Analysis of a ferric leghemoglobin reductase from cowpea (Vigna unguiculata) root nodules

Ferric leghemoglobin reductase (FLbR), an enzyme reducing ferric leghemoglo bin (Lb) to ferrous ib, was purified from cowpea (Vigna unguiculata) root n odules by sequential chromatography on hydroxylapatite followed by Mono-V H R5/5 FPLC and Sephacryl S-200 gel filtration. The purified cowpea FLbR had a specific activity of 216 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for cowpea Lb(3+) and a specific activity of 184 nmol Lb(2+)O(2) formed min (-1) mg(-1) of enzyme for soybean Lb(3+). A cDNA clone of cowpea FLbR was o btained by screening a cowpea root nodule cDNA library. The nucleotide sequ ence of cowpea FLbR cDNA exhibited about 88% similarity with soybean (Glyci ne max) FLbR and 85% with pea (Pisum sativum) dihydrolipoamide dehydrogenas e (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 dedu ced amino acid sequences of cowpea FLbR, soybean FLbR, pea DLDH and other e nzymes in the family of the pyridine nucleotide-disulfide oxido-reductases. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.