Plant Science 154 (2000) 161 – 170
www.elsevier.com/locate/plantsci
Analysis of a ferric leghemoglobin reductase from cowpea (Vigna
unguiculata) root nodules
Peng Luan a, Elena Aréchaga-Ocampo b, Gautam Sarath a, Raúl Arredondo-Peter b,
Robert V. Klucas a,*
b
a
Department of Biochemistry, The Beadle Center, Uni6ersity of Nebraska-Lincoln, Lincoln, NE 68588 -0664, USA
Centro de In6estigación sobre Fijación de Nitrógeno, Uni6ersidad Nacional Autónoma de México, Apartado Postal 565 -A, 62210 Cuerna6aca,
Morelos, Mexico
Received 2 September 1999; received in revised form 9 December 1999; accepted 15 December 1999
Abstract
Ferric leghemoglobin reductase (FLbR), an enzyme reducing ferric leghemoglobin (Lb) to ferrous Lb, was purified from cowpea
(Vigna unguiculata) root nodules by sequential chromatography on hydroxylapatite followed by Mono-Q HR5/5 FPLC and
Sephacryl S-200 gel filtration. The purified cowpea FLbR had a specific activity of 216 nmol Lb2 + O2 formed min − 1 mg − 1 of
enzyme for cowpea Lb3 + and a specific activity of 184 nmol Lb2 + O2 formed min − 1 mg − 1 of enzyme for soybean Lb3 + . A
cDNA clone of cowpea FLbR was obtained by screening a cowpea root nodule cDNA library. The nucleotide sequence of cowpea
FLbR cDNA exhibited about 88% similarity with soybean (Glycine max) FLbR and 85% with pea (Pisum sati6um) dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) cDNAs. Conserved regions for the FAD-binding site, NAD(P)H-binding site,
and disulfide active site were identified among the deduced amino acid sequences of cowpea FLbR, soybean FLbR, pea DLDH
and other enzymes in the family of the pyridine nucleotide-disulfide oxido-reductases. © 2000 Published by Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Cowpea; Dihyrodrolipoamide dehydrogenase; Ferric leghemoglobin reductase; Leghemoglobin; Symbiotic nitrogen fixation; Vigna
unguiculata
1. Introduction
Leghemoglobins (Lbs) are important nodule
proteins that reversibly bind O2 and facilitate its
diffusion to the N2-fixing bacteroids in root nodules. This provides a flux of O2 for rhizobial
respiration, while maintaining O2 at a concentration that does not inactivate the nitrogenase complex [1]. Lb can exist in several different oxidation
states: ferrous (Lb2 + ), ferric (Lb3 + ) or ferryl form
(Lb4 + ), but only Lb2 + is functional. Slight
changes in the physiology of nodules, such as the
Abbre6iations: DLDH, dihydrolipoamide dehydrogenase; FLbR,
ferric leghemoglobin reductase; Lb, leghemoglobin.
* Corresponding author. Tel.: + 1-402-4722932; fax: + 1-4024727842.
E-mail address: [email protected] (R.V. Klucas)
presence of some metal ions, chelators, and toxic
metabolites (nitrite, superoxide radical, peroxides),
may cause the oxidation of functional Lb2 + into
the nonfunctional Lb3 + and Lb4 + [2]. Mechanisms must therefore exist in legume plants for
maintaining Lb in its functional Lb2 + state.
Enzymatic reduction of Lb3 + to Lb2 + has been
hypothesized to exist in legume nodules [3]. A
protein that reduces Lb3 + to Lb2 + was purified
from lupin nodules. It had a molecular weight of
60 kDa, contained FAD as a cofactor, used
NADH as the electron donor, methylene blue as
the electron carrier, and had Km values of 8.7 mM
for NADH and 10 mM for lupin Lb3 + [4,5].
Another protein, FLbR, was purified to homogeneity from soybean root nodules and further
characterized in our laboratory [6,7]. The purified
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PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 2 7 2 - 1
162
P. Luan et al. / Plant Science 154 (2000) 161–170
soybean FLbR was a homodimer with a molecular weight of 110 kDa, contained FAD as a
prosthetic group, and used NAD(P)H as the
electron donor to reduce Lb3 + . It also exhibited
diaphorase activity [6]. The enzyme required O2
at the micromolar levels for the reduction of
Lb3 + to Lb2 + in vitro, had Km values of 7 mM
for NADH, 9.5 mM for soybean Lb3 + , and a
Vmax value for soybean Lb3 + reduction of 499
nmol Lb2 + O2 formed min − 1 mg − 1 [7]. A cDNA
encoding soybean FLbR was cloned and sequenced [8] and subsequently overexpressed in
Escherichia coli [9]. Based on sequence homology, soybean FLbR was shown to be related to
a family of pyridine nucleotide-disulfide oxido-reductases, especially the DLDHs from various
sources [9].
