Superfund Research Program

P450 Systems and Microbial Biodegradation

Project Leaders: John C. Loper, Jagjit S. Yadav
Grant Number: P42ES004908
Funding Period: 1995 - 2001

Project-Specific Links

Final Progress Reports

Year:   1999 

Work completed during 1999-2000 on molecular characterization of catabolic P450 enzyme systems in pollutant-degrading soil fungi Phanerochaete chrysosporium (Pc) and Cunninghamella elegans (Ce) and a marine yeast Debaryomyces hansenii (Dh), led to three publications. Of the five P450 alkane hydroxylase genes isolated from Dh, two genes (CYP52A12 and 13) were characterized (see GENE, 226:139-146,1999). P450 oxidoreductase (CPR) gene cloned from the two fungi (Ce and Pc) was further characterized. Xenobiotic induction experiments in C. elegans showed that the CPR gene transcript is inducible by n-tetradecane (BBRC, 268:345-353, 2000). Additionally the CYP52A14 gene of Dh has been characterized for publication, the C. echinulata CPR has been sequenced and the data is entered in GenBank. In P. chrysosporium, two differentially terminated cDNAs of the CPR gene were isolated and characterized (CURR. GENETICS, 37:65-73, 2000). Previous enzymatic and gene probe analyses had indicated the presence of at least two P450 monooxygenase genes of interest in P. chrysosporium, one of which had been cloned in part. Using the cloned fragment as probe, the full-length P450 gene has been isolated by screening a ZAP Express genomic library newly constructed for the purpose. Additional clones representing different alleles of the gene have also been isolated. Full sequence of the P450 gene (2389 bp) has been obtained. End portions of the corresponding cDNA were isolated by RT-PCR and sequenced. The cDNA sequence information coupled with grail analysis and examination of the putative splice junctions, have led to the identification of 13 small introns (46-68 bp) in the gene. The isolated gene shows highest sequence similarity to yeast P450 CYP52 genes among known P450s and partial homology to mammalian P450 subfamilies CYP 3A and 4. This is promising as a candidate for a Phanerochaete P450 system active in monooxidation of alkyl side-chains or alkyl-based structures of xenobiotic pollutants. The results in the Pc P450 system forms the basis for further research on understanding the interaction between P450-mediated monooxidation and peroxidase-mediated exo-oxidation for the complete metabolism of xenobiotic structures to CO₂ and for application of Pc - based detoxification and biodegradation of xenobiotic pollutants.