Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase (BbMPD) from Beauveria bassiana

Article abstract of DOI:10.1016/j.carres.2009.09.020

A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fungal entomopathogen Beauveria bassiana by gene cloning, heterogeneous expression and activity analysis. The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the 935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42 kDa) showed less than 75% sequence identity to 17 fungal MPDs documented and shared two conserved domains with the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-β-d-galactopyranoside at 20 °C after 5-h growth at 37 °C. The purified BbMPD exhibited a high catalytic efficiency (k_cat/K_m) of 1.31 × 10⁴ mM^-1 s^-1 in the reduction of the highly specific substrate d-fructose-6-phosphate to d-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 °C and pH 7.0 and was much more sensitive to Cu²⁺ and Zn²⁺ than to Li⁺ and Mn²⁺. The results indicate a crucial role of BbMPD in the mannitol biosynthesis of B. bassiana.

Full text of DOI:10.1016/j.carres.2009.09.020

Carbohydrate Research 345 (2010) 50–54
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Gene cloning and catalysis features of a new mannitol-1-phosphate
dehydrogenase (BbMPD) from Beauveria bassiana
Zheng-Liang Wang, Sheng-Hua Ying, Ming-Guang Feng *
Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2009
Received in revised form 11 September 2009
Accepted 21 September 2009
Available online 4 November 2009
A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fun-
gal entomopathogen Beauveria bassiana by gene cloning, heterogeneous expression and activity analysis.
The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the
935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42 kDa) showed
less than 75% sequence identity to 17 fungal MPDs documented and shared two conserved domains with
the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the
Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-b-
Keywords:
Mannitol-1-phosphate dehydrogenase
D
-galactopyranoside at 20 °C after 5-h growth at 37 °C. The purified BbMPD exhibited a high catalytic effi-
D-Fructose-6-phosphate
4
ꢁ1 ꢁ1
ciency (kcat/K
phosphate to
m
) of 1.31 ꢀ 10 mM
s
in the reduction of the highly specific substrate D-fructose-6-
NADH
Catalytic efficiency
Beauveria bassiana
D
-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 °C and pH
2
+
2+
+
2+
7.0 and was much more sensitive to Cu and Zn than to Li and Mn . The results indicate a crucial role
of BbMPD in the mannitol biosynthesis of B. bassiana.
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
-Mannitol is a non-cyclic six-carbon polyol that exists as a nat-
dues, and feature a Rossmann-fold coenzyme-binding domain at
the N-terminus with a highly conserved region (KXXXXNXXG)
D
and a large a-helical domain, which provides key elements of sub-
1
7
ural sugar alcohol in living organisms. It is particularly rich in
spores, fruiting bodies, sclerotia and mycelia of filamentous fun-
gi.
strate-binding recognition and catalysis. Several MPDs have been
6
18
characterized from fungi, such as A. niger, Aspergillus parasiticus
1
–3
19
This polyol contributes in weight 3.9% to Beauveria bassiana
and Crypotococcus neoformans. However, none of them is from
4
5
conidia, 20% to Agaricus bisporus mycelia and 10–15% to Aspergil-
entomopathogenic fungi, which are the biocontrol agents of
6
20,21
lus niger condiophores, respectively. Fungal mannitol acts as a res-
arthropod pests.
ervoir of cellular carbohydrates7 and a source of reducing power
,8
Fungal agents formulated for pest control are often exposed to
for the regeneration of NADPH.9 It is also known to be involved
detrimental stresses in the field, such as high temperatures
and solar UV irradiation
22–24
6,10
25–27
in fungal tolerance to environmental stress
lodgement or dispersal.
and in spore dis-
during summer. Recently, intracellular
1
1,12
Plant mannitol may quench reactive
mannitol accumulated in B. bassiana was found to be involved in the
thermotolerance of B. bassiana, a fungal entomopathogen.28 This
study sought to characterize a novel MPD of B. bassiana (BbMPD)
by gene cloning and heterogeneous expression. The recombinant
BbMPD purified from the cell cultures of an engineered Escherichia
coli strain was analyzed for its catalytic activity in the reduction of
oxygen species (ROS) produced by plants in response to the infec-
tion by phytopathogenic fungi.10,13,14
The biosynthesis and metabolism of fungal mannitol were
hypothesized to be a cyclic process.5 Four enzymes are likely
to be involved in the cycle, including NAD -dependent mannitol-
,15
+
1
-phosphate dehydrogenase (EC 1.1.1.17; abbreviated herein as
D-fructose-6-phosphate (D-Fru6P) to D-mannitol-1-phosphate.