The only FLbR that had been characterized to
date is from soybean root nodules. To investigate if this enzyme is present in other legumes
nodules, we used cowpea (Vigna unguiculata)
root nodules for our study. In this work we describe (1) the identification and purification of
FLbR from cowpea root nodules; (2) some of
the important physical and enzymatic properties
of cowpea FLbR; (3) the sequences of the
cDNA encoding cowpea FLbR; and (4) the comparison of deduced cowpea FLbR amino acids
sequence with soybean FLbR and other similar
proteins. The results revealed that the cowpea
FLbR is very similar to the soybean enzyme,
and indeed FLbR may be common to all
legumes root nodules.
2. Materials and methods
2.1. Chemicals
Chemicals were reagent or molecular biology
grade. Bio-Gel-hydroxylapatite, Bio-Gel P6-DG,
silver staining kit, and protein concentration assay kit were purchased from Bio Rad. Prepacked Mono-Q HR5/5 column, Sephacryl S-200
Super Fine (SF), Sephadex G-25 and G-75 were
from Pharmacia. Agarose, bacteria broth, Taq
DNA polymerase, PCR reagents and DNA ligation reagents were from Gibco-BRL. Hybridization materials were from Boehringer Mannheim.
Other chemicals were from Sigma and Fisher unless noted.
2.2. Purification of cowpea FLbR from root
nodules
Germinated cowpea seeds (V. unguiculata, cv.
Blackeye peas, 137-California No. 5) were
inoculated with Bradyrhizobium japonicum USDA
3456 before planting. The bacteria and plants were
grown as described by Becana et al. [10]. Cowpea
root nodules were harvested from 5 weeks old
plants, and stored at −90°C. All purification
steps were carried out at 4°C, essentially following
the procedure of Ji et al. [7]. Active fractions were
collected, pooled, made to a final concentration of
10% with glycerol and stored at −90°C.
Homogeneity of the sample after each purification
step was analyzed by 10% SDS-PAGE stained
using the Bio-Rad Silver-Stain Kit. Protein
concentration was determined using Bio Rad
protein micro-assay procedure and bovine serum
albumin as a standard.
2.3. Isolation of cowpea Lb and soybean Lb
Isolation and oxidization of cowpea and soybean Lb were as described earlier [10,11]. Lb
was stored as Lb3 + at −90°C until use.
2.4. FLbR acti6ity and diaphorase acti6ity assay
FLbR activity was assayed according to the
procedure used by Ji et al. [7] on a Milton Roy
Spectronic
3000
Array
spectrophotometer
equipped with kinetic acquisition software. The
absorbance change was converted to Lb2 + O2
formation rate using the Dn of 10.2 mM − 1 cm − 1
(Lb2 + O2 minus Lb3 + ) at 574 nm. FLbR specific
activity was expressed as nmol Lb2 + O2 formed
min − 1 mg − 1 of protein. Diaphorase activity was
assayed using DCPIP as described by Ji et al. [7].
Diaphorase specific activity was expressed as nmol
DCPIP reduced min − 1 mg − 1 of protein.
2.5. N-Terminal sequencing
Approximately 20 pmol (2 mg) of purified
cowpea FLbR was separated by 10% SDS-PAGE
and then transferred to a polyvinylidene difluoride
membrane (Millipore). The membrane was stained
with Amido Black solution to visualize proteins.
The band corresponding to the FLbR was excised
from the membrane and sequenced on an ABI
P. Luan et al. / Plant Science 154 (2000) 161–170
Instruments Procise 494 Protein Sequencer at the
Protein Core Facility at University of NebraskaLincoln.
2.6. Characterization of cowpea FLbR
Molecular weight of native FLbR was determined using a gel filtration method [12] on a
Sephacryl S-200 SF column. The cowpea FLbR
activity was assayed in different pH buffers to
determine its optimal pH, using cowpea Lb3 + as the
substrate. The buffers used were: 50 mM potassium
phosphate at pH 3.7, 4.8; 50 mM MES at pH 5.5,
6.0, 6.5, 7.0; and 50 mM Tris–HCl at pH 7.5, 8.5.
