CLONING AND CHARACTERIZATION OF A PLASTIDAL AND A MITOCHONDRIAL ISOFORM OF TOBACCO PROTOPORPHYRINOGEN-IX OXIDASE

Citation
I. Lermontova et al., CLONING AND CHARACTERIZATION OF A PLASTIDAL AND A MITOCHONDRIAL ISOFORM OF TOBACCO PROTOPORPHYRINOGEN-IX OXIDASE, Proceedings of the National Academy of Sciences of the United Statesof America, 94(16), 1997, pp. 8895-8900
Citations number
49
Categorie Soggetti
Multidisciplinary Sciences
ISSN journal
00278424
Volume
94
Issue
16
Year of publication
1997
Pages
8895 - 8900
Database
ISI
SICI code
0027-8424(1997)94:16<8895:CACOAP>2.0.ZU;2-Q
Abstract
Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mito chondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids, We cloned two different, full-length tobacco c DNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy, The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus s ubtilis hemY, and to mammalian sequences encoding protoporphyrinogen I X oxidase, One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the s econd cDNA encodes a protein of 504 amino acid residues, Both deduced protein sequences share 27.2% identical amino acid residues, The first in vitro translated protoporphyrinogen IX oxidase could be translocat ed to plastids, and the approximately 53-kDa mature protein was detect ed in stroma and membrane fraction, The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression o f both genes during tobacco plant development, greening of young seedl ings, and diurnal and circadian growth, The mature plastidal and the m itochondrial isoenzyme were overexpressed in E. coli, Bacterial extrac ts containing the recombinant mitochondrial enzyme exhibit high protop orphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide, T he data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochond ria.