Cytochromes P450 (CYP) are heme-thiolate monooxygenases and form one of the largest superfamilies of enzyme proteins. They are found in virtually all organisms and contribute to diverse vital processes, such as carbon source assimilation, biosynthesis of hormones and structural components; other forms can degrade xenobiotics and interact with carcinogens. Although 75 full length CYP genes have been identified in the genome of C. elegans, the individual biological function of the vast majority is mostly unknown yet. Our investigations started with the validation that C. elegans possesses spectrally active CYP proteins, as demonstrated by CO-difference spectrum from microsomal fractions. To complete the monooxygenase system CYP enzymes need a carrier protein used for conveying reducing equivalents coming typically from NADPH. A genomic screen suggested
emb-8 as the only C. elegans gene in question to encode a protein that resembles mammalian NADPH-CYP reductases (CPR). Indeed, the CPR component was easily detectable by measuring the microsomal NADPH-cytochrome c reductase activity and it abolished upon prevention of functional
emb-8 expression. Moreover, CPR expression was found essential for all CYP activities measured in this study. To identify CYP components of specific monooxygenase systems, a systematic gene silencing by RNAi as well as by using cyp-mutant strains was performed. Several pre-selected enzymatic reactions, proved as CYP dependent in C. elegans, were used as screening systems. By doing this we identified CYP-29A3 and CYP-33E2 as the major isoforms contributing to the metabolism of eicosapentaenoic acid (EPA) and arachidonic acid (AA) to specific sets of regioisomeric epoxy- and w-/(w-1)-hydroxy-derivatives. Heterologous expression of both CYP-forms in SF9 insect cells is in progress. Moreover, CYP34A6 and CYP14A isoforms were found to be required for hydroxylation of an ortho-substituted, non-coplanar tetrachlorbiphenyl (PCB52) to C3-, C4- and/or C6-hydroxy-PCB52. This finding shows for the first time the so far only predicted involvement of C. elegans'' CYP forms in biotransformation. Further CYP-forms of subfamily 35A, which were under suspicion to be part of biotransformation, too, could be assigned to the fat storage pathway. Both individual gene silencing of cyp-35A2, 3, 4, and 5 resulted in a dramatic decrease of intestinal fat content of well fed young adults, measured by Nile red vital staining. Our investigation may facilitate future studies on C. elegans'' CYP enzymes, which are much less investigated than its status as model organism lets expect.