Reduction of cowpea and soybean Lb3 + by
cowpea FLbR was followed spectrophotometrically
using an assay mixture that contained 50 mM
potassium phosphate, pH 6.5, 2 mg cowpea FLbR,
500 mM NADH, and various concentrations of
cowpea or soybean Lb3 + (0–50 mM) in a final
volume of 1 ml. Km and Vmax values for cowpea or
soybean Lb3 + were determined by fitting the data
to the Michaelis – Menten equation or Lineweaver–
Burk plot using commercial software (Grafit version 2.0). The Km value for NADH was determined
as above using reaction mixture that contained 50
mM potassium phosphate, pH 6.5, 50 mM cowpea
Lb, 2 mg cowpea FLbR, and various concentrations
of NADH (0 – 500 mM).
2.7. Construction of a cowpea root nodule cDNA
library
Poly(A+) mRNAs were isolated from 0.5 g of
frozen cowpea root nodules, and used as a template
to generate cDNAs. The cDNAs were ligated with
a EcoRI/NotI adapter and subsequently ligated to
the lgt11 arms following the supplier’s procedures
(Pharmacia). The cDNA-lgt11 constructs were
packaged into commercial available phage particles
and then amplified using E. coli strain Y1090
following the suppliers procedures (Pharmacia
Ready-To-Go Lambda Packaging Kit). The cDNA
library was divided into 2-ml aliquots and stored at
− 90°C.
2.8. Obtaining the cDNA sequence of cowpea
FLbR
Two primers, named FLbR Fwd (5%AAATCTCTGTAGACACCA-3%) and FLbR Rvs
163
(5%-GCCTTAGCTCTGCTATTA-3%), were designed based on the soybean FLbR cDNA sequence
(positions 485–502 and 1272–1289, respectively,
from Ji et al. [8]). They were used for the amplification of an internal FLbR fragment by PCR at high
stringency (annealing temperature of 55°C for 1
min) using aliquots of a cowpea root nodule cDNA
library (above) as template. PCR products were
resolved in a 1.2% agarose gel, the band of the
expected size (800 bp) was cut out and extracted
in 10 ml of sterile water using a Gene Clean II Kit
(Bio 101) following the supplier’s manual. The
extracted DNA was cloned in the pCR2.1 vector
(Invitrogen) and sequenced (see below), and then
used as a probe for screening the cDNA library
from cowpea nodules as described by Sambrook et
al. [13]. Positive plaques were picked and eluted into
50 ml of SM solution [13].
Two primers, named l Forward and l Reverse,
were used for the amplification of inserts from the
positive plaques essentially as described by Arredondo-Peter et al. [14]. Amplified inserts were
resolved in a 0.8% agarose gel, extracted using the
Gene Clean II Kit, cloned in the vector pCR2.1, and
subsequently transformed in E. coli InvaF% cells
(Invitrogen) for DNA sequencing. The FLbR
cDNA inserts were fully-sequenced in both directions at the DNA Sequencing Facility of the University of Nebraska-Lincoln. Cowpea FLbR
nucleotide sequence and the deduced polypeptide
sequence were searched for similarity in databases
(GeneBank, EMBL, SwissProt) using programs of
the GCG package (Wisconsin Computer Group,
version 8.0).
3. Results and discussion
3.1. Purification of cowpea FLbR
FLbR was purified to homogeneity from crude
extracts of cowpea root nodules by a four-step
procedure involving ammonium sulfate precipitation, hydroxylapatite, ion exchange and size-exclusion chromatography. The purified cowpea FLbR
had a specific activity of 216 nmol Lb2 + O2 min − 1
mg − 1 of protein, which corresponded to a purification of approximately 1000-fold and a yield of 16%
(Table 1). The hydroxylapatite column was an
important step in the purification which resulted a
60-fold increase in specific activity of FLbR al-
P. Luan et al. / Plant Science 154 (2000) 161–170
164
though approximately 50% of the total activity
was lost. A 40-fold decrease in the ratio of diaphorase to FLbR activity (Table 1) indicated that
many of the other contaminating diaphorases were
removed at this step. On the FPLC anion exchange column, FLbR was eluted as a sharp peak
at about 15% of NaCl gradient (150 mM NaCl,
Fig. 1). The specific activity of the purified cowpea
FLbR was about 50% of that reported for the
soybean enzyme [7]. The difference in the specific
activities between the cowpea and soybean FLbRs
could result from inherent differences in the two
enzymes.
