Molecular properties of membrane-bound FAD-containing D-sorbitol dehydrogenase from thermotolerant gluconobacter frateurii isolated from Thailand

Article abstract of DOI:10.1271/bbb.69.1120

There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQ-SLDH, having pyrroloquinoline quinone (PQQ), and FAD-SLDH, containing FAD and heme c as the prosthetic groups. FAD-SLDH was purified and characterized from the PQQ-SLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQ-SLDH. The oxidation product of FAD-SLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FAD-SLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FAD-SLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.

Full text of DOI:10.1271/bbb.69.1120

Biosci. Biotechnol. Biochem., 69 (6), 1120–1129, 2005
Molecular Properties of Membrane-Bound FAD-Containing
D-Sorbitol Dehydrogenase from Thermotolerant
Gluconobacter frateurii Isolated from Thailand
1
;y
1
2
Hirohide TOYAMA, Wichai SOEMPHOL, Duangtip MOONMANGMEE,
1
1
Osao ADACHI, and Kazunobu MATSUSHITA
1
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
2
Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi,
Prachauthit Road., Tungkru, Bangkok 10140, Thailand
Received December 24, 2004; Accepted March 14, 2005
There are two types of membrane-bound D-sorbitol
dehydrogenase (SLDH) reported: PQQ–SLDH, having
pyrroloquinoline quinone (PQQ), and FAD–SLDH,
containing FAD and heme c as the prosthetic groups.
FAD–SLDH was purified and characterized from the
PQQ–SLDH mutant strain of a thermotolerant Gluco-
nobacter frateurii, having molecular mass of 61.5 kDa,
ism leads to applications in industry for fermentation of
valuable products such as L-sorbose, dihydroxyacetone,
D-gluconate, and keto-D-gluconates.
3
,4)
L-Sorbose is an important intermediate in the indus-
trial production of vitamin C. The membrane-bound
D-sorbitol dehydrogenase (SLDH) has been considered
5
)
to play a main role in L-sorbose fermentation. In
Gluconobacter strains, two types of membrane-bound
D-sorbitol dehydrogenases have been purified and well
characterized. One was purified from G. suboxydans var
5
2 kDa, and 22 kDa. The enzyme properties were quite
similar to those of the enzyme from mesophilic G. oxy-
dans IFO 3254. This enzyme was shown to be inducible
by D-sorbitol, but not PQQ–SLDH. The oxidation
product of FAD–SLDH from D-sorbitol was identified
as L-sorbose. The cloned gene of FAD–SLDH had three
open reading frames (sldSLC) corresponding to the
small, the large, and cytochrome c subunits of FAD–
SLDH respectively. The deduced amino acid sequences
showed high identity to those from G. oxydans IFO
6
)
ꢀ IFO 3254 as flavohemoprotein, containing a cova-
lently bound FAD having three subunits with molecular
mass of 63 kDa (flavoprotein), 51 kDa (cytochrome c),
and 17 kDa (unknown), designated FAD–SLDH. In
addition, another membrane-bound SLDH (PQQ–
SLDH) was recently purified from G. suboxydans var
5
)
ꢀ IFO 3255, having one subunit of 80 kDa and
pyrroloquinoline quinone (PQQ) as the prosthetic group.
Recently, its gene, sldA, composing an operon with sldB,
3254: SldL showed to other FAD-enzymes, and SldC
having three heme c binding motives to cytochrome c
subunits of other membrane-bound dehydrogenases.
7
,8)
was cloned,
identical to glycerol dehydrogenase, found ubiquitous-
and the enzyme was shown to be
9
)
Key words: FAD; sorbitol dehydrogenase; acetic acid
bacteria; Gluconobacter; cytochrome c
ly in Gluconobacter strains, and also to arabitol
0)
1
dehydrogenase and D-gluconate dehydrogenase, yield-
1
1)
ing 5-keto D-gluconate
using molecular biological
Gluconobacter strains are strict aerobes belonging to
the group of acetic acid bacteria able to oxidize many
compounds incompletely to accumulate the oxidized
products in the culture medium by using membrane-
bound dehydrogenases localized on the outer surface of
techniques. The reaction product of this enzyme from D-
sorbitol was determined to be L-sorbose, but that of
FAD–SLDH has not been determined. In Gluconobacter
strains, two membrane-bound D-gluconate dehydrogen-
ases have been found. One is a flavocytochrome
1
,2)
12)
the cytoplasmic membrane.
Such ‘incomplete’ oxi-
producing 2-keto-D-gluconate similar to FAD–SLDH,
dation is called oxidative fermentation and is carried out
by membrane-bound enzymes, like alcohol dehydrogen-
ase (ADH) and aldehyde dehydrogenase (ALDH) in
vinegar fermentation. This character of the microorgan-
and the other is a quinoprotein identical to PQQ–SLDH
that produces 5-keto-D-gluconate, as described above.
