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 nu mber of cDNAs with similarity to known cytochrome P450 sequences. The partial sequence of one of these cDNAs, 5G6, indicated that it was lik ely to encode a full-length cytochrome P450 monooxygenase (cyt P450) s equence. In this paper we describe the complete sequence of this clone , which has been designated CYP71B7 in accordance with the nomenclatur e for the cyt P450 gene superfamily. The cDNA was used to determine th e pattern of expression of the corresponding gene in A. thaliana. Nort hern hybridization analysis indicated that maximal expression of CYP71 B7 occurred in rosette leaves. Weaker hybridizing bands were also dete cted by Northern analysis of RNA from roots, leaves, flowers, and sili ques. No expression could be detected in stem tissue. Southern analysi s indicated that the CYP71B7 gene was likely to exist as a single copy in the genome of A. thaliana. CYP71B7 was expressed episomally in yea st, and microsomes prepared from transgenic yeast exhibited a carbon m onoxide difference spectrum characteristic of cyt P450. Microsomes fro m yeast expressing CYP71B7 were assayed for enzymatic activity with sy nthetic model cyt P450 substrates. Microsomes from yeast cells express ing 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 observ ed with microsomes from cells expressing CYP71B7 with 7-ethoxycoumarin as substrate, Organic hydroperoxides are well known to support cyt P4 50 catalysis in the absence of electrons from NADPH. The yeast microso mes contained high levels of endogenous NADPH-ferricytochrome P450 red uctase (CPR) activity. The data suggest that this A. thaliana cyt P450 , although expressed in an active form, is incapable of accepting elec trons from the endogenous yeast CPR protein. (C) 1997 Academic Press.