3.2. Homogeneity and molecular weight analysis
of cowpea FLbR
Samples collected from each purification step
were subjected to 10% SDS-PAGE and silver
staining (Fig. 2). The sample after the Sephacryl
S-200 step (lane 4) exhibited a single distinct
protein band of about 55 kDa. The molecular
weight for native cowpea FLbR was estimated to
be 110 kDa using the Sephacryl S-200 SF column
(data not shown) and thus appears to be a homodimer. These molecular weights were similar to
those reported for soybean FLbR [7].
Table 1
Purification and specific activities of FLbR from cowpea nodule cytosola
Steps
Total protein DCIP reductase (A) specific activity
(mg)
(U mg−1)b
Crude extract 893
G-25
607
Hydroxyl4.5
apatite
Mono-Q
0.31
S-200
0.15
187
254
368
2289
2565
FLbR (B) specific activity Total FLbR
(U mg−1)c
activity (U)
0.22
0.29
17.3
162
216
Ratio A/Bd
194
176
78
850
875
21.3
50
32
14.1
11.9
a
Purification steps are described in Section 2.
One unit is defined as 1 nmol of DCPIP reduced per min.
c
One unit is defined as 1 nmol of Lb2+O2 formed per min.
d
This is the ratio of DCPIP reductase (diaphorase) specific activity to FLbR specific activity.
b
Fig. 1. Separation of FLbR by an ion-exchange FPLC on a Mono-Q HR5/5 column. The column was equilibrated with Buffer
A (50 mM Tris–HCl, pH 7.5), and eluted with a linear NaCl gradient from 0 to 35% of Buffer B (1 M NaCl, 50 mM Tris–HCl,
pH 7.5) in 50 ml total volume (Buffer A plus Buffer B) at a flow rate of 1 ml min − 1. Cowpea FLbR was eluted at about 15%
of the gradient (150 mM NaCl, as indicated by the arrow).
P. Luan et al. / Plant Science 154 (2000) 161–170
165
3.3. Characterization of cowpea FLbR
Fig. 2. SDS-Polyacrylamide gel electrophoresis of cowpea
FLbR fractions during purification. The gel was silver-stained
to detect proteins. Lane 1, G-25 fraction (50 mg protein); lane
2, hydroxylapatite fraction (10 mg protein); lane 3, Mono-Q
fraction (5 mg protein); lane 4, Sephacryl S-200 fraction (3 mg
protein); and lane 5, 10-kDa ladder (10 mg protein).
The first 20 amino acids on the N-terminus of
the cowpea FLbR were determined to be: A-S-GS-D-E-N-D-V-V-V-I-G-G-G-P-G-G-Y-V. When
this sequence was compared to sequences deposited in the GCG database, it was found to be
100% identical with soybean FLbR, and 95.2%
identical with pea DLDH. This indicates that the
FLbRs and DLDHs are probably highly conserved in legumes.
The purified cowpea FLbR reduced both
cowpea Lb3 + and soybean Lb3 + at comparable
rates in the presence of NADH, forming Lb2 + O2
under aerobic conditions. The enzyme had maximum Lb3 + reduction activity at pH 6.5, and had
no activity at pH values below pH 4.8 or above
pH 8.5. Reactions as a function of time were
monitored spectrometrically for the reduction of
Fig. 3. Reduction of cowpea Lb3 + by FLbR. Reaction mixture contained 50 mM potassium phosphate buffer, pH 6.5, 500 mM
NADH, 50 mM cowpea Lb3 + , and 2 mg purified cowpea FLbR. The reaction was carried out in a 1-ml cuvette at room
temperature, and the spectra were scanned at 5-min intervals.
