JOURNAL OFBIOSCIENCE ANDBIOENGINEEIUNG

Vol. 94, No. 2, 124129.2002

Production of Aldehyde Oxidases by Microorganisms

and Their Enzymatic Properties
AKINORI YASUHARA,l MIHO AKIBA-GOTO,l IUNYA FUJISHIRO; HIROYUKI UCHIDA,
TAKAYIJKI UWAJIMA,3 AND KAZUO AISAKA’*
Tokyo Research Laboratories, Kyowa Hakko Kogvo Co. Ltd., 3-6-6 Asahimachi, Machida-shi, Tokyo 194-8533, Japan,’
Kyowa Medex Co. Ltd., 2-l-l Irtjune, Chuo-ku, Tokyo 104-0042, Japan2 andFact&v of Engineering,
Fukui University, 3-9-l Bunkyo, Fukui 910-8507, Japan’

Received 5 March ZOOZ/Accepted17 May 2002

In order to establish an efftcient process to decompose environmentally toxic ahlehydes, dioxy-

gen-dependent aldehyde oxidase (ALOD) from microorganisms was first sought, and some bac-
teria and actinomycetes were found to produce the enzyme in their cells. MethyZobucillus sp.,
Pseudomonas sp., and Strepomyces moderutus were selected as the representative ALOD-produc-
ing strains, and their enzymes were partially purified and characterized. The three ALODs could
oxidize a wide range of aldehydes including formaldehyde, aliphatic aldehydes, and aromatic al-
dehydes, though their preferences differ depending on their producing strains. The other enxy-
matic properties were also determined with regard to their producing strains. Methylobucilhs sp.
ALOD had the most acidic optimum pH for its activity and stability, and Pseudomonas sp. ALOD
had the highest stability against heat treatment. Three native ALODs had molecular weights
ranging from 140 to 148 kDa, and were composed of three subunits of different sixes: large (85 to
88 kDa), medium-sized (37 to 39 kDa), and small (18 to 23 kDa).

[Key words: aldehyde oxidase, formaldehyde, Methylobacillus sp., Pseudomonas sp., Streptomyces moderatus]

Aldehyde oxidase (aldehyde : oxygen oxidoreductase,
ALOD; EC 1.2.3.1) catalyzes the oxidation of various alde-
hydes to their corresponding carboxylic acids with the re-
duction of molecular oxygen to hydrogen peroxide accord-
ing to Eq. 1.
Aldehyde+O,+H,O + acid+H,O, (1)
ALOD is present in the organs of various animal species
(1). For example, bovine ALOD is expressed at high levels
in the liver and lung, and has been implicated in the de-
toxification of certain types of environmental pollutants and
xenobiotics (2). Furthermore, in animals, ALOD may also
play a role in biosynthetic processes such as retinoic acid
synthesis (3). In plants, ALOD seems to be involved in hor-
mone biosyntheses, such as those of indole-3-acetic acid (4)
and abscisic acid (5).
In microorganisms, however, ALODs using molecular
oxygen as an electron acceptor have not yet been reported.
However, aldehyde oxidoreductase using 2,6-dichlorophe-
nol-indophenol as an electron acceptor has been isolated
and characterized from a sulfate-reducing bacterium of the
genus Desulfovibrio (6, 7). Furthermore, aldehyde dehydro-
genases (EC 1.2.1.3, 1.2.1.4, and 1.2.1.5) that use NAD
and/or NADP as a coenzyme have already been reported in
some microorganisms. Enzymes in Escherichia coli (8),
* Corresponding author. e-mail: kaisaka@kyowa.co.jp
phone: +81-(0)42-725-2555 fax: +81-(0)42-726-8330
Abbreviation: ALOD, aldehyde oxidase.
Saccharomyces cerevisiae (9), and Aspergillus nidulans ( 10)
are involved in the catabolism of ethanol, and those in
Pseudomonas sp. (11) and Vibrio harveyi (12) are involved
in the metabolisms of aromatic aldehydes and long-chain
aliphatic aldehydes, respectively. The oxidation of vanillin
(4-hydroxy-3-methoxybenzaldehyde) to vanillic acid (4-hy-
droxy-3-methoxybenzoic acid) has also been reported in
Streptomyces viridosporus (13), although the enzymatic
properties have not been fully investigated.
In recent years, some aldehydes have gained increasing
attention as environmental pollutants. For example, formal-
dehyde and acetaldehyde are hazardous air pollutants that
are known to cause nasal cancer in test animals (14, 15).
Therefore, it is of great significance to develop biotechno-
logical methods for removing contaminant aldehydes from
environment. In such a case, an oxygen-dependent ALOD is
better than NAD(P)-dependent enzymes from an economi-
cal standpoint, because it does not require any expensive
cofactors. In the present study, we perform the screening of
a novel oxygen-dependent ALOD in microorganisms. Fur-
thermore, we purify some microbial ALODs, and compare
their properties among the producing strains.
MATERIALS AND METHODS
Screening The distribution of aldehyde oxidase (ALOD)
was examined using 46 strains of bacteria, 66 strains of actino-
mycetes, 67 strains of yeasts, and 173 strains of molds in our cul-
ture collection. The bacteria were inoculated into yeast bouillon
medium (pH7.0) consisting of 2% bouillon and 0.5% yeast ex-