Analogically, it is possible that FAD–SLDH might
produce not L-sorbose but D-fructose, differently from
y
To whom correspondence should be addressed. Tel/Fax: +81-83-933-5859: E-mail: hirot@yamaguchi-u.ac.jp
Abbreviations: SLDH, D-solbitol dehydrogenase; PQQ, pyrroloquinoline quinone; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase;
FDH, D-fructose dehydrogenase; AcB, sodium acetate buffer; tat, twin-arginin translocation; 2KGDH, 2-keto-D-gluconate dehydrogenase; GADH, D-
gluconate dehydrogenase

FAD-Sorbitol Dehydrogenase from Thermotolerant Gluconobacter
1121
PQQ–SLDH.4) Since nowadays D-fructose is produced
resuspended with 10 mM sodium acetate buffer (AcB,
pH 5.0) at a concentration of 3 ml/g-wet cells, and
passed twice through a French pressure cell press
(American Instruments Co., Silver Spring, MD.,
U.S.A.) at 16,000 psi. After centrifugation at 6,000 rpm
for 10 min to remove the intact cells, the supernatants
were ultracentrifuged at 40,000 rpm for 60 min. The
resultant precipitate was resuspended with McIlvaine
buffer (McB, pH 5.0) and used as a membrane fraction.
EDTA treatment of the membrane fraction was done as
1
3)
industrially from D-glucose by glucose isomerase as a
reversible reaction, the theoretical yield of D-fructose
never exceeds 50% of the starting material without
separation of the product. Hence it is advantageous if the
enzyme which oxidizes D-sorbitol irreversibly to pro-
duce D-fructose is found and applied in oxidative
4
)
fermentation.
In this study, screening of D-fructose-producing
microorganisms in Gluconobacter IFO strains and
thermotolerant strains from Thailand was performed
with both growing and resting cells. Thermotolerant
strains were included because of their potential advant-
1
0)
described previously, and the treated membrane was
resuspended with McB, pH 5.0.
1
4)
age in industry. Furthermore, EDTA treatment was
performed with the membrane fraction to distinguish
Qualitative and quantitative analyses of ketohexose.
D-Fructose concentration was measured by FDH as
described above. Qualitative analysis of ketohexose was
performed using thin layer chromatography (TLC)
analysis. Samples were spotted on a silica gel plate 60
(Merck) and developed with a solvent reagent contain-
ing ethyl acetate:acetic acid:methanol:deionized water
(6:1.5:1.5:1). After the silica gel plate was dried, phenol
sulfuric reagent was spread to visualize color develop-
ment. Identification of the reaction product was also
done by HPLC equipped with Aminex HPX-87P (7:8 ꢁ
300 mm, Bio-Rad). The mobile phase was deionized
1
0)
FAD–SLDH from PQQ–SLDH. Among these strains,
the isolated strain THD32 classified as Gluconobacter
frateurii appeared to have high activity of FAD–SLDH,
and hence this enzyme was purified from the PQQ–
SLDH disrupted mutant of the THD32 strain. Then
molecular cloning of the gene of FAD–SLDH was done
using primers designed from N-terminal amino acid
sequences of the subunits.
Materials and Methods
ꢀ
water and the column temperature was 66 C. Elution
Materials. All other chemicals were obtained from
commercial sources. Yeast extract was a kind gift from
the Oriental Yeast Co., Ltd.
was monitored by absorption at 210 nm. When analyzed
at a flow rate at 0.6 ml/min, L-sorbose and D-fructose
were detected at 14.9 and 17.9 min respectively. For
quantitative measurement of the total amount of keto-
1
0)
Bacterial strains and growth conditions. Glucono-
bacter IFO strains and thermotolerant strains (some are
in Ref. 14) newly isolated from Thailand were used in
this experiment. These strains were maintained on
hexose, resorcinol was used, as described previously.
Enzyme assay. Dehydrogenase activities with D-
sorbitol, glycerol, D-mannitol, D-arabitol, ethanol, and
acetaldehyde were measured by the ability to reduce
potato-CaCO agar slant, which was prepared by adding
3
6
)
2
% agar and 0.5% CaCO3 to a potato medium consisting
potassium ferricyanide, as described previously. One
unit of enzyme activity was defined as the amount of
enzyme which catalyzed 1 mmol of substrate oxidation
per min under the above conditions, which was
equivalent to 4.0 absorbance at 660 nm.
of 5 g of D-glucose, 10 g of yeast extract, 10 g of
polypeptone, 20 g of glycerol, and 100 ml of potato
extract, filled to 1-liter with tap water. Preculture was
done in 5 ml of potato medium with shaking for 24 h at
ꢀ
3
0 C, and transferred to 100 ml of the appropriate
medium in a 500 ml Erlenmeyer flask, and then cultured
for another 24 h. Bacterial growth was monitored with a
Klett-Summerson photoelectric colorimeter with a red
filter.