+
MPD), NADP -dependent mannitol dehydrogenase (EC 1.1.1.138),
mannitol-1-phosphate phosphatase (EC 2.7.1.1) and hexokinase
2. Results and discussion
(
EC 3.1.3.22). The biosynthesis can be achieved via the dephosphor-
ylation of mainnitol-1-phosphate by the MPD-catalyzed reduction
2.1. The cloned gene BbMPD
of fructose-6-phosphate.2 Almost all fungal MPDs are classified
1
6
into polyol-specific, long-chain dehydrogenases and reductases.
These enzymes, which are composed of subunits of 350–560 resi-
A full-length 3049-bp fragment of the target gene BbMPD was
obtained by degenerate PCR and two runs of DNA walking. This
fragment consisted of a 1334-bp open reading frame (ORF) with
a 158-bp intron and the 935-bp upstream and 780-bp downstream
regions. The intron-free 1176-bp ORF encoded a 391-aa protein
*
Corresponding author. Tel./fax: +86 571 88206178.
E-mail address: mgfeng@zju.edu.cn (M.-G. Feng).
0
008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.09.020

Z.-L. Wang et al. / Carbohydrate Research 345 (2010) 50–54
51
(
Fig. 1a) with a predicted molecular weight of 42.8 kDa and an
tium rubrum, Magnaporthe grisea and Neurospora crassa (Fig. 1b).
This protein shared two conserved domains with the fungal MPD
family at the N- and C-terminus (Fig. 1c), respectively.
isoelectric point of 5.08.
2
.2. The deduced protein sequence of BbMPD
2
.3. The expression and purification of BbMPD
The protein deduced from the gene BbMPD (GenBank accession
number: GQ344502) showed no more than 75% sequence identity
to 17 fungal MPDs documented in the Genbank protein database,
based on BLASTP analysis online, and was phylogenetically close
to the MPDs of Botryotinia fuckeliana, Chaetomium globosum, Euro-
BbMPD was well expressed as a recombinant protein in the cul-
ture of engineered E. coli BL21 induced by 0.5 mM isopropyl 1-thio-
b- -galactopyranoside (IPTG) for 16 h at 20 °C after 5-h incubation
to log-growth phase at 37 °C. The expressed protein was purified to
D
Figure 1. Analysis of the gene BbMPD cloned from B. bassiana 2860. (a) The nucleotide sequence of BbMPD and the deduced protein sequence. The upper-case nucleotide
sequence is the 1176-bp open reading frame (ORF) encoding a 391-aa protein, while the lower-case fragments are a 158-bp intron and the 935-bp upstream and 780-bp
downstream regions (both shown in part). Two upstream boxes (tata and caat) and a putative poly(A) tail signal sequence (aaaaaa) in downstream are underlined. The shaded
ORF region (GAGNIG) is a coenzyme-binging motif. (b) A phylogenetic tree of the BbMPD and the MPDs from other 17 fungi, constructed with the bootstrap values of 1000
replications (shown at the nodes) by means of a neighbour-joining method. The three entries in parentheses after the name of each fungal species are the GenBank accession
number of the corresponding gene, the MPD sequence length (i.e., number of amino acids), and the percent identity to the BbMPD sequence. (c) Two conserved domains of
BbMPD revealed in BLASTP analysis (shown as the two bars). (d) SDS–PAGE profiles of the recombinant BbMPD expressed in the cell culture of the BbMPD-transformed E. coli
BL21. Lane 1: the culture lysate. Lane 2: the soluble fraction in the lysate. Lane 3: the purified BbMPD. M: molecular marker.

5
2
Z.-L. Wang et al. / Carbohydrate Research 345 (2010) 50–54
homogeneity by a HisTrap affinity column, as was shown by a sin-
gle band of 42 kDa in the SDS–PAGE profile (Fig. 1d). This is well in
agreement with the predicted molecular size of the deduced
protein.