124
VOL.94,2002
tract. The actinomycetes were inoculated into Bennett’s medium
(ATCC medium 174). The molds and yeasts were inoculated into
YM medium (ATCC medium 200). For meth~o~-~similating
bacteria and yeasts, 1% methanol was added to each medium. Cul-
tivation was carried out at 28’C for l-5 d with shaking in test tubes
(2.0 x20 cm) with 10 ml of each medium. The cells were collected
by centrifugation at 10,OOOxg for 15 min or by filtration. The har-
vested cells were washed with 10 mM potassium phosphate buffer
(PH 7.0) and suspended in the same buffer. The cell suspensions of
bacteria and a~tinomycetes were dis~pted by sonication for 5 min,
and those of molds and yeasts were homogenized for IO min with
0.25 mmd, glass beads in a homogenizer. After centrifugation at
15,OOOxg for 15 min, the resultant supernatants were used as en-
zyme preparations.
Assay of aldebyde oxidase ALOD activity was assayed by
measuring hydrogen peroxide generated during the oxidation of
aldehydes. The standard reaction mixture contained I5 mM form-
aldehyde, 50 mM potassium phosphate buffer (pH 7.0), 0.36 mM
4-aminoantipyrine, 6.3 mM phenol, 7 units/ml peroxidase, and en-
zyme in a total volume of 2.0 ml. After incubation at 37°C for S-
15 min, the formation of the quinoneimine dye was estimated by
measuring the absorbance at 500nm with a spectrophotometer
(16). One enzyme unit was defined as the amount of enzyme that
produces 1 ,umol of hydrogen peroxide per min.
Purification of aldehyde oxidases All operations were car-
ried out at 4°C and 1OmM potassium phosphate buffer (pH7.0)
was used.
The culture broths (2400ml each) from Methylobacillus sp.,
Pseudomonas sp. and Streptomyces moderotus were centrifuged at
12,000xg for I5 min to collect each cell. The cells were disrupted
by sonication, and centrifuged at 12,000 xg. (NH&SO, were added
to the resulting supernatants (cell-free extracts) up to 60% satu-
ration, and the precipitates were collected by centrifugation at
12,OOOxg for 15 min, and dialyzed against the buffer overnight.
The dialyzed enzyme solutions were applied onto a column (5 x 15
cm) of DEAE-Toyopearl 650M, which was pre-equilibrated with
the buffer. After the column was washed with the buffer, the
enzyme was eluted with a linear gradient of NaCl from 0 to 0.5
M. The active fractions were combined, and concentrated with an
Amicon YMlO membrane (Millipore, Bedford, MA, USA). Then,
(NH,),SO, was added to the enzyme solution up to 30% satura-
tion, and applied onto a column (3 x 15 cm) of phenyl-Toyopearl
650M, which was pre-equilibrated with the buffer containing
30% saturated ~H~)*SO~. After the column was washed with 30%
~H~)~SO~-saturated buffer, the enzyme was eluted with a reverse
linear gradient of (NH&SO, saturation from 30% to 0%. The ac-
tive fractions were combined, dialyzed against the buffer, and con-
centrated with a YMlO membrane. The concentrates were used as
the sample enzymes for determining their enzymatic properties.
ALODs from ~ethy~obaciz~~s sp., Pseudomonas sp., and S.
moderator were pained 590-fold with 19.5% yield, 538-fold with
20.9% yield, and 107-fold with 12.2% yield, respectively. The pu-
