Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

• Published in: Biochemical journal 2007-03, Vol.402 (3), p.575-580
• Main Authors: Heinemann, Ilka U, Diekmann, Nina, Masoumi, Ava, Koch, Michael, Messerschmidt, Albrecht, Jahn, Martina, Jahn, Dieter
• Format: Article
• Language: English
• Subjects: Amino Acids - genetics; Amino Acids - metabolism; Binding Sites; Kinetics; Models, Molecular; Mutation - genetics; Nicotiana - enzymology; Nicotiana - genetics; Protein Structure, Tertiary; Protoporphyrinogen Oxidase - chemistry; Protoporphyrinogen Oxidase - genetics; Protoporphyrinogen Oxidase - metabolism; Protoporphyrins - chemistry; Protoporphyrins - metabolism; Substrate Specificity
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj20061321

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent K(m) value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 muM, with a V(max) of 4.27 muM.min(-1).mg(-1) and a catalytic activity k(cat) of 6.0 s(-1). Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe(392) in substrate ring A stacking. Mutations of Leu(356), Leu(372) and Arg(98) increased k(cat) values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased K(m) values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure-function relationship underlying detrimental human enzyme defects.