Purification and characterization of aldoxime dehydratase of the head blight fungus, Fusarium graminearum

Article abstract of DOI:10.1271/bbb.69.2254

Fungal aldoxime dehydratase (Oxd) of Fusarium graminearum MAFF305135 was purified and characterized for the first time from its overexpressing Escherichia coli transformant. The enzyme showed about 20% identity with known Oxds, and had similar enzymatic properties with nitrilase-linked Oxd from the Bacillus strain. It belongs to a group of phenylacetaldoxime dehydratases (EC 4.99.1.7), based on its substrate specificity and kinetic analysis.

Full text of DOI:10.1271/bbb.69.2254

Biosci. Biotechnol. Biochem., 69 (11), 2254–2257, 2005
Note
Purification and Characterization of Aldoxime Dehydratase
of the Head Blight Fungus, Fusarium graminearum
y
Yasuo KATO and Yasuhisa ASANO
Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University,
5180 Kurokawa, Toyama 939-0398, Japan
Received June 29, 2005; Accepted July 9, 2005
Fungal aldoxime dehydratase (Oxd) of Fusarium
graminearum MAFF305135 was purified and charac-
terized for the first time from its overexpressing Esche-
richia coli transformant. The enzyme showed about
20% identity with known Oxds, and had similar
enzymatic properties with nitrilase-linked Oxd from
the Bacillus strain. It belongs to a group of phenyl-
acetaldoxime dehydratases (EC 4.99.1.7), based on its
substrate specificity and kinetic analysis.
Fusarium graminearum (telemorph Gibberella zeae),
which causes Fusarium head blight of wheat and barley,
contains a gene encoding an Oxd homolog (protein
code: 78267.1) at location 165270–166361 on chromo-
some 4 of that strain [accession no. AACM01000259].
The present work reports cloning and overexpression of
this gene in E. coli. The protein encoded by the gene
was purified in a His6-tagged form and subsequently
characterized. This is the first report on purification and
characterization of Oxd from eukaryotes.
Key words: aldoxime dehydratase; aldoxime-nitrile
pathway; nitrilase; Fusarium graminearum;
characterization
F. graminearum Schwabe MAFF (gene bank of the
Ministry of Agriculture, Foresty, and Fisheries of Japan)
305135 was used as DNA source since the original
genome-sequenced strain F. graminearum PH-1 could
not be obtained. The strain was cultured on a potato-
dextrose agar medium as described previously.2) The
putative Oxd was originally annotated as a hypothetical
protein, but we found remarkable (20–21%) sequence
identities of the protein with known bacterial Oxds, as
shown in Fig. 1. To obtain the complete coding region
for the gene, PCR-amplification was performed. Since
the genome database of F. graminearum suggests that
the Oxd homolog gene does not contain introns, we
amplified the gene directly from the genome of the strain
with the following primers designed from its open
reading frame: OxdFG-F (50-TAAGGATCCGCTTCG-
GTCACGTTTCCC-30) contained an engineered BamH1
site (underlined) and 50-region of the gene (ATG start
codon was deleted), while OxdFG-R (50-CTACCATGG-
CTACCACTCTATCGGCTT-30) had an engineered
NcoI site (underlined) and 30-region of the gene
including stop codon (italics). The vector used for
expression, pUC18-His, was modified pUC18, such that
a His6-tag and short polypeptides derived from its multi-
cloning sites (MHHHHHHGMASMTGGQQMGRDP)
were placed at N-terminal Met position of the Oxd
homolog. PCR was performed in reactions (30 ml)
containing 0.1–5 mg chromosomal DNA of F. grami-
nearum extracted as described previously,17) 30 pmol
each of the primers, 300 mM dNTPs, 1 ꢀ PCR buffer, and
2.5 units of Blend-Taq polymerase (Toyobo, Osaka,
We have been studying microbial aldoxime metabo-
lism and have found from gene sequences and enzy-
matic evidence that aldoxime is metabolized into the
corresponding carboxylic acids through nitrile by a
combination of a novel enzyme, aldoxime dehydratase
(Oxd; EC 4.99.1.-), and nitrile-hydrolyzing enzymes
such as nitrilase (Nit), and nitrile hydratase (NHase)
and/or amidase. The pathway involving the enzymes
was named the ‘‘aldoxime-nitrile pathway’’.1–10) Among
these enzymes, we found that Oxd is unique because it
contains heme as a prosthetic group despite its simple
catalytic activity, involving
a dehydration reac-
tion.3,6,7,9–11) Some reaction mechanisms of Oxds have
been elucidated,10–13) but their details are still unknown.