rities of the final preparations obtained were suggested to be in the
range of 30-50% from the results of SDS-PAGE.
In order to clarify its subunit composition, further purification
of the ALOD from Methylobacillus sp. was carried out. The par-
tially purified enzyme obtained above was further purified by Re-
source Q (6 ml) anion exchange c~omatography and Superdex
2OOpg (26160) gel filtration, which were carried out essentially
under the same conditions described above. The SDS-PAGE of
the final preparation gave three major protein bands and a few con-
taminant-like protein bands, as shown in Fig. 1.
Molecular weight estimation Gel filtration was carried out
with a TSK-gel G3000SW column (0.75 x60 cm; Tosoh, Tokyo) at
a flow rate of 0.3 mVmin with 200 mM pot~sium phosphate buffer
(pH 7.0) containing 0.1 M NaCl as eluant. The elution profiles of
MICROBIAL ALDEHYDE OXIDASE 125
Ml234
(kDa1
94
+ Large
67
43
+ Medium
30
20
+ Small
14
FIG. I. SDS-PAGE pattern of purified aidehyde oxidase from
~ethyZobociI~~ssp. Lane M, Contained molecular mass marker pro-
teins; lanes l-4, contained purified ~e~~~Q~~cj~~~~sp. aldehyde oxi-
dase (5, 10,25, and 50 pg, respectively).
the enzymes were determined by assaying the oxidase activity of
the eluates. The molecular weights of the enzymes were calcu-
lated from the mobilities of standard proteins obtained &om Orien-
tal Yeast, Osaka. SDS-PAGE was carried out by the method of
Laemmli with 0.1% SDS and 12.5% polyacrylamide gel (1 mm
thick). The molecular weights of the enzymes were calculated
from the relative mobilities of standard proteins (Amersham Bio-
sciences, Piscataway, NJ, USA).
Chemicals Formaldehyde was prepared by heating para-
fo~aldehyde (Sigma Chemicals, St. Louis, MO, USA) at 100°C
for 3 h, because commercial fo~aldehyde solution contains ap-
proximately 8% methanol for stabilization. The other aldehydes
and all other chemicals were of the highest analytical grade avail-
able. DEAE-Toyopearl 650M and phenyl-Toyopearl 650M were
obtained from Tosoh, and Resource Q (6 ml) and Superdex 200 pg
(26160) were obtained from Amersham Biosciences.
RESULTS
Production of aldehyde oxidase by microorganisms
The distribution of ALOD was examined using formalde-
hyde as a substrate against approximately 3.50 strains of mi-
croo~~isms in our culture collection. The fo~aldehyde~
oxidizing enzymes were found in several kinds of micro-
organisms, and were classified into two classes, depending
on their substrate specificity. That is, the first class included
enzymes that could oxidize only formaldehyde among the
aldehydes tested, but could efficiently oxidize lower pri-
mary alcohols such as methanol and ethanol. The second
class included enzymes that could oxidize various kinds of
aldehydes, but could not oxidized alcohols at all. The first
class of enzymes seemed to be alcohol oxidases, and was
produced by molds such as Cylindrocarpon didymum KY392
and U&ago crus-galli KY2708 (data not shown). On the
other hand, the second class of enzymes seemed to be real
ALODs, and was found in cells of some strains of the genus
Pseudomonas, methanol-assimilating bacteria such as the
126 YASUHARA ET AL. J. BIOSCI.BIOENG.,