Determination of protein concentration. Protein con-
centration was measured by a modified version of the
Lowry method. Bovine serum albumin was used as
the standard protein.
1
6)
Screening of D-fructose producing strains in Gluco-
nobacter. Screening of D-fructose producing strains was
performed using growing cells, resting cells, and
membrane fractions of Gluconobacter strains. Produc-
tion of D-fructose was determined by the enzymatic
method with D-fructose dehydrogenase (FDH), which
was a kind gift from Toyobo, after removal of cells or
membrane by centrifugation.
Purification of FAD–SLDH from G. frateurii THD 32.
ꢀ
All steps of purification were carried out at 4 C. The
membrane fraction was suspended in 10 mM AcB
(pH 5.0), and was adjusted to 10 mg of protein/ml. To
solubilize the membrane proteins, Triton X-100, KCl,
and D-sorbitol were added at final concentrations of 1%,
0.1 M, and 0.1 M, respectively, and gently stirred in an ice
bath for 90 min. Solubilized enzyme obtained by cen-
trifugation at 40,000 rpm for 90 min was dialyzed against
10 mM AcB (pH 5.0) containing 0.1% Triton X-100,
1
5)
Preparation of membrane fraction. Cells were har-
vested by centrifugation at 9,000 rpm for 10 min and
washed once with distilled water. The washed cells were
25 mM D-sorbitol, and 5 mM MgCl (buffer A) for 6 h, by
2
exchanging the buffer twice. Precipitate was removed by

1122
H. TOYAMA et al.
ꢀ
centrifugation at 9,000 rpm for 20 min, and the super-
natant was put onto a CM-cellulose column equilibrated
with buffer A. The enzyme activity was passed though
the column, whereas large portions of alcohol dehydro-
genase and aldehyde dehydrogenase were adsorbed on
this column. The active fractions were combined and put
into a dialysis tube, and dialyzed against 10 mM AcB
with liquid nitrogen and stored at ꢂ80 C. The com-
petent cells were thawed on ice just before use and
mixed with a DNA solution, and then transferred into a
cold gene pulser cuvette. Introduction of DNAs into the
competent cells were achieved by applying 1.8 kV
electric pulse to the cuvette with a Gene Pulser (Bio-
Rad). After pulsing, 1 ml of ice-cold potato medium was
immediately added to the cuvette. Then the suspension
was transferred into a test tube and incubated for 6 h at
(
pH 4.5) containing 0.1% Triton X-100, 25 mM D-
sorbitol, and 5 mM MgCl (buffer B) for 6 h, and this
2
ꢀ
was repeated by changing the dialysis buffer. The
dialysate was put onto a CM-cellulose column equili-
brated with buffer B. The column was washed with
buffer B and major impurities were removed, and then
elution of the enzyme was done with a linear gradient of
KCl (0–0.1 M). Enzyme activity was eluted at about
30 C with shaking. After incubation, 100 ml of the cell
suspension was spread on an agar plate medium
containing 1% glycerol, 1% sorbitol, 0.1% yeast extract,
0.1% polypeptone, and kanamycin (50 mg/ml), and
ꢀ
incubated at 30 C.
3
0 mM KCl. The active fractions were put into a dialysis
DNA techniques. Restriction enzyme digestion, DNA
ligation, and other DNA modifications were performed
according to the vendor’s recommendations. Preparation
of plasmid DNA from E. coli strains and other general
molecular biology techniques were done as described by
tube and concentrated by embedding it in sucrose pow-
der, and then dialyzed against 5 mM MES–NaOH buffer
(
pH 6.0) containing 0.1% Triton X-100, 25 mM D-
sorbitol, and 5 mM MgCl2 (Buffer C) for 8 h, by ex-
changing the buffer twice. The dialysate was put onto a
DEAE-cellulose column equilibrated with buffer C. Al-
dehyde dehydrogenase activity was removed by washing
with the same buffer while the FAD–SLDH was eluted
at about 30 mM KCl in the linear gradient (0–0.1 M).
1
9)
Sambrook et al. Genomic DNA of Gluconobacter
strains was isolated from cells grown to a mid-
exponential phase in D-sorbitol medium by a method
2
0)
of Marmur with some modifications. PCR reaction
was performed using the Ready.To.Go/PCR Bead Kit
(
Amersham Biosciences). Disruption of PQQ–SLDH
1
1)
SDS–Polyacrylamide gel electrophoresis (SDS–
PAGE). SDS–PAGE was done on 12.5% (w/v) acryl-
amide slab gel by the method described by Laemmli.
was confirmed by PCR, as described previously. To
obtain the gene fragment of FAD–SLDH, the primers
SLDH1 (TCG(C)GCCGAT(C)GTCGTCATC(G)GT)
and SLDH2 (TGG(A)CAG(C)GCT(C)TCGCAA(G)TC-
G(A)CC) were used. PCR was performed with 25 cycles
under the following conditions: denaturation for 2 min at
1
7)
The following calibration proteins with the indicated
molecular masses were used as references for measure-
ment of molecular mass: phosphorylase b (94 kDa),
bovine serum albumin (68 kDa), ovalbumin (43 kDa),
carbonic anhydrase (31 kDa), and lysozyme (14.4 kDa).
