Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site.
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.
Heinemann
Ilka U
IU
Institute of Microbiology, Technical University Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
Diekmann
Nina
N
Masoumi
Ava
A
Koch
Michael
M
Messerschmidt
Albrecht
A
Jahn
Martina
M
Jahn
Dieter
D
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D050768:Protoporphyrinogen Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011524:Protoporphyrins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D011524:Protoporphyrins; D050768:Protoporphyrinogen Oxidase