CATALYTIC SITE IN FURANOCOUMARIN METABOLIC P450S

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Furanocoumarins are naturally occurring secondary plant metabolites which are extremely phototoxic because when photoactivated they react directly and irreversibly with pyrimidine bases in DNA. The photobiologic properties of these furanocoumarins have been exploited for the treatment of a wide variety of diseases even though the mechanisms for their metabolism have not been well established. Considerable effort invested in documenting metabolic detoxification of furanocoumarins in mammals, birds and insects has suggested that the primary metabolites for xanthotoxin, a linear furanocoumarin, are identical in insects and a variety of vertebrate species and yet little is known, in vertebrate systems, about the P450 amino acid determinants (P450s) responsible for these catabolisms. P450s which exist in some herbivorous insects are capable of detoxifying linear and angular forms of these furanocoumarins and thus, represent prototypes for the catalytic domains existing in comparable vertebrate monooxygenases. The specific reactivities for CYP6B1 cloned from Papilio polyxenes (black swallowtail) indicate that these insect larvae express at least two furanocoumarin-metabolic P45Os: CYP6B1, which principally metabolizes linear furanocoumarins, and another CYP6B1-related P450, which metabolizes angular furanocoumarins. Characterization of the CYP6B1 alleles and the CYP6B1-related variant will be undertaken in order to define catalytic site reactivities in furanocoumarin-metabolic P45Os. Natural and mutant CYP6B1 variants will be used to identify amino acids which discriminate between linear and angular derivatives. The objectives are: 1) To fully define the substrate specificities of the CYP6B1v1/1v2 alleles using a series of natural and synthetic linear furanocoumarins; 2) To clone and express cDNAs encoding the other naturally occurring isozymes, including a CYP6B1-related variant which maybe responsible for metabolizing angular furanocoumarins, and to define the substrate specificities for each variant; 3) To predict and refine the active site tertiary structure for CYP6B1 by comparison of its structure with the vertebrate CYP2A5, which also metabolizes the linear furanocoumarin xanthotoxin, and with the natural CYP6B1 variants; 4) To test the tertiary structure predictions by mutagenizing amino acids potentially in the catalytic site. These specific aims should molecularly define amino acids in this insect P450 critical for modulating substrate specificity towards linear and angular furanocoumarins and provide information on the evolution of furanocoumarin resistances in insects as well as vertebrates which also metabolize these highly toxic compounds.