Proteins were stained with 0.1% Coomassie Brilliant
Blue R-250. Cytochrome c was stained by heme-
ꢀ
ꢀ
94 C, annealing for 2 min at 55 C, and extension for
ꢀ
1 min at 72 C. DNA fragments obtained by PCR were
isolated by agarose gel eletrophoresis and purified with
QIA Quick Gel Extraction Kit (Qiagen), and then cloned
into pGEM-T Easyꢀ vector (Promega).
1
8)
catalyzed peroxidase activity.
Determination of N-terminal amino acid sequences of
the purified FAD–SLDH. After SDS–PAGE, the proteins
in the gel were transferred electrophoretically onto a
polyvinylidene difluoride membrane at 100 mA for 4 h.
The proteins were stained with Coomassie Brilliant Blue
G-250, destained with 50% methanol, and dried, and
then the stained bands were cut off. The N-terminal
amino acid sequence was analyzed with a protein
sequencer PPSQ-21 (Shimadzu).
Southern hybridization. Chromosomal DNA was
digested with suitable restriction enzymes and electro-
phoresed in agarose gel, and then transferred to a
þ
Hybond N membrane (Amersham Biosciences) by
capillary blotting. The DNA bands were then fixed to the
membrane by exposure to UV light for 5 min. Hybrid-
ization and detection was carried out with the ECL
Direct Nucleotide Labeling System (Amersham Bio-
sciences) according to the protocol provided by the
supplier.
Electroporation method. Competent cells of Gluco-
nobacter strains were obtained by the following method:
A preculture (1 ml) was transferred into 100 ml of 1%
D-sorbitol medium in a 500 ml Erlenmeyer flask with
Colony hybridization. Colonies of the gene library
constructed from the chromosomal DNA of G. frateurii
THD 32 in pUC119 were grown on an LB agar plate
containing 50 mg/ml of ampicillin, and transfered to a
ꢀ
shaking at 30 C for 12 h. Cells were chilled on ice for
0 min before harvesting by centrifugation at 4,000 rpm
þ
3
for 10 min at 4 C, and washed twice with ice-cold 10%
Hybond-N membrane and lysed. Hybridization and
ꢀ
detection were performed using the DIG System (Roche
Diagnostics) according to the protocol provided by the
supplier.
(w/v) glycerol. The final cell pellet was resuspended
with 0.5–1 ml of ice-cold 10% (w/v) glycerol. The cell
suspension (40 ml each) was frozen in an Eppendorf tube

FAD-Sorbitol Dehydrogenase from Thermotolerant Gluconobacter
1123
Nucleotide sequence analysis. Plasmids for sequenc-
ing were prepared with a QIA Prep Spin Miniprep Kit
the large subunit of PQQ–SLDH from G. suboxydans
1
1)
IFO 3255 disrupted by the kanamycin-resistant gene.
Disruption and absence of the wild-type sldA gene was
confirmed by PCR (data not shown).
(Qiagen). Sequencing was performed using ABI Prism
10 (Applied Biosystems). Sequence data were analyzed
3
using Genetyx-Mac (Software Development) and Clone
Manager (Scientific and Educational Software). Homol-
ogy search analysis and alignment were performed with
BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The
sequence described in this study has been deposited
with DNA Data Bank of Japan (DDBJ) under accession
no. AB192961.
The mutant strain grew at almost the same rate as the
wild-type strain; the growth rate was slightly slower on
the medium with D-sorbitol plus D-gluconate or glycerol,
but better on the medium with D-sorbitol alone, when
each carbon source was added at a concentration of 1%
(w/v). Among the three media used, growth yield of
both the wild-type and the mutant strain was best on the
medium with D-sorbitol plus D-gluconate (data not
shown). SLDH activity (the sum of PQQ–SLDH and
FAD–SLDH activities) was highest in both wild-type
and mutant cells grown on the medium with D-sorbitol
alone, while enzyme activities with other substrates did
not significantly change among four media tested
(Fig. 1), indicating that D-sorbitol is an inducer for
FAD–SLDH. Activity with D-arabitol disappeared in the
sldA mutant and was unchanged in the wild-type strain
grown on different carbon sources (Fig. 1), indicating
that PQQ–SLDH was constitutively expressed. When
the concentration of D-sorbitol in the growth medium
Results
Isolation of thermotolerant strains which produce D-
fructose and EDTA-tolerant SLDH
Screening of D-fructose-producing strains was per-
formed in Gluconobacter species including IFO strains
and isolated thermotolerant strains from Thailand. All
the strains examined (more than 100 strains), accumu-
lated only small amounts of D-fructose in the medium,
less than 5% of the D-sorbitol used, that is, <0:5 g/l, in
both growing- and resting-cell conditions. The remain-
ing part of D-sorbitol appeared to be converted to L-
sorbose in all strains tested, because large amounts of
ketohexoses were detected by resorcinol test (data not
shown). Perhaps the oxidation rate from D-sorbitol to D-
fructose was much slower than that to L-sorbose by
PQQ–SLDH. Hence, several strains producing relatively
high amounts of D-fructose were selected to examine
FAD– or PQQ–SLDH activity at the membrane level.