In order to obtain and compare biochemical properties
of Oxds from various sources, it is important to identify
characteristics of Oxd from eukaryotes, since Oxds have
been purified and characterized only from bacterial
sources such as Bacillus, Pseudomonas, and Rhodococ-
cus.3,6,7,9,11) It has been reported that strains belonging to
the genus Fusarium are effective producers of Nit.14–16)
Nit was used as a potential catalyst to produce mono-
carboxylic acid from di- or tri-nitriles.14,15) We have
clarified that the Fusarium strain has both Oxd and Nit
activities,2) but no enzymatic studies including the
primary structure on Fusarium Oxd have been carried
out. We searched genome databases and found that
y
To whom correspondence should be addressed. Tel: +81-766-56-7500; Fax: +81-766-56-2498; E-mail: asano@pu-toyama.ac.jp
Abbreviations: Oxd, aldoxime dehydratase; PAOx, phenylacetaldoxime; IAOx, indoleacetaldoxime

Aldoxime Dehydratase of Fusarium graminearum
2255
1
10
20
30
40
50
60
70
80
90
100
110
120
130
OxdRE MESAIGEHLQCPRTLTRRVPDTYTPPFPMWVGRADDALQQVVMGYLGVQFRDEDQRPAALQAMRDIVAGFDLPDGPAHHDLTHHIDNQGYENLIVVGYWKDVSSQHRWSTSTPIASWWESEDRLSDG-LGFFREI
OxdRG MESAIGEHLQCPRTLTRRVPDTYTPPFPMWVGRADDTLHQVVMGYLGVQFRGEDQRPAALRAMRDIVAGFDLPDGPAHHDLTHHIDNQGYENLIVVGYWKDVSSQHRWSTSPPVSSWWESEDRLSDG-LGFFREI
OxdK
OxdA
OxdB
MESAIDTHLKCPRTLSRRVPDEYQPPFAMWMARADEHLEQVVMAYFGVQYRGEAQRAAALQAMRHIVESFSLADGPQTHDLTHHTDNSGFDNLIVVGYWKDPAAHCRWLRSAPVNAWWASEDRLNDG-LGYFREI
MESAIDTHLKCPRTLSRRVPEEYQPPFPMWVARADEQLQQVVMGYLGVQYRGEAQREAALQAMRHIVSSFSLPDGPQTHDLTHHTDSSGFDNLMVVGYWKDPAAHCRWLS-AEVNDWWTSQDRLGEG-LGYFREI
----------------KNMPENHNPQANAWTAEFPPEMSYVVFAQIGIQSKS---LDHAAEHLGMMKKSFDLRTGPKHVDRALHQGADGYQDSIFLAYWDEPETFKSWVADPEVQKWWSGKKIDENSPIGYWSEV
OxdFG -----------------------------MLRSRFPASHHFTVSVFGCQYHSEAPSVEKTELIGRFDKLIDSAAIH-----VEHLEQNDVPSKIWMSYWESPQKFKQWWEKDDTASFWASLP----DDAGFWRET
: : : *
*
:
:
:
*
:
:
: **
*
*
:
*: :
*
140
150
160
170
180
190
200
210
220
230
240
250
260
OxdRE VAPRAEQFETLYAFQED-LPGVGAVMDGISGEINEHGYWGSMRERFPISQTDWMQASG----ELRVIAGDPAVGGRVVVRG-HDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMDFLRDNGPAVGCYSNRF
