Expression of CYP71B7, a cytochrome P450 expressed sequence Tag from Arabidopsis thaliana.
The systematic sequencing of anonymous cDNA clones (expressed sequence tags or ESTs) from the plant Arabidopsis thaliana has identified a number of cDNAs with similarity to known cytochrome P450 sequences. The partial sequence of one of these cDNAs, 5G6, indicated that it was likely to encode a full-length cytochrome P450 monooxygenase (cyt P450) sequence. In this paper we describe the complete sequence of this clone, which has been designated CYP71B7 in accordance with the nomenclature for the cyt P450 gene superfamily. The cDNA was used to determine the pattern of expression of the corresponding gene in A. thaliana. Northern hybridization analysis indicated that maximal expression of CYP71B7 occurred in rosette leaves. Weaker hybridizing bands were also detected by Northern analysis of RNA from roots, leaves, flowers, and siliques. No expression could be detected in stem tissue. Southern analysis indicated that the CYP71B7 gene was likely to exist as a single copy in the genome of A. thaliana. CYP71B7 was expressed episomally in yeast, and microsomes prepared from transgenic yeast exhibited a carbon monoxide difference spectrum characteristic of cyt P450. Microsomes from yeast expressing CYP71B7 were assayed for enzymatic activity with synthetic model cyt P450 substrates. Microsomes from yeast cells expressing CYP71B7 or those from control cells exhibited no detectable NADPH-supported 7-ethoxycoumarin or 7-ethoxyresorufin deethylase activities. However, in the presence of cumene hydroperoxide, activity was observed with microsomes from cells expressing CYP71B7 with 7-ethoxycoumarin as substrate. Organic hydroperoxides are well known to support cyt P450 catalysis in the absence of electrons from NADPH. The yeast microsomes contained high levels of endogenous NADPH-ferricytochrome P450 reductase (CPR) activity. The data suggest that this A. thaliana cyt P450, although expressed in an active form, is incapable of accepting electrons from the endogenous yeast CPR protein.
Maughan
J A
JA
Biology Department, University College London, United Kingdom.
Nugent
J H
JH
Hallahan
D L
DL
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004579:Electron Transport; D005470:Fluorometry; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017421:Sequence Analysis; D013053:Spectrophotometry
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D003577:Cytochrome P-450 Enzyme System; C105888:CYP71B7 protein, Arabidopsis; D015660:7-Alkoxycoumarin O-Dealkylase; D019363:Cytochrome P-450 CYP1A1