TABLE 1. Distribution of aldehyde oxidase in microorganisms

Aldehyde oxidase activity” (mu/ml)

Microorganism
Initial mediumb Improved medium
Methylobacillus extorquens DSM1337 4.4 ND’
Methylobacillus sp. KY4400 6.4 18.0”
Methylophaga thalassica ATCC33 146 0.5 ND
Pseudomonas parvonacea KY3991 23.0 ND
I? stutzeri IF012695 24.0 ND
Pseudomonas sp. KY4690 26.0 115.0*
Streptomyces moderatus ATCC23443 9.4 41.3’
S. ochraceiscleroticus ATCCl5814 2.0 ND
S. rimosus ATCC 10970 6.2 ND
a Aldehyde oxidase activity was assayed using formaldehyde as a substrate, and is indicated as mu per ml of broth.
b Each initial medium is described in Materials and Methods.
c Yeast bouillon medium supplemented with 1% sucrose, 0.2% glutamate, 0.2% aspartate, 0.2% alanine, and 0.01% MnSO,.7H,O was used.
* Yeast bouillon medium supplemented with 1% maltose, 0.2% valine, 0.2% leucine, 0.2% isoleucine, and 0.01% MnSO,.7H,O was used.
c Bennett’s medium supplemented with 0.5% glucose was used.
f ND means “not determined”.

genera Methylobacillus and Methylobacterium, and the ALODs were examined, and were expressed relative to that
genus Streptomyces (Table 1). Of these, Methylobacillus against formaldehyde (Table 2). Methylobacillus sp. ALOD
sp. KY4400, Pseudomonas sp. KY4690, and S. moderatus showed a high activity against formaldehyde. Pseudomonas
ATCC23443 were selected as representative strains for tI.rr- sp. ALOD showed a high activity against aliphatic alde-
ther study, because of their higher activities and stable re- hydes. S. moderatus ALOD showed high activities against
producibilities. both aliphatic and aromatic aldehydes. On the other hand,
The effect of various kinds of carbon, nitrogen, and metal all of the three ALODs did not show an activity against
compounds on the production of ALOD by three strains of xanthine, purine, and IV’-methylnicotinamide, which are sub-
microorganisms was investigated. First the three strains strates of xanthine oxidase (EC 1.1.3.22), and lower pri-
were cultivated in each basal medium described in Materi- mary alcohols such as methanol and ethanol, which are sub-
als and Methods, which was supplemented with ten metal
salts of 0.01% concentration, and the ALOD activities of
TABLE 2. Substrate specificity of three microbial aldehyde oxidases
their cell-free extracts were assayed. The addition of man-
ganese ion resulted in about 3.2-fold, 1S-fold, and 0.91-fold Relative activityb
increase in the enzyme production of Methylobacillus sp., Aldehyde” Methylo-
Pseudomonasd StreptomyceSe
Pseudomonas sp., and S. moderatus, respectively. On the bacillus’
other hand, the addition of molybdenum ion, which is Formaldehyde 1.0 1.0 1.0
known to be a cofactor in animal and plant ALODs (17), did Acetaldehyde 0.35 3.5 1.7
not affect the enzyme production in all the strains. However, Propionaldehyde 0.23 3.4 2.2
Butylaldehyde 0.31 3.6 2.6
the addition of tungsten ion, which is known to have an
Valeraldehyde 0.56 3.6 3.5
antagonistic effect on MO-dependent enzymes (18, 19), re- Hexylaldehyde 0.74 3.6 4.3
sulted in the reduction of their enzyme production to 6.1%, Heptylaldehyde 0.76 3.5 4.8
5.7%, and 16% in Methylobacillus sp., Pseudomonas sp., Octylaldehyde 0.64 3.5 4.3
and S. moderatus, respectively. Decylaldehyde 0.41 0.52 4.0
Furthermore, the addition of sugar and amino acids to the Phenylacetaldehyde 0.26 1.3 2.3
Cimramaldehyde 1.7 2.4 4.1
basal medium resulted in the increase in the enzyme pro-
Benzaldehyde 1.3 1.6 4.1
duction, because of their growth-stimulating effects. On the p-Hydroxybenzaldehyde 2.0 2.5 3.2
other hand, the addition of various aldehydes did not show a Salicylaldehyde 0.22 1.2 2.6
positive effect on the enzyme production. Finally, the im- p-Anisaldehyde 1.8 1.9 5.0
proved cultivating conditions, which are described in Table Veratraldehyde 1.2 2.2 6.1
1, brought about 2.8-fold, 4.4-fold, and 4.4-fold increase in Acrolein 0.08 1.4 1.3
the enzyme production, compared with those in the initial Crotonaldehyde 0.61 2.3 3.4
Citral 2.1 2.6 4.3
screening (Table 1). DL-Glyceraldehyde 0.28 0.77 0.73
Properties of microbial aldehyde oxidases In order to Glutalaldehyde 0.18 0.64 0.20
determine the enzymatic properties of the mirobial ALODs, 2-Ethylbutylaldehyde 0.51 3.5 1.7
a brief purification was carried out. The cell-free extracts Indol-3-carboxyaldehyde 0.20 0.08 1.0
of the three strains were purified using ammonium sulfate BThe standard concentration of the aldehydes used was 15 mM, and
fractionation, anion exchange and hydrophobic chromato- aldehydes with lower solubility were used at the saturation concentra-
graphies, as described in Materials and Methods. Then, the tion.
b The activity is expressed relative to that against formaldehyde.
partially purified enzymes were used for the characteriza- c Aldehyde oxidase from Methylobacillus sp. KY4400.
tion of each ALOD. * Aldehyde oxidase from Pseudomonas sp. KY4690.
The oxidizing acitivities against various aldehydes of three e Aldehyde oxidase from Streptomyces moderatus ATCC23443.
VOL. 94,2002 MICROBIAL ALDEHYDE OXIDASE 127

