Correct identification of the chloroplastic protoporphyrinogen IX oxidase N-terminus places the biochemical data in frame.
Maize (Zea mays) protoporphyrinogen IX oxidase (PPO: EC 1.3.3.4) possesses a chloroplast transit peptide (CTP) that delivers the enzyme into the chloroplast. The cleavage site yielding the mature protein was predicted by using the ChloroP software and by comparing conserved regions of the available plant PPO sequences. In parallel, the processed NH(2)-terminus of native PPO was identified experimentally by microsequencing the immunoprecipitated plant PPO from maize etioplasts. The cleavage sites identified using the bioinformatic approaches did not match the experimental result. The three sequences have been cloned and expressed in bacteria and their kinetics were compared in order to understand if the generated proteins had biochemically relevant differences. Recombinant PPO corresponding to the native PPO accumulated at higher level and was more active than the two homologues. A cysteine present in the CTP seems to be able to modify the redox state of the enzyme and to be responsible for the alteration of the kinetic features. In contrast, the sensitivity to different herbicides was unaffected by modifications at the NH(2)-terminus, suggesting that the mode of action is non-competitive and that the NH(2)-terminus is involved in the recognition of the natural substrate.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000378:metabolism*; D050768:Protoporphyrinogen Oxidase; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology
D017931:DNA Primers; D011994:Recombinant Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase