Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP(+) reductase from plant chloroplasts.
Fusion proteins of rat cytochrome P4501A1 with maize ferredoxin I (Fd) and pea ferredoxin NADP(+) reductase (FNR), the last electron transfer proteins of the photosynthetic channel in plant chloroplasts, were obtained by gene fusion in the yeast expression vector pAAH5N. The encoded fusion proteins P4501A1-Fd, P4501A1-FNR, P4501A1-Fd-FNR and P4501A1-FNR-Fd were produced in microsomes of the yeast Saccharomyces cerevisiae AH22. Enzymatic assays were carried out in vitro with the isolated microsomes. P4501A1-Fd-FNR and P4501A1-FNR-Fd were found to catalyze P450-monooxygenase activities towards 7-ethoxycoumarin and the herbicide chlortoluron. P4501A1-Fd-FNR was the most efficient enzyme as measured in vitro in ferricyanide and cytochrome c reductions, as well as P450-monooxygenase assays. Apparent K(m) and k(cat) of P4501A1-Fd-FNR were 70 microM and 7800 min(-1) for NADPH, 13.2 microM and 51.1 min(-1) for 7-ethoxycoumarin, and 21.3 microM and 23. 8 min(-1) for the herbicide chlortoluron, respectively. Fd in P4501A1-Fd-FNR fusion enzyme was found to be a limiting factor compared to P4501A1 fused to the yeast NADPH-cytochrome P450 reductase, an artificial enzyme described previously. The efficiency of electron transfer in the P4501A1 fusion proteins and a possible in vivo molecular coupling of Fd and FNR with microsomal cytochrome P4501A1 produced in plant chloroplasts are discussed.
Lacour
T
T
Department of Biological and Environmental Sciences, Faculty of Agriculture, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan.
Ohkawa
H
H
D015660:7-Alkoxycoumarin O-Dealkylase / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002736:Chloroplasts / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005292:Ferricyanides / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009249:NADP / Q000737:chemistry; D018532:Pisum sativum; D010671:Phenylurea Compounds / Q000378:metabolism; D010957:Plasmids; D015202:Protein Engineering; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D003313:Zea mays
D003374:Coumarins; D005292:Ferricyanides; D010671:Phenylurea Compounds; D011993:Recombinant Fusion Proteins; C007931:hexacyanoferrate III; C017299:7-ethoxycoumarin; D009249:NADP; D015660:7-Alkoxycoumarin O-Dealkylase; D019363:Cytochrome P-450 CYP1A1; D005287:Ferredoxin-NADP Reductase; C007033:chlortoluron