TABLE 3. K,,, values of three microbial aldehyde oxidases

to various aldehydes

K- fmM)
Aldehyde Methylo-
Pseudomonasb Streptomycesc
bacillus”
Formaldehyde 4.2 21.2 3.6
Acetaldehyde 1.4 1.4 0.13
Hexylaldehyde 1.8 0.15 0.26
Sal~~yialdehyde 0.07 0.70 0.34
Acrotein 0.48 1.4 0.74
Citral 0.12 2.1 0.06
2-Ethylbutylaldehyde 0.57 3.5 2.2
a Aldehyde oxidase from Methylobacillus sp. KY4400.
b Aldehyde oxidase from Pseudomonas sp. KY4690. FIG. 3. Effects of pH on the activity and stability of three micro-
’ Aldehyde oxidase from Streptomyces moderatus ATCC23443. bial aldehyde oxidases. (a) Each enzyme reaction was carried out
under the standard conditions except that 20 mM universal buffers of
various pHs were used. As the color intensity of the product is depen-
dent on the pH, appropriate corrections were made using hydrogen
strates of alcohol oxidase (EC 1.1.3.13). The affinities for peroxide as a standard product. (b) Each enzyme was treated using 20
seven representative aldehydes of the three ALODs were mM universal buffer of various pHs for 1.5min at 55°C for Methylo-
examined (Table 3). Methylobacillus sp. ALOD showed bacillus sp. ALOD, at 80°C for Pseudomonas sp. ALOD, and at 60°C
for S. moderatus ALOD. Then, the residual enzyme activity was
high affinities for formaldehyde (the smallest aldehyde), assayed under the standard conditions (pH 7.0). The results are ex-
salicylaldehyde (an aromatic aldehyde), and acrolein and pressed as a percentage of the activity of each native enzyme. Open
citral (double-bond-confining aldehydes). Pseudomonas sp. circles, ~ethy~obacill~s sp. ALOD; closed circles, Pseudomonas sp.
ALOD showed high aftinity for hexylaldehyde (a long aii- ALOD; open triangles, S. moderutus ALOD.
phatic aldehyde), but showed rather low afftnity for form-
aldehyde (the smallest aldehyde). S. moderatus ALOD active at temperatures higher than that suggested based on
showed high affinities for formaldehyde and acetaldehyde its thermal stability, and Pseudomonas sp. ALOD was high-
(small aldehydes), and acrolein and citral. Furthermore, ly stable against heat denaturation.
among the aldehydes tested, acrolein showed a strong sub- The effect of pH on the enzyme activity was examined
strate inhibition at a concentration more than 2 mM only for (Fig. 3a). Methylobacillus sp. ALOD showed an optimum
Methylobacillus sp. ALOD. pH of approximately 3.0 to 4.0. On the other hand, the en-
The effect of temperature on the enzyme activity was zymes from Pseudomonas sp. and S. moderatus showed
examined (Fig. 2a). The apparent optimum temperatures for optimum pHs of approximately 6.0 to 7.0 and 5.0 to 6.0, re-
a IO-min reaction were 50°C 45”C, and 30°C for ~ethylo- spectively, and showed no or only little activity at pH 3.0,
b~~i~~us sp., Pseudomonus sp., and S. moderatus, respec- The pH stabilities of the enzymes were examined (Fig. 3b).
tively. The thermal stabilities of the enzymes were exam- ~ethy~obacillus sp. ALOD was stable between pH 3.0 and