OxdRG VAPRAEQFETLYAFQDD-LPGVGAVMDGVSGEINEHGYWGSMRERFPISQTDWMQASG----ELRVVAGDPAVGGRVVVRG-HDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMDFLRDNGPAVGCYSNRF
OxdK
OxdA
OxdB
SAPRAEQFETLYAFQDN-LPGVGAVMDRISGEIEEHGYWGSMRDRFPISQTDWMKPTS----ELQVIAGDPAKGGRVVVLG-HGNLTLIRSGQDWADAEAEERSLYLDEILPTLQDGMDFLRDNGQPLGCYSNRF
SAPRAEQFETLYAFQRDNLPGVGAVMDSTSGEIEEHGYWGSMRDRFPISQT-WMKPTN----ELQVVAGDPAKGGRVVIMG-HDNIALIRSGQDWADAEAEERSLYLDEILPTLQDGMDFLRDNGQPLGCYSNRF
TTIPIDHFETLHSGENYD-NGVSHFVPIKHTEVHE--YWGAMRDRMPVSASSDLESPLG--LQLPEPIVRESFGKRLKVTA-PDNICLIRTAQNWSKCGSGEREYYIGLVEPTLIKANTFLRENASETGCISSKL
OxdFG FSLPATRAMYEGTGKDA---YGFGHCGSLIPLTTKTGYWGAYRSRMTPDFEGDTFSSPIPTYADQSVPADKIRPGRVRITDFPDNLCMVVEGQHYADMGEREREYWNENFDGLTKQWVTNVVTAGHEQGMVIARA
*** :
*
* :
*:
:
* : : : :
:
*
:
**
:
:
*
:
270
280
290
300
310
320
330
340
350
360
370
380
390
400
OxdRE VRNIDIDGNFLDLSYNIG---------------HWASLDQLERWSESHPTHLRIFTTFFRVAAG-----LSKLRLYHEVSVFDAADQLYEYINCHPGTGMLRDAVTIAEH-------------------------
OxdRG VRNIDIDGNFLDLSYNIG---------------HWASLDQLERWSESHPTHLRIFTTFFRVAEG-----LSKLRLYHEVSVFDAADQLYEYINCHPGTGMLRDAVITAEH-------------------------
OxdK
OxdA
OxdB
VRNIDLDGNFLDVSYNIG---------------HWRSVEKLERWTESHPTHLRIFVTFFRVAAG-----LKKLRLYHEVSVSDAKSQIFGYINCHPQTGMLRDAQVSPA--------------------------
VRNIDLDGNFLDVSYNIG---------------HWRSLEKLERWAESHPTHLRIFVTFFRVAAG-----LKKLRLYHEVSVSDAKSQVFEYINCHPHTGMLRDAVVAPT--------------------------
VYEQTHDGEIVDKSCVIG---------------YYLSMGHLERWTHDHPTHKAIYGTFYEMLKRHD--FKTELALWHEVSVLQSKDIELIYVNCHPSTGFLPFFEVTEIQEPLLKSPSVRIQ-------------
OxdFG CHGFAGEKKLGATNGPVNGIFPGLDYVHQAQILIWQDISKMEHIGRYDQTHVKLRRDFMKAYGPGGEMEGGDLLLWVDLGILKKDEIDAEYVGCYESTGFLKLDKGQFFKVESTAGSKLPSFFDEPIESKPIEWP
** *: **
:
*
:
:
*
*
*
:
*
*
Fig. 1. Amino Acid Sequence Comparison of Oxds from Rhodococcus sp. N-771 (OxdRE), R. globerulus A-4 (OxdRG), Pseudomonas sp. K-9
(OxdK), P. chlororaphis B23 (OxdA), Bacillus sp. OxB-1 (OxdB), and F. graminearum MAFF305135 (OxdFG, this study).
Identical and similar amino acids are indicated by asterisks and colons respectively. Conserved distal and proximal histidine residues are
boxed by solid and dotted lines respectively.
