Plant Physiol, January 2002, Vol. 128, pp. 300-313
Molecular Cloning, Functional Characterization, and Subcellular
Localization of Soybean Nodule Dihydrolipoamide
Reductase1,2
Jose F.
Moran,*
Zhaohui
Sun,
Gautam
Sarath,
Raúl
Arredondo-Peter,
Euan K.
James,
Manuel
Becana, and
Robert V.
Klucas
Departamento de Nutrición Vegetal, Estación
Experimental de Aula Dei, Consejo Superior de Investigaciones
Científicas, 50080 Zaragoza, Spain (J.F.M., M.B.); Department
of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska
68588 (Z.S., G.S., R.V.K.); Laboratorio de Biofísica y
Biología Molecular, Facultad de Ciencias, Universidad
Autónoma del Estado de Morelos, Avenida Universidad 1001, Colonia
Chamilpa, 62210 Cuernavaca, Morelos, Mexico (R.A.-P.); and Centre for
High Resolution Imaging and Processing, MSI/WTB Complex, School of Life
Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom (E.K.J.)
Nodule ferric leghemoglobin reductase (FLbR) and leaf
dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules.
The cDNA is 1,779 bp in length and codes for a precursor protein
comprising a 30-residue mitochondrial transit peptide and a 470-residue
mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum
sativum) leaf mitochondria DLDH (89% identity). The cDNA
encoding the mature protein was overexpressed in Escherichia
coli. The recombinant enzyme showed Km
and kcat values for ferric leghemoglobin
that were very similar to those of DLDH. The transcripts of FLbR-2 were
more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an
unknown protein of approximately 70 kD localized in the nodule cytosol.
Immunogold labeling was also observed in the mitochondria, cytosol, and
bacteroids of soybean nodules. The similar biochemical, kinetic, and
immunological properties, as well as the high amino acid sequence
identity and mitochondrial localization, draw us to conclude that
FLbR-2 is soybean DLDH.
1
This work was supported by the National Science
Foundation (grant no. OSR-92552255) and the U.S. Department of
Agriculture-Cooperative State Research Education and Extension Service
(grant no. 95-37305-2441). Access to the BioCad workstation was
provided by the Center for Biotechnology at the University of Nebraska,
Lincoln, funded through the Nebraska Research Initiative. J.F.M. was
the recipient of a postdoctoral contract from the Ministry of Education
and Culture (Spain).
2
This is journal paper no. 12,643, Agricultural Research
Division, University of Nebraska.
*
Corresponding author; e-mail jmoran{at}eead.csic.es; fax
34-976-716145.
© 2002 American Society of Plant Physiologists