With the selected strains, EDTA treatment of the
membrane fraction was performed to eliminate PQQ–
SLDH activity. Most of the strains showed SLDH
activity sensitive to EDTA treatment, while some strains
such as THD32 were resistant, like G. suboxydans var ꢀ
IFO 3254, which previously was used to purify FAD–
SLDH (data not shown). Even with the membrane
fraction of strain THD32 treated with EDTA, D-fructose
was not produced by D-sorbitol oxidation; instead, a
substantial amount of L-sorbose was detected (data not
shown). For further analysis of this ‘EDTA-resistant’
enzyme, which is assumed to be FAD–SLDH, purifica-
tion of this enzyme was addressed from strain THD32.
ꢀ
This strain grew at 37 C at which mesophilic strains
1
4)
cannot grow. According to the 16S rDNA analysis,
this strain is classified as G. frateurii (S. Moonmang-
mee, personal communication).
Fig. 1. Enzyme Activities in the Membrane Fractions of the Wild-
Type and PQQ–SLDH Mutant Strains of G. frateurii THD32 Grown
on Different Media.
Purification of FAD–SLDH from G. frateurii THD32
From the membrane fraction of strain THD32,
purification of SLDH was attempted. Partially purified
SLDH, having three main bands of 61.5, 52, and 22 kDa
on SDS–PAGE, was obtained, but its purity was not
improved by further column chromatographies (data not
shown). Hence we decided to purify the enzyme from
the PQQ–SLDH mutant constructed using a plasmid
pSUP202-sldA::Km, a suicide plasmid with the gene of
Wild-type (A) and the PQQ–SLDH-disrupted mutant (B) of
G. frateurii THD32 were grown to late exponential phase on each
medium containing 0.3% polypeptone, 0.3% yeast extract and
different concentration of sugar alcohol [1.0% each of D-sorbitol and
D-gluconate (Sor-Gln), 1.0% each of D-sorbitol and glycerol (Sor-
Gly), 1.0% glycerol (Gly), or 1.0–7.0% D-sorbitol (1%, 3%, 5%, and
7
%)]. Dehydrogenase activities were measured with ferricyanide as
the electron acceptor, as described in ‘‘Materials and Methods’’, with
glycerol (gray), D-mannitol (white), D-sorbitol (hatched), and D-
arabitol (black) as substrate.

1124
H. TOYAMA et al.
Table 1. Purification Summary of FAD–SLDH from the PQQ–SLDH Mutant of G. frateurii THD32
Total protein
mg)
Protein
(mg/ml)
Total activity
(U)
Specific activity
(U/mg)
Recovery
(%)
Purification
Fold
Step
(
Membrane
1512
291
80
10.43
1.73
0.42
0.15
0.06
1228
1278
851
0.81
4.40
10.7
32.8
109
100
104
69
1
5
Solubilization
CM-cellulose (1)
CM-cellulose (2)
DEAE-cellulose
13
40
134
21
3
679
326
55
27
Catalytic properties of the purified enzyme
The substrate specificity of the purified enzyme was
examined. Among the substrates tested, D-sorbitol was
the best and D-mannitol was oxidized at 5% of the
reaction rate of D-sorbitol. The other polyols, such as
glycerol, ribitol, D- and L-arabitol, dulcitol, and myo-
inositol, were inert. The Km and Vmax values for D-
sorbitol were calculated to be 20.4 mM and 93.5 U/mg
respectively when ferricyanide was used as an electron
acceptor. The optimum pH and temperature for the
reaction of the purified enzyme was about pH 4.5 and at
ꢀ
Fig. 2. SDS–PAGE Analysis of the Purified FAD–SLDH from the
PQQ–SLDH Mutant of G. frateurii THD32.
2
5 C. These characters are quite similar to the enzyme
6)
from G. suboxydans var ꢀ IFO 3254. The enzyme
appeared to be less active and stable at higher temper-
atures even though it was obtained from a thermotoler-
ant strain (data not shown). We prepared the PQQ–
SLDH mutant of G. suboxydans var ꢀ IFO 3254 using
pSUP202-sldA::Km. Its thermostability as to SLDH
activity in the membrane fraction was found to be
similar to that of the mutant of the THD32 strain (data
not shown), indicating no significant difference between
enzymes from the two strains.