Table 1
The effects of different agents on BbMPD activity
Agents testeda Concd (mM) MPD activity (U mgꢁ1
)
% Relative activityb
Control
CuCl
ZnCl
CaCl
673.7 ± 2.1
452.9 ± 7.5
372.0 ± 12.5
432.0 ± 2.9
463.3 ± 6.6
509.0 ± 4.9
462.5 ± 1.1
134.1 ± 0.4
363.8 ± 1 5.4
623.9 ± 6.1
598.7 ± 1.7
100
2
2
2
67.2 ± 1.6
55.3 ± 1.9
64.1 ± 0.4
68.8 ± 0.8
75.6 ± 0.7
68.7 ± 0.8
19.9 ± 0.1
54.0 ± 2.3
92.6 ± 0.9
88.9 ± 0.3
2
.4. The catalysis features of purified BbMPD
2
2
10
10
50
50
1
10
50
50
MgCl
LiCl
MnCl
SDS
DTT
EDTA
2
The catalytic activity of the purified BbMPD was proven to
+
strictly depend on the coenzymes NAD /NADH but not on
2
b-ME
a
SDS: sodium dodecyl sulfate; DTT: DL-Dithiothreitol; EDTA: ethylenediamine-
tetraacetic acid; b-ME: b-mercaptoethanol.
b
Estimates relative to the control excluding a tested agent in the reaction system.
+
NADP /NADPH. The protein exhibited high substrate specificity to
both
was very low or undetectable when
fructose-1,6-phosphate or -glucose-6-phosphate was used as a
D-Fru6P and NADH in the reduction reaction. Its activity
D
-fructose-1-phosphate,
D-
D
substrate in the presence of either NADH or NADPH.
The kinetic properties for the purified BbMPD to catalyze the
reduction of D-Fru6P in the presence of NADH are illustrated in Fig-
ure 2. The non-linear Michaelis–Menten equation for the reduction
2
was fitted very well (r = 0.998). As a result of the fit, the parame-
ters K
m
and Vmax (±SEM) were estimated as 0.892 ± 0.045 mM and
Figure 2. Effects of
activity of purified BbMPD (y) in the reaction system of 0.3 mM NADH at 30 °C and
pH 7.0 [non-linear fit to y = Vmaxx/(K + x)]. Error bars: SD.
D
-fructose-6-phosphate (
D
-Fru6P) concentration (x) on the
ꢁ1
9
1
1
13.5 ± 14.9 U mg , generating a catalytic efficiency (kcat/K
m
) of
4
ꢁ1 ꢁ1
.31 ꢀ 10 mM
s
for the reduction of D-Fru6P to D-mannitol-
m
-phosphate.
The catalytic activity of the purified BbMPD in the reduction
reaction was maximal at the regime of 30 °C (Fig. 3a) and pH 7.0
Fig. 3b) but dropped greatly after pre-incubation at 40–45 °C or
(
at a pH outside of the neutral point. The enzyme retained high
activity during storage for two-weeks at 4 °C.
2
.5. The effects of 10 tested agents on BbMPD activity
Sodium dodecyl sulfate (SDS) was most influential on the
BbMPD activity among the tested 10 agents (Table 1). Up to 80%
of its activity was inhibited by only 1 mM SDS in the reaction sys-
tem. Only slight activity change resulted from the inclusion of
<
50 mM EDTA (ethylenediaminetetraacetic acid) or b-mercap-
2
+
2+
+
2+
toethanol. The effects of Mg , Ca , Li and Mn on the enzyme
activity were inconspicuous at 5 mM but became significant above
that concentration. However, the enzyme activity was suppressed
by 34%, 45% and 46% at 2 mM Cu or Zn and 10 mM D,L-dithio-
threitol (DTT), respectively.
2
+
2+
3
. Conclusion and remarks
The enzyme BbMPD characterized from B. bassiana in this study
is apparently a new MPD. The gene encoding this enzyme was pro-
ven to be the 1334-bp ORF gene with a 158-bp intron. The deduced
3
91-aa protein shared two conserved domains with the fungal
MPD family, but its sequence was only 56–74% similar to those
known from 17 other fungi. The BbMPD purified from the culture
of E. coli BL21 transformed with the cloned gene exhibited high
NADH-dependent activity in the reduction of D-Fru6P to D-manni-
tol-1-phosphate, which is apparently crucial to the biosynthesis of
B. bassiana mannitol.
BbMPD differs in substrate specificity from the MPDs of C. neo-
1
9
2
29
formans, Pyrenochaeta terrestris and Sclerotinia sclerotiorum
Figure 3. Effects of temperature and pH on the activities of purified BbMPD in the
reduction of -fructose-6-phosphate. (a) Temperature effect at fixed pH 7.0. (b) pH
effect at fixed 30 °C. Error bars: SD.