ined (Fig. 2b). The enzymes from Methylobacilius sp., pH 5.0. In contrast, the enzymes from Pseudomonas sp. and
Pseudomonas sp., and S. moderatus showed residual ac- S. moderutus were stable between pH 5.0 and pH 9.0.
tivities of more than 90% at 6O”C, 80°C and 70°C, when The effects of SH reagents and chelating agents on the
treated at pH 7.0 for 15 min. ~ethy~obacil~us sp. ALOD was ALOD activity were examined. The addition of p-chloro-
mercuri~~oate at 1 mM caused 88%, 94%, and 90% in-
hibitions of the activities of enzymes from ~ethyloba~i~~us
sp., Pseudomonas sp., and S. moderatus, respectively, but
iodoacetate, iodoacetamide, and N-ethylmaleimide did not
inhibit the enzyme activities at the same concentration (data
not shown). Chelating agents such as EDTA, o-phenanthro-
line and ~,ff’-dip~idyl had no effect on the enzyme activity
(data not shown).
The molecular weights of the above ALODs were deter-
mined to be 142 kDa for the Methylobacillus enzyme, 140
kDa for the Pseudomonas enzyme, and 148kDa for the
0-
20 30 40 50 60 70 OOW 60 100
Streptomyces enzyme, as determined by gel filtration. On
TeBRerature (“cl Temperature CC) the other hand, SDS-PAGE of the highly purified ~ethy~o-
bac~lius sp. ALOD revealed that the enzyme was composed
FIG. 2. Effects of temperature on the activity and stability of three
microbial aldehyde oxidases. (a) Each enzyme reaction was carried out
of three different subunits: large (88 kDa), medium-sized (38
for 1Omin under the standard conditions except for the incubation kDa) and small (18 kDa) (Fig. 1). By analogy with Methylo-
temperature, which is indicated in the figure. (b) Each enzyme was bacillus sp. ALOD, 85-, 39-, and 19-kDa proteins for Pseu-
treated for 15 min in 10 mM potassium phosphate buffer (pH 7.0), and domonas sp. ALOD, and 86-, 37-, and 23-kDa proteins for
then the residual enzyme activity was assayed under the standard con-
S. moderatus ALOD were presumed to be the subunits of
ditions. The results are expressed as a percentage of the activity of
each native enzyme. Open circles, ~ethy~obac~~~~~sp. ALOD; closed ALODs from these microorg~isms.
circles, Pseudomonas sp. ALOD; open triangles, S. moderatus ALOD.
128 YASUHARA ET AL. J. BIOSCI.BIOENG.,

TABLE 4. Comparison of aldehyde oxidases with their refated enzymes

Native Subunit
Electron Subunit
Enzyme Source molecular molecular Ref.
acceptor structure
mass &Da) mass(es) &Da)
ALOD h4ethylo&acillus sp. 0, 142 88,38,18 .- czgr This
ALOD P~e~~orno~~sp. 0, 140 85,39, 19 al% This
ALOD S. m~era~s 02 I48 86,37,23 a& This
ALOD Bovineliver 0, 300 147 a2 2
ALOD Maize 4 300 I47 a, 20
ALOR D. gigas DCIP 200 97 a2 21
ALDH S. cerevisiae NADP 54.6 9
XDHIXOD Cow’s milk NAD/O, 290 150 CL, 24
XDH p ptida 86 NAD 550 91.0,46.2 a&, 25
XDH E. barkeri NADP 530 81,30, 17.5 a@,~~ 26
ALOD,Aldehydeoxidase;ALOR,aIdehydeoxidoreductase;ALDH,aldehydedehydrognase;XDH,xanthinedehydrogenase;XOD,xanthine
oxidase; DCIP,2,6-dichlorophenol indophenol.