Ten mg of purified FAD–SLDH was applied and stained with
Coomassie Brilliant Blue R-250.
was increased (1–7%), the activity with D-sorbitol
increased, although no significant difference in growth
was observed, whereas the other enzyme activities did
not change very much (Fig. 1). Thus it is clear that
FAD–SLDH is induced with D-sorbitol in the medium.
The membrane fractions were prepared from mutant
cells grown on 1% D-sorbitol alone, and purification was
carried out as described in ‘‘Materials and Methods’’. A
summary of purification is shown in Table 1. The
enzyme was purified 134-fold with 27% recovery, and
the purified enzyme exhibited a specific activity of
Identification of the oxidation product from D-sorbitol
by purified FAD–SLDH
The oxidation of D-sorbitol with purified FAD–SLDH
was carried out by coupling with purified ubiquinol
2
2)
1
09 U/mg of protein. The purified enzyme showed a
oxidase of Acetobacter
ꢀ
in the presence of quinol
(Q H ) for 8 h at 25 C with gentle shaking. Only a
typical spectrum with absorption maxima at 415, 521,
and 551 nm, corresponding to a reduced cytochrome c,
which is consistent with the fact that the enzyme was
purified in the presence of D-sorbitol. Three protein
bands with molecular masses of 61.5 kDa, 52 kDa, and
2
2
small amounts of D-fructose (less than 0.1% of the initial
amount of D-sorbitol) was detected by FDH assay, while
a resorcinol test showed a high amount of ketohexose
(about 7% of the initial D-sorbitol). The reacted product
had similar mobility to L-sorbose on thin-layer chroma-
tography, after it was developed with L-sorbose and D-
fructose as standards. The product was also analyzed by
HPLC with an Aminex HPX-87P column. The peak of
the reaction product appeared at the same retention time
as that of L-sorbose, but not that of D-fructose. These
results clearly indicate that the oxidation product of D-
sorbitol by the purified FAD–SLDH from G. frateurii
THD32 was not D-fructose but L-sorbose.
Furthermore, the membrane fraction from the PQQ–
SLDH disrupted strain was used to confirm the oxidation
product from D-sorbitol. The reaction product of D-
sorbitol with the membrane fraction was compared to
the standard of L-sorbose and D-fructose by TLC. Only
one oxidation product, corresponding to L-sorbose, was
2
2 kDa were found on SDS–PAGE (Fig. 2). Under UV
light, the large and middle protein bands showed
fluorescence on SDS–PAGE gel before staining, and
the former protein band showed much more intense
fluorescence after the gel was treated with performic
acid, suggesting that the large subunit contains cova-
lently bound flavin and that the covalent linkage might
2
1)
be on a cystein residue. The 52 kDa protein band
was found to be stained by heme staining after SDS–
PAGE (data not shown), indicating that it is a cyto-
chrome c. The N-terminal amino acid sequences were
determined as follows: large subunit, SSNSFSADV-
VIVGSGV; cytochrome c subunit, EDQATTIXRXA-
YXA (X: unidentified); small subunit, EETKPPLASR-
DEYERFFEV.

FAD-Sorbitol Dehydrogenase from Thermotolerant Gluconobacter
1125
Fig. 3. Schematic Representation of the Gene Fragment Obtained in This Study.
The gray box represents the gene fragment obtained by PCR with SLDH1 and SLDH2 as primers. Orf1 and Orf2 are not complete.
detected (data not shown), indicating that FAD–SLDH is
also involved in D-sorbitol oxidation to produce L-
sorbose in the cytoplasmic membrane. It was also
confirmed that little activity if any to produce D-fructose
is present in the membrane fraction.
It has twin-arginine sequence and thus might be used in
2
5)
the twin-arginine translocation (tat) system. On the
other hand, the N-terminal amino acid sequence of the
large subunit was found without any signal sequence,
suggesting that the large subunit might be translocated
together with the small subunit by the tat system. SldS
encodes 197 amino acids and the calculated molecular
weight of the mature protein is 22,306 Da.
Cloning of the gene encoding membrane-bound
FAD–SLDH from G. frateurii THD32
Two oligonucleotide primers were synthesized: a
forward primer, named SLDH1, was designed from the
N-terminal amino acid sequence of subunit I, while a
reverse primer, named SLDH2, was designed according
to the result of alignment of amino acid sequences of
cytochrome c subunits of several membrane-bound
dehydrogenases (see Fig. 5). PCR amplification was
performed under the conditions described in ‘‘Materials
and Methods’’. A band of expected size, about 1.7 kb,
was obtained and confirmed to be the part of the gene of
SLDH by nucleotide sequencing. The PCR product was
used as a probe for Southern hybridization, and 3.1 kb
and 2.9 kb of the HindIII fragments were assumed to
contain the whole part of the gene cluster (Fig. 3). These
fragments were isolated from the library by colony
hybridization. The two plasmids obtained were se-
quenced, and finally, a 6,183 bp-nuleotide sequence
containing the complete structural genes of FAD–SLDH
was confirmed.