P. Luan et al. / Plant Science 154 (2000) 161–170
166
Table 2
Kinetic properties of cowpea FLbR, soybean FLbR and pig
DLDH
Km (mM)
Cowpea FLbR
10.4
Cowpea
Lb3+
Soybean
12.4
Lb3+
NADH
57
Soybean FLbR a,b
Soybean
9.2
Lb3+
NADH
46
Lipoamide 716
Pig DLDH b,c
Soybean
28
Lb3+
NADH
73
Lipoamide 430
Vmax
Kcat (s−1)
(U mg−1)
Kcat/Km
(M−1 s−1)
221a
3.1
298
185a
2.5
201
NA
NA
450
16 000
NA
350
25 000
NA
6.2
220
NA
1.1
344
NA
NA
674
4.8
NA
40
4.5
NA
a
One enzyme unit is defined as 1 nmol of Lb2+O2 formed
min−1 mg−1.
b
Determined by Ji et al. [9].
c
One enzyme unit is defined as 1 nmol NADH oxidized
min−1 mg−1.
cowpea Lb3 + (Fig. 3) and soybean Lb3 + . The
absorption at 541 and 574 nm, which was contributed by Lb2 + O2, increased as a function of
time, whereas the absorption at 627 nm resulting
from Lb3 + decreased. Two isosbestic points at 525
and 588 nm were present in the reduction of
cowpea Lb3 + . The spectroscopic characteristics
for the reduction of cowpea Lb3 + by cowpea
FLbR were similar to those reported for the soybean enzyme [7,9].
The Km and Vmax values of cowpea FLbR for
cowpea Lb3 + reduction were determined to be
10.4 mM and 221 U mg − 1, respectively. The corresponding Km and Vmax values for soybean Lb3 +
reduction by cowpea FLbR were determined to be
12.4 mM and 185 U mg − 1, respectively. The Km
value for NADH by the cowpea FLbR was determined to be 57 mM (Table 2). These values are
similar to those reported for soybean FLbR [7,9].
The Kcat (6.2 s − 1) and Kcat/Km values (674 M − 1
s − 1) of soybean enzyme were about twofold
greater than the cowpea enzyme, 3.1 s − 1 and 298
M − 1 s − 1, respectively (Table 2). The Km value of
pig DLDH for soybean Lb3 + (28 mM) was more
than twofold higher than those of cowpea FLbR
(12.4 mM) and soybean FLbR (9.2 mM) for Lb3 +
(Table 2). The catalytic efficiencies (Kcat/Km) of
cowpea FLbR for cowpea Lb3 + (298 M − 1 s − 1)
and soybean FLbR for soybean Lb3 + (674 M − 1
s − 1) were about eight- and 17-fold higher than
that of pig DLDH (40 M − 1 s − 1) (Table 2). Conversely, the affinity of pig DLDH for lipoamide
was higher than the two FLbRs. These data suggest that dehydrogenation of lipoamide is most
efficiently catalyzed by DLDH, and reduction of
Lb3 + is most efficiently catalyzed by FLbRs.
Thus, although DLDH and FLbRs exhibit many
similarities in their enzyme kinetics, they are expected to function differently in vivo.
3.4. cDNA sequence of cowpea FLbR
A cDNA fragment of 802 bp was amplified
from a cowpea root nodule cDNA library by PCR
using the FLbR Fwd and FLbR Rvs primers.
DNA sequencing showed that the 802-bp PCRfragment encoded for a cowpea FLbR. Thus, this
fragment was PCR-labelled by incorporating Dig11-dUTP [15] and used as probe for screening the
above cowpea cDNA library. About 5×105 recombinant phage plaques were screened, resulting
in seven positive plaques that were numbered c1
through c7. The inserts from these plaques were
amplified by PCR using l primers [14]. Inserts
from plaques c4 and c6 (named clones 4 and 6)
were about 1.8 kb in length and thus they were
subcloned for DNA sequencing. Sequence comparison showed that clones 4 and 6 are copies of
the same cDNA, and that they code for the same
protein. Comparison with sequences deposited in
the GenBank database revealed that clones 4 and
6 have high similarity ( \80%, see below) to the
soybean FLbR, and other DHLD sequences, and
thus that they code for a cowpea FLbR.
The complete cowpea FLbR cDNA consists of
1797 nucleotides with an open reading frame of
1569 bases (Fig. 4). The poly(A+)11 tail is present
150 bases after the stop codon position. The deduced polypeptide sequence has 523 amino acids
with a predicted molecular weight of 56 kDa,
which corresponds well to the observed value of 55
P. Luan et al. / Plant Science 154 (2000) 161–170
167
Fig. 4. Nucleotide and deduced amino acid sequences of cowpea FLbR. The experimentally determined N-terminal amino acid
sequence of the purified enzyme is underlined in the amino acid sequence. The deduced leader sequence is indicated in italics
(amino acid residues 1–30). The cysteine residues hypothesized to form the active disulfide bond are asterisked.