Table 1. Comparison of the Properties of Oxds from Bacillus sp. OxB-1 (OxdB), R. erythropolis N-771 (OxdRE), R. globerulus A-4 (OxdRG),
P. chlororaphis B23 (OxdA), Pseudomonas sp. K-9 (OxdK), and F. graminearum MAFF305135 (OxdFG, this study)
Properties
OxdBa
OxdREa
OxdRGa
OxdA
OxdKa
OxdFGa
Molecular weight (Mr)
Native
Sequence
42,000
40,972
1
80,000
44,794
2
80,000
44,817
2
76,400
40,127
2
85,000
44,511
2
34,100
44,070
1
Number of subunits
Soret peak (nm) (ferric form)
(ferrous form)
Specific activityb
Optimum pHd
407
432
409
428
409
428
408
408
428
420
431
428
851
7.0
562
8.0
633
8.0
197c
5.5c
2.25
7.0
24.8
5.5
Temp. (ꢁC)e
30
6.5–8.0
<45
30
6.0–9.5
<40
30
6.0–9.5
<40
45c
20
5.5–6.5
<30
25
4.5–8.0
<20
Stability pHd
6.0–8.0
<40
Temp. (ꢁC)e
aAs His6-tagged form at the N-terminus.
bEnzyme activity for Z-PAOx (U/mg) was measured under anaerobic conditions.
cn-Butyraldoxime was used as a substrate.
dThe effects of pH on enzyme activity and stability were investigated in several 0.1 M buffers at various pHs using Z-PAOx as the substrate.
eThe effects of temperature on enzyme activity and stability were investigated at various temperatures between 20 ꢁC and 80 ꢁC in 0.1 M KPB (pH 7.0).
Japan) according to a previously described method.8)
The resulting amplified band (1 kbp) was extracted from
an agarose gel, digested with BamH1 and NcoI, and
ligated with pUC18-His vector to obtain pOxdFG for
further sequencing and overexpression. We sequenced
PCR-amplified fragments, and found 35 differences on
the nucleic acid sequence from that found in the genome
database: that caused 10 amino acid replacements and
25 silent mutations. These results might be due to
differences between the strains used for DNA isolation
and genome sequencing. The nucleotide sequence
of the gene identified in this study was deposited in
DDBJ/EMBL/GenBank databases under accession
no. AB214653.
The protein was expressed in E. coli JM109 cells
harboring pOxdFG in LB medium, and aldoxime
dehydration activity was measured as described for the
other Oxds.3–10) Cell-free extract of the transformant
exhibited stoichiometric dehydration of Z-phenylacet-
aldoxime (Z-PAOx) into phenylacetonitrile at pH 7.0. It
was clear that the gene (oxd) coding for Oxd had a new
primary structure, and we tentatively named the protein
OxdFG. The transformant was grown under various
culture conditions (medium volume, temperature, me-
dium, etc.) as we previously examined for Oxds of
Bacillus sp. OxB-1 (OxdB),5) R. erythropolis N-771
(OxdRE),7) and Pseudomonas sp. K-9 (OxdK),9) and Z-
PAOx dehydration activity was measured. The highest
activity was seen when the strain was grown at about
30 ꢁC in a high medium volume of Terrific medium,18)
as we had seen when expressing other Oxds.5,7,8) It
reached about 4,000 U/l culture.
OxdFG was purified to homogeneity from the E. coli
strain in its N-His6-tagged form using a Co-chelated
Talon affinity column (BD Bioscience, Palo Alto, CA),
as previously described for Oxds,9,10) and was charac-
terized. Table 1 summarizes the properties of OxdFG,
and those of Oxds from other sources. Purified OxdFG
showed a single band on SDS–PAGE, in agreement with
its Mr deduced from the gene sequence. The native Mr of

2256
Y. KATO and Y. ASANO
Table 2. Kinetic Parameters of N-His6-Tagged OxdFG from F. graminearum MAFF305135
Km
Vmax
Vmax=Km
(U/mg/mM)
Relative
activity (%)a
Aldoxime
Arylalkylaldoxime
(mM)
(U/mg)
Z-Phenylacetaldoxime
E=Z-2-Phenylpropionaldoxime
Z-3-Phenylpropionaldoxime
E=Z-Indoleacetaldoxime
E=Z-Mandelaldoxime
E=Z-4-Phenybutyraldoxime
Alkylaldoxime
3.52
3.71
2.76
1.46
1.70
1.79
28.2
18.1
20.4
19.3
2.32
14.1
8.01
4.88
7.39
13.2
1.36
7.88
100
68.6
104
125
16.1
79.2
E=Z-n-Butyraldoxime
E=Z-n-Valeraldoxime
E=Z-Isovaleraldoxime
E=Z-n-Capronaldoxime
2.87
10.1
2.66
20.4
88.8
23.1
3.60
7.11
8.79
8.68
4.48
107
341
92.7
17.6
0.802
aMeasured at 5 mM concentration. The enzyme activity for Z-PAOx dehydration activity was taken to be 100%. The relative activities for the other aldoximes are as
follows: Z-cinnamaldehyde oxime (17.9%), E=Z-propionaldoxime (10.6%), E=Z-isobutyraldoxime (5.97%), and E=Z-cyclohexanecarboxaldehyde oxime (11.6%).