The coding region of the large subunit was started at
position 1,917 with the ATG codon. The gene sldL
encoded a polypeptide of 545 amino acid residues with a
calculated molecular mass of 60,075 Da. The deduced
amino acid sequence of subunit I was found to have the
sequence GSGVAG at a position between the 15th and
the 20th residues, corresponding to the binding motif of
FAD (GXGXXG).
The ORF corresponding to the cytochrome c subunit,
sldC, started at position 3,547. A possible SD sequence,
AGGAGA, was found at 6 nt upstream of the start
codon. The gene encoded a 478 amino acid protein with
a molecular mass of 51,057 Da. The N-terminal amino
acid sequence of the cytochrome c subunit was found
inside this ORF, and the 31 amino acid residues were
confirmed to be a typical sec-dependent signal se-
2
6)
quence.
The deduced amino acid sequence was
revealed to have three CXXCH sequence motives
serving as heme c binding sites.
The nucleotide and amino acid sequences of FAD–
SLDH and the flanking regions are shown in Fig. 4.
Three open reading frames (ORFs) corresponding to the
small, large and cytochrome c subunits were found and
named sldSLC. They might be in the same transcrip-
tional unit. A rho-dependent terminator-like sequence
was found at position 4,997–5,034. The upstream of this
operon was found to be an AraC-like transcriptional
regulator2 which is possibly involved in transcriptional
control of the sldSLC gene. The downstream sequences
showed identity to transposase, which is responsible for
A protein similarity search on data base by the
BLAST program revealed that the large subunit is
almost identical to the large subunit of FAD–SLDH
from G. oxydans IFO 3254 (98% identity), and also
similar to many FAD-dependent enzymes of the so-
called glucose-methanol-choline oxidoreductase family,
including glucose dehydrogenase from Burkholderia
cepacia (49%), and 2-keto-D-gluconate dehydrogenase
(2KGDH) from Pantoea agglomerans (formally Erwinia
herbicola) (44%). The deduced amino acid sequences of
the small subunit showed identity to the small subunits
of FAD–SLDH from G. oxydans IFO 3254 (79%), and
the small subunit of P. agglomerans 2KGDH (18%).
The predicted amino acid sequence of sldC showed
considerable identity to the cytochrome c subunits of
SLDH of G. oxydans IFO 3254 (98%) 2KGDH of
P. agglomerans (42%), and GDH of B. cepacia (42%),
and to those of D-gluconate dehydrogenase (GADH) of
3)
2
4)
transposon movement.
The GTG (nt 1,305) might be the start codon of sldS,
and a possible Shine-Dalgarno sequence was found at
2 bp upstream of this start codon. The N-terminal
1
amino acid sequence of this small subunit determined by
the protein sequencer was found inside in this ORF, and
the signal sequence was confirmed to be 41 amino acids.

1126
H. TOYAMA et al.
Fig. 4. Nucleotide and Amino Acid Sequences of sldSLC.
The nucleotide and amino acid sequences of sldSLC are shown. A probable ribosome-binding sequence prior to each gene is shown in
boldface. The rho-dependent terminator-like sequence is indicated by arrows. The amino acid sequences from subunits of the purified FAD–
SLDH detected by an amino acid sequencer are underlined.
Fig. 5. Alignment of SldC with Cytochrome c Subunits from Several Membrane-Bound Dehydrogenases.
Putative signal sequences are italicized. In the bottom line, completely conserved amino acids among the sequences aligned are shown by
capital letters, and the conserved amino acids among them with one exception are shown by small letters. From top to bottom, accession numbers
to DDBJ are: AB192961 for SldC THD32 from G. frateurii THD32; AB039821-3 for SldC 3254 from G. oxydans IFO3254; AF068066-3 for
2KGDH Pant from P. agglomerans; AF493970-1 for GDH Burkh from B. cepacia; AE016786-183 for PP3382 from P. putida KT2440;
U97665-3 for GADH Ecyp from P. cypripedii; AB086012-2 for ADH Apast from A. pasteurianus; M58760-1 for ADH Goxy from
G. suboxydans, and Y08696-2 for AldF Geuro from G. europaeus.

FAD-Sorbitol Dehydrogenase from Thermotolerant Gluconobacter
1127
E. cypripedii (36%), PP3382 of Pseudomonas putida
KT2440 (35%), ADH of A. pasteurianus (34%), ADH
of G. oxydans (34%), and ALDH of A. pasteurianus
is incorporated in the cytoplasm.29) The mechanism of
covalent attachment of FAD is not certain; it is probably
a spontaneous reaction after incorporation into the apo-
2
1)
(31%) (Fig. 5).