This enzyme was highly specific to the substrate
D
-Fru6P with a
D
4
ꢁ1 ꢁ1
catalytic efficiency of 1.31 ꢀ 10 mM
s , whereas the three

Z.-L. Wang et al. / Carbohydrate Research 345 (2010) 50–54
53
above-mentioned MPDs could catalyze the interconversion of glu-
cose-6-phosphate to sorbitol-6-phosphate. Its catalytic efficiency
is equivalent to 222-fold of that of the Aspergillus fumigatus MPD,
A PCR-amplified 470-bp fragment was gel-purified and cloned into
pGEM-T-Easy (Promega) for sequencing at Invitrogen (Shanghai,
0
0
China). The 5 - and 3 -regions of the target gene were then cloned
4
ꢁ1 ꢁ1
ꢁ1 ꢁ1
TM
which was reported as 5.9 ꢀ 10 M
s
(i.e., 59 mM
s
) in
by two runs of DNA walking with the SpeedUp Premix Kit II. All
the reduction of the same substrate.16 The optima of pH 7.0 and
DNA samples were sent to Invitrogen (Shanghai, China) for
sequencing.
3
0 °C for BbMPD to catalyze the D-Fru6P reduction were similar
30
to those for most of the fungal MPDs except the C. neoformans
The gene-encoding protein sequence was compared with those
of all fungal MPDs in the GenBank protein database by BLASTP
analysis online (http://www.ncbi.nlm.nih.gov/). Protein sequence
MPD, which was most active at the higher temperature of
1
9
3
7 °C. The new enzyme was also featured with the same depen-
35
dence on NADH in the reductive reaction as were the MPDs of A.
alignment was carried out using the software MEGA 4.0, generat-
ing a phylogenetic tree for the aligned MPD sequences from differ-
ent fungi.
6
,16
fumigatus and A. niger.
BbMPD falls in the same MPD group of B. fuckeliana, C. globosum,
E. rubrum, M. grisea and N. crassa with 71–74% sequence identity.
Such long-chain dehydrogenases and reductases are featured with
a conserved coenzyme-binding domain (GXGXXG) at the N-termi-
4.3. Construction and transformation of expression plasmid
3
1
nus, and their catalytic activities are generally independent of
metal cofactors. Perhaps for this reason, the BbMPD activity was
not affected significantly by including <50 mM EDTA, a metal che-
The RNA of B. bassiana 2860 was isolated from 2-day colonies
grown on SDAY plates. Reverse transcription was performed in
20
l
L of reaction buffer containing 2
l
L of RNA and 1
l
L of each
0
late, in the
D
-Fru6P reduction system. This is different from what
of the reagents oligo(dT)17 primer [5 -GACTCGAGTCGACATCG
A(T)17-3 ], dNTPs, Rnase inhibitor and reverse transcriptase. The
0
was observed on the C. neoformans MPD with a zinc-binding do-
19
main (CXXCXXCXC) not located at the N-terminus. The inhibitary
final mixture was sequentially incubated at 25 °C for 5 min, 42 °C
for 60 min and 70 °C for 15 min. The resultant cDNA was used as
a PCR template. The target gene BbMPD was then PCR-amplified
2
+
2+
effects of Cu and Zn on the enzyme activity were non compet-
itive, because the suppression occurred at the low concentration of
2
+
2+
0
2
mM Cu or Zn . The noncompetitive inhibition was in accord
using the pfu DNA polymerase, the sense primer BbMPD-N (5 -GG
3
2
0
with that of an MPD from the red algae Caloglossa continua, but
GAATTCCATATGGCTCCCAAGGCTGTTCATTTC-3 ) with NdeI restric-
2
+
0
it is clearly distinguished from the insignificant effects of Zn
tion site and the anti-sense primer BbMPD-X (5 -CCGCTCGAGC
2
+
19
0
and Mg observed for the C. neoformans MPD.
TCGCTGTCGGCCTGAAC-3 ) with XhoI. The PCR product digested
with NdeI/XhoI was inserted into the NdeI/XhoI-linearized pET-
2
9b(+), generating the plasmid pET-29b-BbMPD. This plasmid
4
4
. Experimental
was finally transformed into BL21, and putative transformants
were taken from colonies grown on the selective LB plates for
identification.