the enzyme was estimated to be about 31,400, according
to the results of gel filtration chromatography with
Superdex200 (Amersham, Uppsala, Sweden), indicating
that the enzyme existed in a monomeric form, like
OxdB.3) The enzyme showed an orange color and had
heme b as a prosthetic group, as seen by heme-extraction
and analysis, as described previously.3,6,7,9) The absorp-
tion maximum (Soret peak) of the purified OxdFG was
at 420 nm, which was shifted to 431 nm by reduction
with Na2S2O4. The Soret peak of the reduced enzyme
was further shifted to 424 nm by bubbling CO gas.
These results suggest that the enzyme was purified in a
ferric form, but its absorption spectrum was different
from those of Oxds from various sources, whose Soret
peak of ferric form is about 407–408 nm.6,7,9,10) The
differences in Soret peak between OxdFG and other
Oxds presumably derive from diversity in the structure
of Oxds, although the exact reason is not clear. The
effects of temperature on enzyme stability and activity
were lower than those of known Oxds. The enzyme
showed highest activity at about pH 5.5, which is close
to that of Oxd of P. chlororaphis B23 (OxdA).11) The
specific activity of the enzyme toward Z-PAOx was
24.8, which is 20 times lower than that of OxdB,
OxdRE, or OxdRG,9,10) even when measured under
anaerobic conditions by five independent experiments,
but the reasons for the low activity of OxdFG remain
unclear at the present time. Eighty to 95% of OxdFG
activity was inhibited by the presence of 1 mM carbonyl
reagents such as NH2OH and phenylhydrazine, electron
donors such as trimethylhydroquinone, p-phenylenedi-
amine, and dimethoxybenzidine, and by some metal ions
such as Fe3þ, Agþ, and Cuþ, as seen with other
Oxds.3,6,7,9) Enzyme activity was nearly doubled by
adding thiol compounds such as DTT, mercaptoethanol,
cysteine, and thioglycerol, and ferrous ions such as
ferrocyanide and FeSO4. As shown in Table 2, the
enzyme was active toward various arylalkyl- and alkyl-
aldoximes, converting them to the corresponding ni-
triles. Arylaldoximes such as benzaldoxime, pyridine-2-
aldoxime, pyridine-3-aldoxime, and pyridine-4-aldox-
ime were inert as substrates, as was also seen with
known Oxds. Table 2 also shows values for Vmax and
Km, determined from Lineweaver-Burk plots of the
kinetic data. Based on a comparison of Vmax=Km values,
the enzyme preferentially acts on arylalkylaldoximes.
Therefore, we propose tentatively that it can be
categorized into a group of ‘‘phenylacetaldoxime dehy-
dratase (EC 4.99.1.7)’’.
From studies on the heme environments of OxdB10)
and OxdA,12) His306 and His320 residues were confirmed
to be the catalytic residue located in the distal heme
pocket of the Oxds, respectively, by alanine-scanning
mutagenesis. As shown in Fig. 1, however, the histidine
residue (boxed) is not present in OxdFG, suggesting that
catalytic residues located at the distal heme pocket of
OxdFG are different from those of the other Oxds. On
the other hand, another histidine (boxed by a dotted
line), which corresponds to His299 of OxdA, been
proposed to be the histidine residue located in the
proximal heme pocket of OxdA,12) is present in OxdFG.