168
Table 3
Conserved domains in cowpea FLbRa
in the FAD-binding domain
37–66
N D V V V
·
·
37–66
·
·
·
I
38–67
·
·
·
·
42–71
T ·
A ·
·
28–56
– ·
·
·
I
6–35
T Q ·
·
·
21–50
Y ·
Y L ·
5–34
T ·
T I
A
I
·
·
·
·
L
·
·
G
·
·
·
·
·
·
·
G
·
·
S
·
·
·
·
G
·
·
·
·
·
·
·
P
·
·
·
·
·
S
S
G
·
·
·
A
A
·
·
G
·
·
·
·
·
·
·
Y
·
·
·
·
·
L
I
V
·
·
·
·
S
A
A
A
·
·
·
·
·
S
S
A
·
·
·
·
·
·
I
I
·
·
·
·
F
R
N
K
·
·
·
·
R
R
R
A
·
·
·
·
C
·
·
S
A
A
A
A
A
A
A
Q
·
·
·
·
D
E
M
L
·
·
·
·
·
·
Y
G
·
·
·
·
·
·
·
L
·
F
F
F
·
A
Q
K
·
·
·
N
E
R
·
T
·
·
·
·
·
A
C
T
·
·
V
A
V
A
A
C
·
·
·
·
I
V
L
I
·
·
·
V
V
V
·
E
·
·
·
·
·
·
·
Conserved sequences
FLbR-cowpea
FLbR-soybean
DLDH-pea
DLDH-human
DLDH-yeast
DLDH-E. coli
GSHR-human
GSHR-E. coli
in the disulfide active
67–96
K R G
67–96
·
·
·
69–98
·
·
·
73–102 ·
N E
58–87
·
·
·
37–66
R Y N
52–80
S H K
36–64
·
N E
Conserved sequences in the NAD(P)H
FLbR-cowpea
196–225 S S
FLbR-soybean
196–225 ·
·
DLDH-pea
198–227 ·
·
DLDH-human
203–232 ·
·
DLDH-yeast
194–223 ·
·
DLDH-E. coli
163–192 D ·
GSHR-human
173–202 D ·
GSHR-E. coli
161–190 T ·
a
site
T
·
A
·
K
·
–
–
L
·
·
·
·
·
·
·
G
·
·
·
·
·
·
·
G
·
·
·
·
·
·
·
T
·
·
·
·
V
·
·
Cc
·
·
·
·
·
·
·
L
·
·
·
·
·
V
V
N
·
·
·
·
·
·
·
V
·
·
·
·
·
·
·
G
·
·
·
·
·
·
·
Cc
·
·
·
·
·
·
·
I
·
·
·
·
·
V
V
P
·
·
·
·
·
·
·
S
·
·
·
·
·
·
K
K
·
·
·
·
·
·
·
A
·
·
·
·
·
T
V
L
·
·
·
·
·
M
M
L
·
·
·
·
·
W
W
H
·
·
N
N
·
N
·
S
·
·
N
N
V
T
A
S
·
·
·
·
A
A
A
H
·
·
·
·
K
V
Q
M
·
·
Y
L
V
H
I
Y
·
·
·
F
I
S
R
H
·
·
·
·
E
E
E
E
·
·
M
Q
·
F
A
A
·
·
·
M
·
M
I
domain
T G
·
·
·
·
·
·
·
·
·
S
D ·
D ·
A
·
·
·
·
·
F
F
L
·
·
·
·
·
F
F
A
·
·
S
S
E
·
Q
L
·
·
·
·
·
·
·
T
S
S
K
K
K
P
E
E
·
·
K
·
·
A
·
I
·
·
V
·
V
L
L
P
·
·
·
·
·
·
·
K
·
·
E
·
E
E
G
K
·
·
·
R
R
R
R
L
·
·
M
·
·
V
S
V
·
·
·
T
L
A
·
V
·
·
·
I
·
·
I
I
·
·
·
·
M
V
V
G
·
·
·
·
·
·
·
A
·
·
·
G
G
·
S
G
·
·
·
·
·
·
·
Y
·
·
V
I
I
·
·
I
·
·
·
·
·
·
·
G
·
·
·
·
·
A
A
L
·
·
V
·
·
V
V
E
·
·
·
·
·
·
·
M
·
·
L
·
·
L
·
G
·
·
·
·
·
A
A
S
·
·
·
·
T
G
G
V
·
·
·
·
·
·
I
Identical amino acids are shown as dots, gaps are shown as dashes.
