Analysis of a ferric leghemoglobin reductase from cowpea (Vigna ...

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Table 3 Conserved domains in cowpea FLbR a Conserved sequences in the FAD-binding domain FLbR-cowpea 37–66 N D V V V I G G G P G G Y V A A I K A S Q L G L K T T C I E FLbR-soybean · · · · · · · · · · · · · · · · A b 37–66 · · · · · · · · · · · · · DLDH-pea I · · · · · b 38–67 · · · · · · · · · · · · · A · · · F · · · · · · DLDH-human 42–71 T · · · S · · · · · · · · · · · A · · · F · · V · · · b A · · DLDH-yeast 28–56 – · · · I · · · · · A · · · · · · · · A · · · F N · A · V · b DLDH-E. coli · L · · · · A b 6–35 T Q · · · · S · · F R C A D · · · E · V I V · GSHR-human L · · · · · S · · L A S · R R · A b 21–50 Y · Y E · · A R A A V V · GSHR-E. coli b 5–34 T · T I A · · · · S · · I A S I N R · A M Y · Q · C A L · · Conserved sequences in the disulfide active site L G G T CcL N V G Cc FLbR-cowpea 67–96 K R G T I P S K A L L H S S H M Y H E A FLbR-soybean 67–96 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · DLDH-pea 69–98 · · · A · · · · · · · · · · · · · · · · · · · · · · · · · · DLDH-human 73–102 · N E · · · · · · · · · · · · · · · · · · N N · · Y · · M · DLDH-yeast 58–87 · · · K · · · · · · · · · · · · · · · · · N N · · L F · Q M DLDH-E. coli 37–66 R Y N · · · · V · · · · · · · · · · · · · · V A K V I E · · GSHR-human 52–80 S H K – · · · · · V · · · · V · · · T M W N T A V H S E F M GSHR-E. coli 36–64 · N E – · · · · · V · · · · V · K · V M W · A A Q I R E A I Conserved sequences in the NAD(P)H domain FLbR-cowpea 196–225 S S T G A L A L T E I P K K L V V I G A G Y I G L E M G S V FLbR-soybean 196–225 · · · · · · · · S · · · · · · · · · · · · · · · · · · · · · DLDH-pea 198–227 · · · · · · · · S · · · · · · · · · · · · · · · · · · · · · DLDH-human 203–232 · · · · · · S · K K V · E · M · · · · · · V · · V · L · · · DLDH-yeast 194–223 · · · · · · S · K · · · · R · T I · · G · I · · · · · · · · DLDH-E. coli 163–192 D · · S · · E · K · V · E R · L · M · G · I · · · · · · T · GSHR-human 173–202 D · D · F F · · P A L · E R V A · V · · · · · A V · L A G · GSHR-E. coli 161–190 T · D · F F Q · E · L · G R S · I V · S · · · A V · · A G I a Identical amino acids are shown as dots, gaps are shown as dashes. b Sequences are cited from Ji et al. [8]. c Hypothesized disulfide cysteines. 168 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 Lb 3+ 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 P. Luan et al. / Plant Science 154 (2000) 161–170 169 enzyme may be common to all legumes. As hypothesized for soybean FLbR [6,11], FLbRs in cowpea and other legumes probably reduce Lb 3+ in vivo to maintain adequate levels of functional Lb 2+ form. Acknowledgements This work was supported in part by Grants from the National Science Foundation (no. OSR- 92552255), 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 Tecnología (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 io, 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.
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