We found that the proximal histidine residue has been
highly conserved among Oxds even from eukaryotes,
and that it is indispensable for heme-binding. Further
studies are needed to identify crucial catalytic residues
of OxdFG.
Genome sequencing analysis of F. graminearum PH-
1 revealed that the oxd gene exists together with genes
coding for Nit (protein code: 78265.1), and most
probably for its regulatory protein (protein code:
78266.1) in the genome of the strain, as we observed
in Bacillus sp. OxB-13) and P. syringae [AE016853].9)
Nit of F. graminearum had 37.1 and 39.7% identities
with those of Bacillus sp. and P. syringae respectively.
A possible regulatory protein of F. graminearum also
showed 19.6 and 18.1% identities respectively with
those of the strains although they belong to different
biological species: eukaryotes and prokaryotes. The
characteristics of OxdFG are similar to those of OxdB in
substrate specificity and subunit structure. Based on

Aldoxime Dehydratase of Fusarium graminearum
2257
Aldoxime dehydratase co-existing with nitrile hydratase
and amidase in iron-type nitrile hydratase producer
Rhodococcus sp. N-771. J. Biosci. Bioeng., 97, 250–259
(2004).
these results, we speculate that the oxd gene coding for
OxdFG has evolved from an ancestor gene together with
genes of Nit and regulatory proteins, as we have
observed in bacterial strains, in which NHase and Oxd
genes are suggested to be co-evolved.4,6,7,9)
8) Kato, Y., Yoshida, S., and Asano, Y., Polymerase chain
reaction for identification of aldoxime dehydratase in
aldoxime- or nitrile-degrading microorganisms. FEMS
Microbiol. Lett., 246, 243–249 (2005).
9) Kato, Y., and Asano, Y., Molecular and enzymatic
analysis of the ‘‘aldoxime-nitrile pathway’’ in the
glutaronitrile degrader Pseudomonas sp. K-9. Appl.
Microbiol. Biotechnol., in press.
10) Kobayashi, K., Yoshioka, S., Kato, Y., Asano, Y., and
Aono, S., Regulation of aldoxime dehydratase activity
by redox-dependent change in the coordination structure
of the aldoxime-heme complex. J. Biol. Chem., 280,
5486–5490 (2005).
11) Oinuma, K., Hashimoto, Y., Konishi, K., Goda, M.,
Noguchi, T., Higashibata, H., and Kobayashi, M., Novel
aldoxime dehydratase involved in carbon-nitrogen triple
bond synthesis of Pseudomonas chlororaphis B23:
sequencing, gene expression, purification, and character-
ization. J. Biol. Chem., 278, 29600–29608 (2003).
12) Konishi, K., Ishida, K., Oinuma, K., Ohta, T.,
Hashimoto, Y., Higashibata, H., Kitagawa, T., and
Kobayashi, M., Identification of crucial histidines
involved in carbon-nitrogen triple bond synthesis by
aldoxime dehydratase. J. Biol. Chem., 279, 47619–47625
(2004).
Mahadevan et al. reported some primitive studies on
indoleacetaldoxime (IAOx) dehydratase (EC 4.99.1.6)
of Gibberella fujikuroi (anamorph of F. moniliforme),19)
and showed that the enzyme was activated by dehy-
droascorbic acid, ascorbic acid, and PLP, and was
specific for IAOx. Since IAOx dehydratase has been
partially purified and is poorly characterized, we cannot
compare the properties of IAOx dehydratase with those
of OxdFG, reported in this study. But the fact that
OxdFG acts on various arylalkyl- and alkylaldoximes
and is not activated by these compounds allowed us to
show that OxdFG is the first example of fungal Oxd
purified and characterized, and is different from the
reported IAOx dehydratase. It is our interest to clarify
the physiological functions of the aldoxime-nitrile path-
way in the fungal strains to determine the genetic and
enzymatic associations between pathogenic fungi and
plants.
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