD was done with
R
(0) 184
(0) 153
(100)
(83)
various concentrations of NAD and 100 m
M
ethanol, while that
for NADH with various concentrations of NADH and 100 m
S
＋
(0)
(0) 138
2.30 (1)
(75)
M
R
acetaldehyde, as described as the forward and reverse reactions,
respectively, in Materials and Methods.
Cyclic alcohol
Cyclobutanol
Cyclopentanol
Cyclohexanol
Cyclooctanol
Benzylalcohol
Aldehyde
Formaldehyde
Acetaldehyde
Propionaldehyde
Butylaldehyde
Valeraldehyde
Anisaldehyde
0.93 (119) 11.9
(73)
(0)
(0) 13.2
(0)
(8)
8.73 (5)
0.23 ( 1)
(7)
6.43 (4)
0.02
(3)
0
0
0
1.26
º
ADH I
ADH II
Substrate
0.36 (46)
0.32 (41)
0.25 (32)
or cofactor
Km
Vmax
Km
Vmax
(m
M
)
(U mg)
(m
M
)
(U mg)
W
W
0
(0)
Ethanol
Acetaldehyde
NAD
2.3
0.3
1.2
0.2
9.0
375
55.9
500
7.3
24
0.04
0.03
0.04
222
0.05
90
0.07
(9)
2.23 (14)
0.92 (
º
1)
*
19.5 (2500) 364
19.9 (2551) 223
33.4 (4282) 96.6 (589) 888
2.78 (356) 20.4 (124)
2.91 (281) 4.28 (26)
(2219) 337
(1360) 551
(183)
(299)
(483)
NADH
7.04 (4)
2.76 (2)
a
dialyzed against 20 m
M
HEPES buŠer at pH 7.5 in
The activity with ethanol of the crude extract and of ADH I were de-
ˆned as 100
z.
the absence or presence of EDTA are shown in Table
4. Metal analyses revealed the presence of 1.225 and
b
The activity with
R
-(–)-2-hexanol of ADH II was deˆned as 100
M
z.
*
Measured with 100 m acetaldehyde at the ˆnal concentration.
＋
0.995 mol of Zn2 per mol of ADH I subunit in the
absence and presence of EDTA, respectively. The
data suggest that each subunit of ADH I contains 1
mole of Zn2＋, but ADH II seemed not to contain any
speciˆc metal, at least after dialysis. The eŠects of
metal ions and EDTA on the activity of NAD-depen-
dent ADHs were also studied. The activity of ADH II
ADH II, the decreased enzyme activity was recovered
by the addition of 1 m
M
Mg2＋, but some more inhibi-
＋
tion was observed by the addition of 1 m
M
Zn2
.
was inhibited around 67
z
by EDTA treatment,
EDTA fol-
Discussion
which was done by a dialysis against 5 m
M
lowed by an additional dialysis against the buŠer to
remove EDTA, while ADH I activity was not inhibit-
ed at all by the same treatment. Although data are
not shown, however, the enzyme activity of ADH I
In this study we present the puriˆcation and
characterization of two NAD-dependent ADHs from
A. pasteurianus CN6-2 mutant strain. Two NAD-
dependent ADHs, one with a preference for primary
alcohols (ADH I), and the other for secondary alco-
hols (ADH II), were detected in ethanol-grown A.
pasteurianus CN6-2 mutant strain. The two NAD-
dependent ADHs were separated from each other by
was inhibited 45
z
by the direct addition of 1 m
M
＋
EDTA in the reaction mixture. Addition of Mg2 or
Zn2＋ did not aŠect the enzyme activity of the EDTA-
treated ADH I. In contrast, in the EDTA-treated

NAD-Dependent Alcohol Dehydrogenases from Acetobacter Strain
Table 4. Metal Content in NAD-Dependent ADHs from A.
963
ondary alcohols such as
R
-(–)-2-hexanol or
R
-(–)-2-
pasteurianus CN6-2 Mutant Strain
EDTAa
enzyme)
octanol, which is a very rare case in the secondary
alcohol-speciˆc ADHs so far described. The other
ADHs in this family that have a preference for sec-
ondary alcohols showed the optimal activity with 2-
propanol or 2-butanol and have rather poor activity
with long-chain secondary alcohols greater than
－
＋
EDTAb
m enzyme)
Enzyme
(
m
m
(
m
M WM
MWM
＋
＋
＋
＋
＋
＋
Zn2
Mg2
Fe2
Zn2
Mg2
Fe2
ADH I
ADH II
1.255 0.080 0.035 0.995 0.145 0.035
0.075 0.061 0.020 0.150 0.123 0.020
2-hexanol.17–21) However, the selectivity for
R-(–)-
a
stereoisomers of ADH II is similar to almost all
short-chain ADHs known. ADHs reacting with sec-
ondary alcohol are obtained from methylotrophic
bacteria or yeast.22–24) Comamonas terrigena oxidize
Both ADHs were dialyzed against 20 m
M 2-[4-(2-Hydroxyethyl)-1-
piperazinyl] ethanesulfonic acid (HEPES) buŠer at pH 7.5 in the ab-
sence of EDTA.
b
Both ADHs were dialyzed against 20 m
presence of 5 m EDTA.