protein. SldS showed similarity to the small subunits
of GADH and 2KGDH, of which the function is also
unknown. Kondo et al. have suggested that the 20 kDa
small subunit of ADH from Acetobacter pasteurianus
has a role in the stability of the dehydrogenase subunit
and functions as a molecular coupler to the cytochrome
Discussion
We failed to find a membrane-bound enzyme oxidiz-
ing D-sorbitol to D-fructose. Our first assumption was
that FAD–SLDH might produce D-fructose from D-
3
0)
c subunit on the cytoplasmic membrane, but the small
subunit has no similarity to SldS in amino acid
sequence. Although the small subunit of ALDH from
4)
sorbitol, but it came out wrong and it is clear that this
enzyme produces L-sorbose from D-sorbitol. Since both
PQQ–SLDH and FAD–SLDH produce the same oxi-
dized product from D-sorbitol and pass electrons to
ubiquinone in the cytoplasmic membrane, it appears that
they have no functional differences physiologically.
Furthermore, it is obvious that all Gluconobacter strains
have PQQ–SLDH, which is responsible for producing
dihydroxyacetone from glycerol, and this character is
used for classical identification of Gluconobacter
strains, whereas FAD–SLDH is found only in certain
Gluconobacter strains like THD32. This raises the
question of the significance and rare existence of
FAD–SLDH. We found a transposase-like gene next to
the sldSLC gene in G. frateurii THD32. Perhaps the
gene cluster encoding FAD–SLDH and the regulator
gene might be transferred from one microorganism to
others in a certain environment, leading to the appear-
ance of this enzyme only in some Gluconobacter strains.
It is clear that FAD–SLDH is inducible and has higher
specific activity, while expression of PQQ–SLDH
appears constitutive, as shown in Fig. 2. No significant
difference was found in growth on D-sorbitol between
the wild-type and the PQQ–SLDH mutant strains. Hence
it would be interesting further to analyze the advantage
of having FAD–SLDH in addition to PQQ–SLDH.
There is an NAD-dependent enzyme that catalyzes
reversible oxidoreduction between D-sorbitol and D-
fructose. This enzyme exists in Gluconobacter strains
3
1)
acetic acid bacteria contains iron-sulfur clusters, the
amino acid sequence of SldS does not have such a
binding motif.
Interestingly, many of membrane-bound enzymes
found in Gluconobacter strains, including FDH, GADH,
and 2KGDH, are known to contain cytochrome c
2
)
subunits similar to those of ADH and ALDH. Thus,
the cytoplasmic membrane of Gluconobacter strains
contains high amounts of cytochrome c, and these
enzymes are also assumed to pass electrons to ubiq-
uinone. From bacteria other than acetic acid bacteria,
membrane-bound GADH and 2KGDH from E. cypri-
3
2)
33)
pedii and P. agglomerans or P. citrea respectively
have been reported to contain similar cytochrome c
subunits. These cytochrome c subunits show high
identity to each other in amino acid sequence (Fig. 5).
Moreover, many similar cytochrome c genes are found
in the genome sequences of several bacteria (data not
shown). According to the data of kinetic and structural
analyses of ADH, the cytochrome c subunit is believed
to contain membrane-binding and ubiquinone- and
3
4)
ubiquinol-reacting sites, although no segments with
successive hydrophobic amino acid residues are found in
the sequence. It is interesting to consider the evolution
of these cytochromes c, since cytochromes c found in
respiratory chains in many aerobic organisms, in
mitochondria for example, have high redox potential
and cannot easily reduce ubiquinone energetically.
2
7)
and has been purified from G. suoxydans IFO3257.
The small amount of D-fructose detected in the culture
supernatant of several strains might be provided by this
enzyme, although this ought to be confirmed. If this is
the case, NAD–SLDH might be applicable for D-
fructose fermentation. There is a report about another
type of membrane-bound SLDH having three subunits
similar to ADH with PQQ as the prosthetic group. But
this enzyme appears not to exist in strain THD32,
judging from the substrate specificities of the membrane
fractions in wild-type and the PQQ–SLDH mutant
strains in Fig. 2.
The deduced amino acid sequence of the cytochrome
c subunit has a typical sec-dependent signal sequence.
On the other hand, the large subunit does not have such a
signal sequence but the small subunit has a tat-depend-
ent signal sequence. Therefore, the large subunit appears
to be translocated together with the small subunit by a
Acknowledgments
This work was carried out under the collaboration of
the Core University Program between Yamaguchi
University and Kasetsart University, supported by the
Scientific Cooperation Program of the Japan Society for
the Promotion of Science (JSPS) and the National
Research Council of Thailand (NRCT).
2
8)
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