DISCUSSION three subunits. Similar observations were made between the

When the cell-free extracts of various kinds of microor-
ganisms were screened for oxidative activity against form-
aldehyde, some bacteria and actinomycetes were found to
be able to catalyze the dioxygen-dependent oxidation of
formaldehyde and many other kinds of aldehydes. Current-
ly, the oxygen-de~ndent ALOD is known in animals (2)
and plants (20), but not in microorganisms, although alter-
native aldehyde-metabolizing enzymes such as NAD and/or
NADP-dependent aldehyde dehydrogenases (9-12) and 2,6-
dichrorophenol indophenol-dependent aldehyde oxidoreduc-
tases (20-23), are known in some microorganisms.
The production of ALODs by micr~~~isms was lim-
ited in dis~bution, and involved low and constitutive for-
mation. Therefore, the roles of ALODs are of interest in
connection with the aldehyde metabolism in microorga-
nisms. Mammalian and plant ALODs are well known to be
molybdo-iron-sulfur flavoproteins (17). We do not know
whether the microbial ALODs obtained here are molybdo-
iron-sulfur enzymes or not. However, although the addition
of molybdate to the growth media did not essentially affect
the production of microbial ALODs, the addition of tungs-
tate strongly inhibited the production of the enzymes. These
results suggest that these microbial ALODs are molybdo-
proteins, because tungstate has been proved to show an
~~gonistic effect on the inco~oration of molybdate into
molybdate-dependent enzymes ( 18, 19).
ALODs from the microorganisms were composed of three
subunits that differ in sizes, and were in the ranges of 85-
88 kDa for the large ones, 37-39 kDa for the medium-sized
ones, and 18-23 kDa for the small ones. The molecular
masses of the native enzymes were in the range of 140-
148 kDa, which indicated that the microbial ALODs were
heterotrimers. On the other hand, animal and plant ALODs
are known to be homodimers, consisting of two 147-150
kDa subunits (2, 20), as summarized with other related en-
zymes in Table 4. These results indicate that the total molec-
ular mass of the three subunits from the microorg~sms is
about the same as that of the subunits of the animal and
plant ALODs. Therefore, it is suggested that in animal and
plant enzymes all the functional units are loaded into one
subunit, but in microbial enzymes they are separated into
one subunit (150 kDa) of x~thine dehy~ogen~e (XDH)
from cow’s milk (24), the two subunits (91 .O and 46.2 kDa)
of XDH from Pseudomonas putidu (25) and the three sub-
units (81, 30, and 17.5 kDa) of XDH from Eubacterium
barkeri (26) (Table 4). It remains to be solved why these
microbial enzymes tend to be divided into smaller and more
than one subunit.
The properties of microbial AL,ODs were dependent on
their producing strains. Methylobacillus sp. ALOD showed
high activity and stability in the acidic-pa region, and gave
high activity against formaldehyde.~Pseudomonus sp. ALOD
was characterized by its extremely high heat stability, but
showed only limited activity and a%nity against formalde-
hyde. S. modera~s ALOD showed low optimum tempera-
ture for the oxidation reaction, although its heat stability
was rather high. Therefore, the enzyme from S. moderutus
seemed to be a kind of intermediate between those from
Mthylobacillus sp. and Pseudomonas sp. These results give
us the following two important oppo~ities. First, different
ALODs give us a chance to determine the structural charac-
teristics of this type of enzyme to clarify the differences of
these enzymes. Second, distinctive ALODs give us a chance
to create more superior enzymes by combining each good
point into one unit with the methods such as the family
DNA shuffling (27). Therefore, the cloning of the ALOD
genes is now under way.
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