Sequences are cited from Ji et al. [8].
c
Hypothesized disulfide cysteines.
b
P. Luan et al. / Plant Science 154 (2000) 161–170
Conserved sequences
FLbR-cowpea
FLbR-soybeanb
DLDH-peab
DLDH-humanb
DLDH-yeastb
DLDH-E. coli b
GSHR-humanb
GSHR-E. coli b
P. Luan et al. / Plant Science 154 (2000) 161–170
kDa. By comparing the deduced N-terminal sequence to the mature protein sequence, we found
the existence of a 32-amino acid leader peptide.
This leader peptide is rich in basic and hydroxylated amino acids, and deficient in acidic residues
which is highly similar to that of soybean FLbR
[9]: the first 20 amino acids are identical, and only
four amino acids are different in the last 10 amino
acids.
Comparison of the sequence of cowpea FLbR
cDNA to known sequences revealed striking similarities to soybean FLbR cDNA [8] (88%), pea
DLDH (EC 1.8.1.4) gene [16] (85%) and other
DLDH genes [17,18], glutathione reductase (EC
1.6.4.2) [19], and mercuric reductase (EC 1.16.1.1)
[20] (20 – 60%). All of these enzymes belong to the
pyridine nucleotide-disulfide oxido-reductase family [21]. Of the 523 deduced amino acids of cowpea
FLbR, 445 were identical to soybean FLbR [8];
441 identical to pea DLDH [16]; 275 identical or
less to other enzymes in this family [17–20].
Pileup (GCG package) analyses of the amino
acid sequences for the FLbR and the pyridine
nucleotide-disulfide oxido-reductase family enzymes [21]showed that important residues and
functional domains for FAD-binding, disulfide active site and NAD(P)H-binding were highly conserved (Table 3). The FAD-binding domain in
cowpea FLbR is essentially identical (residue 37–
66) to soybean FLbR, only three residues different
from pea DLDH, and 6–18 residues different
from the other enzymes in this class (Table 3). The
disulfide-active site of cowpea FLbR is proposed
to be located from residue 67 to 96, which is
identical to soybean FLbR, one amino acid different from pea DLDH, and 6–17 amino acids different from the others. The region for the
NAD(P)H-binding domain in cowpea FLbR is
identified from residue 196 to 225, which is one
residue different from soybean FLbR and pea
DLDH, and 9 – 18 residues different from the others. All of these sites are located on the N-terminal
half of the protein. The presence of a high homology in the functional domains and cofactor-binding domains suggests a similar origin and enzyme
mechanism for these proteins. However, the kinetic constants and catalytic efficiencies for Lb3 +
reduction by the FLbRs are very different from
DLDHs, indicating that the two are not identical
and may have different mechanisms. The existence
of FLbR in cowpea and soybean suggests that this
169
enzyme may be common to all legumes. As hypothesized for soybean FLbR [6,11], FLbRs in
cowpea and other legumes probably reduce Lb3 +
in vivo to maintain adequate levels of functional
Lb2 + form.
Acknowledgements
This work was supported in part by Grants
from the National Science Foundation (no. OSR92552255), and the U.S. Department of Agriculture Cooperative State Research, Education and
Extension Service (no. 95-37305-2441) to R.V.
Klucas, the Center for Biotechnology, University
of Nebraska-Lincoln funded through the Nebraska Research Initiative to Gautam Sarath, and
the Consejo Nacional de Ciencia y Tecnologı́a
(project number 25229-N), México, to Raúl
Arredondo-Peter.
References
[1] C.A. Appleby, Leghemoglobin and Rhizobium respiration, Annu. Rev. Plant Physiol. 35 (1984) 443 – 478.
[2] M. Becana, R.V. Klucas, Oxidation and reduction of
leghemoglobin in root nodules of leguminous plants,
Plant Physiol. 98 (1992) 1217 – 1221.
[3] C.A. Appleby, Properties of leghaemoglobin in 6i6o, and
its isolation as ferrous oxyleghaemoglobin, Biochim. Biophys. Acta 188 (1969) 222 – 229.
