Nucleotide sequences of cDNA clones encoding ferrochelatase from barley and cucumber.

Abstract

Ferrochelatase is the enzyme that catalyzes the last step in the heme biosynthetic pathway. It catalyzes the insertion of iron (Fez+) into the tetrapyrrole ring of protoporphyrin IX to generate protoheme. Recently, we isolated mutants of Escherichia coli that were sensitive to visible light (Miyamoto et al., 1991; Nakahigashi et al., 1991), and these mutants [designated visA (=hemH) mutants] were shown to be the result of a mutation in the visA (=hemH) gene, which encodes ferrochelatase. The nucleotide sequences of genes that encode ferrochelatases from six different sources are available (E. coli, Miyamoto et al., 1991; Bacillus subtilis, Hansson and Hederstedt, 1992; Bradyrhizobium japonicum, Frustaci and O’Brian, 1992; yeast, Labbe-Bois, 1990; mouse, Taketani et al., 1990; human, Nakahashi et al., 1990), but none have been reported from higher plants. Here we report the isolation and the sequences of cDNAs that encode a ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) from barley (Hordeum vulgare L. cv Svalof‘s Bonus) and a ferrochelatase from cucumber (Cucumis sativus L. cv Aonagajibai). Both cDNAs were isolated from libraries in phage vector Xgtll. To screen the clones, we used a deletion mutant of E. coli (strain VSZOO), in which part of the gene for ferrochelatase had been deleted. Because VS200 mutants produce no heme, they cannot grow well even on complete medium. Therefore, we isolated large colonies of VS200 cells that formed among the tiny colonies after infection by the clones in the cDNA libraries of barley and cucumber. Phages obtained from the large colonies were tested for their ability to improve the poor growth of VS200 bacteria. To determine the nucleotide sequence of the cDNA insert in each selected phage clone, an EcoRI fragment was recloned into plasmid vector pUCl18 and serial-deletion clones were constructed by the step-wise method of Yanisch-Perron et al. (1985). The insert of a phage clone from the barley cDNA library was 1728 bp long and included a single open reading frame that encoded a polypeptide of 484 amino acids (Table I). Coincidentally, the insert from the cucumber cDNA library