.1. Microbial strains, cultures and reagents
The fungal strain, B. bassiana 2860 (ARSEF accession number;
RW Holley Center for Agriculture and Health, Ithaca, NY, USA)
was grown on the plates of Sabouraud dextrose agar containing
4
.4. Expression and purification of BbMPD
A colony identified as a positive transformant by PCR was trans-
(
w/v) 4% glucose, 1% peptone, 2% agar and 1% yeast extract (SDAY).
ferred into 100 mL of kanamycin-inclusive LB medium and shaken
by 150 rpm for 5 h at 37 °C for the liquid culture to reach ca. 0.6 at
OD600. The expression of BbMPD was then induced for 16 h at 20 °C
by adding 0.5 mM IPTG to the LB culture. The cells in the culture
were harvested by a 10-min centrifugation at 12,000g. These were
then resuspended in 50 mL of 20 mM sodium phosphate (pH 7.3),
followed by 20-min sonication at 220–240 W in an ice bath (cycles
of 3 s running and 3 s interval). The sonicated suspension was then
centrifuged (12,000g) for 20 min at 4 °C. The supernatant was fil-
The strains E. coli DH5 and E. coli BL21 (DE3) pLysS from Invitro-
gen (Shanghai, China) were used for vector construction and pro-
tein expression, respectively. These were cultured at 37 °C in
a
Luria–Bertani (LB) medium containing 100
kanamycin mL in terms of their resistance type.
l
g ampicillin or 50
lg
ꢁ1
Restriction endonucleases, T4 DNA ligase, Taq DNA polymerase,
pfu DNA polymerase, reverse transcriptase from Promega (Madi-
TM
son, WI, USA) and DNA Walking SpeedUp
Premix Kit II from
Neuro-Hemin Biotech Co. (Hangzhou, China) were used for gene
cloning. The plasmid pET-29b(+) from Invitrogen was used for pro-
tein expression in BL21. Enzyme cofactors (NADH and NADPH) and
tered through a 0.22-lm membrane of Millex HPF PVDF (GE
Healthcare, Shanghai, China) and then loaded onto a HisTrap affin-
ity column, which was pre-equilibrated with the loading buffer
D
-fructose-6-phosphate (D-Fru6P) were purchased from Sigma (St.
(
5
pH 7.3) of 20 mM sodium phosphate, 5 mM imidazole and
00 mM NaCl. The expressed BbMPD was eluted by the same buf-
Louis, MO, USA).
fer (pH 7.3) containing 500 mM imidazole. The purified fraction
was finally desalted, pooled and analyzed through SDS–PAGE.
The concentration of the active protein was assayed using bovine
4
.2. Gene cloning and sequence analysis of BbMPD
+
The amino acid sequence of the NAD -dependent A. niger MPD
36
serum albumin as standard.
gene MPD (GenBank accession number: AAL89587) was retrieved
from the protein database of the National Center for Biotechnology
Information (NCBI). A BLASTP search of this sequence (http://
www.ncbi.nlm.nih.gov/BLAST/) generated similar sequences from
Alternaria alternate (AAQ63948), B. fuckeliana (ABC84125), N. crassa
4
.5. Activity assays of purified BbMPD
The activity of the purified BbMPD was spectrophotometrically
assayed at 30 °C for the OD340 change in the oxidation of NADH.
The standard reaction for the reduction of -Fru6P as a substrate,
(
EAA33240), Phaeosphaeria nodorum (AAT11122) and Paracoccidi-
D
oides brasiliensis (AAO47089). Such sequences were aligned with
the software ClustalW.33 To clone the gene BbMPD, two conserved
regions of the sequences were used to design the degenerate prim-
ers BbMPD-F (5 -AAYATHGGNCGNGGNTTYGTNGC-3 ) and BbMPD-
R (5 -CGNGGNACDATCCKRTCDATNGC-3 ). The genomic DNA of B.
unless mentioned otherwise, was in a total volume of 1 mL of solu-
tion containing 20 mM sodium phosphate buffer (pH 7.0), 0.3 mM
NADH, 2 mM
D-Fru6P and 10 lg of BbMPD. One unit of MPD activ-
0
0
ity was defined as 1
reduction reaction, and the total activity was expressed as U mg
l
mol of NADH consumed per min in the
0
0
ꢁ1
3
4
bassiana 2860 was extracted following a documented method.
protein.

5
4
Z.-L. Wang et al. / Carbohydrate Research 345 (2010) 50–54
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reduction of -Fru6P were determined in the same 1-mL reaction
system containing 0.05–5.0 mM -Fru6P (x) and 0.3 mM NADH.
The Michaelis–Menten constant (K ) and the maximal reaction
velocity (Vmax) were estimated by the nonlinear fitting of the ob-
served activities (y) to the equation y = Vmaxx/(K + x) using the
software GRAPHPAD Prism (San Diego, CA, USA). The catalytic effi-
ciency was then computed as kcat/K , where kcat was the ratio of
the fitted Vmax over the concentration of the tested enzyme.
Six metal ions, three reducing agents and one metal chelate (Ta-
ble 1) were assayed separately for their effects on the BbMPD
activity in the reaction volume. The optimal temperature and pH
8.
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