[4] V.L. Kretovich, S.S. Melik-Sarkisyan, N.F. Bashirova,
A.F. Topunov, Enzymatic reduction of leghemoglobin in
lupin nodules, J. Appl. Biochem. 4 (1982) 209 – 217.
[5] L.I. Golubeva, A.F. Topunov, S.S. Goncharov, K.B.
Aseeva, V.L. Kretovich, Production and properties of a
homogeneous preparation of metleghemogloin reductase
of lupine root nodule cytosol, Biokhimiya 53 (1998)
1478 – 1482 English translation.
[6] L.L. Saari, R.V. Klucas, Ferric leghemoglobin reductase
from soybean root nodules, Arch. Biochem. Biophys. 231
(1984) 102 – 113.
[7] L. Ji, S. Wood, M. Becana, R.V. Klucas, Purification
and characterization of soybean root nodule ferric
leghemoglobin reductase, Plant Physiol. 96 (1994) 32–37.
[8] L. Ji, M. Becana, G. Sarath, R.V. Klucas, Cloning and
sequence analysis of a cDNA encoding ferric
leghemoglobin reductase from soybean nodules, Plant
Physiol. 104 (1994) 453 – 459.
[9] L. Ji, M. Becana, G. Sarath, L. Shearman, R.V. Klucas,
Overproduction in Escherichia coli and characterization
of soybean ferric leghemoglobin reductase, Plant Physiol.
106 (1994) 203 – 209.
[10] M. Becana, M.L. Salin, L. Ji, R.V. Klucas, Flavin-mediated reduction of ferric leghemoglobin from soybean
nodules, Planta 183 (1991) 575 – 583.
170
P. Luan et al. / Plant Science 154 (2000) 161–170
[11] H.K. Jun, G. Sarath, F.W. Wagner, Detection and
purification of modified leghemoglobins from soybean
root nodules, Plant Sci. 100 (1994) 31–40.
[12] M.G. Murray, C. Ross, Molecular weight estimations of
some pyrimidine-metabolizing enzymes from pea cotyledons by gel filtration, Phytochemistry 10 (1971) 2645 –
2648.
[13] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, 1989, pp. 2.60 –
2.121.
[14] R. Arredondo-Peter, A. Bonic, G. Sarath, R.V. Klucas,
Rapid PCR-based detection of inserts from cDNA libraries using phage pools or direct phage plaques and
lambda primers, Plant Mol. Biol. Rep. 13 (1991) 138 –
146.
[15] Y.H. Lu, S. Negré, P. Leroy, M. Bernard, PCR-mediated
screening and labeling of DNA from clones, Plant Mol.
Biol. Rep. 11 (1993) 345–349.
[16] S.R. Turner, R. Ireland, S. Rawsthorne, Purification and
primary amino acid sequence of the L subunit of glycine
decarboxylase. Evidence for a single lipoamide dehydrogenase in plant mitochondria, J. Biol. Chem. 267 (1992)
7745–7750.
.
[17] P.E. Stephen, H.M. Lewis, M.G. Darlison, J.R. Guest,
Nucleotide sequence of the lipoamide dehydrogenase of
E. coli K12, Eur. J. Biochem. 135 (1983) 519 – 527.
[18] J. Bourguignon, D. Macherel, M. Neuburger, R. Douce,
Isolation, characterization, and sequence analysis of a
cDNA clone encoding L-protein, the dihydrolipoamide
dehydrogenase component of the glycine cleavage system
from pea-leaf mitochondria, Eur. J. Biochem. 204 (1992)
865 – 873.
[19] R.L. Krauth-Siegel, R. Blatterspiel, M. Saleh, E. Schilts,
R.H. Schirmer, R. Untucht-Grau, Glutathione reductase
from human erythrocytes, the sequence of the NADPH
domain and of the interface domain, Eur. J. Biochem.
121 (1982) 259 – 267.
[20] R.A. Laddaga, L. Chu, T.K. Misra, S. Silver, Nucleotide
sequence and expression of the mercuric-resistance
operon from Staphylococcus aureus plasmid pI258, Proc.
Natl. Acad. Sci. USA 84 (1987) 5106 – 5110.
[21] C.H. Williams, Lipoamide dehydrogenase, glutathione
reductase, thioredoxin reductase and mercuric ion reductase-family of flavoenzyme transhydrogenases, in: F.
Muller (Ed.), Chemistry and Biochemistry of Flavoenzymes, CRC Press, Boca Raton, FL, 1992, pp. 121–211.