M HEPES buŠer at pH 7.5 in the
M
preferentially the
Rhodococcus erythropolis enzyme is speciˆc for the
-(
)-stereoisomer.26) Interestingly, there is no
R
-(–)-stereoisomer,25) whereas the
S
＋
DEAE-Toyopearl column chromatography. N-termi-
nal amino acid sequence analysis of the NAD-depen-
dent ADH I showed signiˆcant similarities with the
long-chain ADHs family (350 amino acid residues
per subunit), but the NAD-dependent ADH II
belongs to the short-chain ADHs family (250 amino
acid residues per subunit).3) The general properties of
these enzymes are similar to those of other NAD-
dependent ADHs described. Metal analysis of NAD-
report on NAD-dependent ADHs with an extremely
high speciˆc activity towards longer-chain secondary
alcohols (C6-C8).
Km and Vmax values of ADH I and also of ADH II
for ethanol and acetaldehyde (Table 2), clearly show
both enzymes favor the reduction of acetaldehyde to
ethanol, but not the oxidation of ethanol to acetalde-
hyde. However, since both ADH I and ADH II are
induced 12 and 20 times, respectively, when the
mutant strain was grown in the medium containing
ethanol, where the ethanol concentration was
＋
dependent ADHs showed that only Zn2 was a sig-
niˆcant component, present as one atom per subunit
of ADH I but not in ADH II. Consequently, native
reduced from 4
z to 2z without producing acetic
＋
ADH I contained 3 Zn2 atoms per molecule. The
acid (unpublished data), they seem to work for
ethanol assimilation. As for the ethanol oxidation,
ADH I is more favorable than ADH II because ADH
I has a lower Km value and higher Vmax for ethanol
than ADH II. However, since the Km value of ADH
＋
Zn2 seems to be tightly bound in the active site of
the enzyme, since EDTA treatment did not remove
the metal from the enzyme. This situation of ADH I
is similar to other long-chain ADH family such as
ADH of propane-grown Pseudomonas ‰uorescens
NRRL B-1244,9) and NAD-dependent ADH of ther-
II for acetaldehyde is very high, 24 m , compared
M
with that of ADH I, the reverse reaction is relatively
unlikely to occur at low levels of acetaldehyde. Thus,
ADH II may work for ethanol oxidation to acetalde-
hyde even when acetaldehyde is accumulated at some
level. This notion may be supported by the ˆnding
that Km values for NAD and NADH of ADH II,
especially for NAD, are extremely low compared
with those of ADH I. Thus, ADH II seems to be
mainly involved in ethanol assimilation under the
conditions that the NAD level is low and acetalde-
hyde is relatively high, which may occur at the second
stage when A. pasteurianus CN6-2 is grown on
ethanol medium. In contrast, ADH I seem to work
for ethanol assimilation at the initial stage of the cell
growth in the presence of ethanol, where ethanol
concentration of the cell is high enough but that of
acetaldehyde is marginal.
＋
moacidophilic archaebacterium,10) where Zn2 in the
enzyme is stable against EDTA treatment. Although
ADH II seems not to contain any signiˆcant level of
＋
Zn2 or Mg2＋, the enzyme activity is decreased by
＋
EDTA treatment but recovered by Mg2 treatment.
In addition, ADH II activity could be stabilized by
the addition of 1 m MgCl2 in the column buŠer dur-
M
＋
ing the puriˆcation. Thus, Mg2 seems to work for
maintaining the enzyme stable and active. Physical
characterization of ADH I showed that the native
enzyme is probably a trimer of identical subunits,
unlike other long-chain ADHs.3) Whereas, the native
enzyme of the ADH II is probably a dimer of
identical subunits, which is similar to dimeric ADHs
isolated from Pseudomonas sp.,9) Rhodococcus
11)
12)
rhodochrous
,
and Zymomonas mobilis
.
ADH I
shows a preference for short-chain primary alcohols,
and also has weaker but signiˆcant activity on longer-
chain primary alcohols, and some activity on
secondary alcohols only for short-chain secondary
alcohols. This is similar to almost all of the known
long-chain ADH family from yeast and other
microorganisms.13–16) In contrast, ADH II is rather
unique in showing a preference for long-chain sec-
Acknowledgments
The authors wish to express sincere thanks to Dr.
R. Murakami, Faculty of Science, Yamaguchi
University for her kind support on the measurement
of metal content by ICP-AES. This work was sup-
ported by the Thailand Research Fund (TRF) under

964
P. CHINNAWIROTPISAN et al.
12) Wills, C., Kraoˆl, P., Londo, D., and Martin, T.,
Characterization of the two alcohol dehydrogenases
The Royal Golden Jubilee Ph.D. Program and
Association of International Education, Japan
(AIEJ). A part of this work was done by collabora-
of Zymomonas mobilis
210, 775–785 (1981).
13) Dickinson, F. M., and Dalziel, K., The speciˆcities
and conˆgurations of ternary complexs of yeast and
liver alcohol dehydrogenases. Biochem. J., 104,
165–172 (1967).
14) Dalziel, K., and Dickinson, F. M., The kinetics and
mechanism of liver alcohol dehydrogenase with pri-
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. Arch. Biochem. Biophys.,
tion in
a
Core University Program between
Yamaguchi University and Kasetsart University sup-
ported by the Society for the Promotion of Science
(JSPS) and the National Research Council of
Thailand (NRCT).
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