Cytochrome P450cam isolated from the soil bacterium Pseudomonas putida catalyses the hydroxylation of camphor to 5-exo-hydroxy camphor and further to 5-ketocamphor. Unexpectedly, we have also observed the formation of the reduction product, borneol in our enzymatic assays performed under shunt conditions using meta-chloro perbenzoic acid (m-CPBA) or with the complete P450 system under low O2 conditions. Under shunt conditions using m-CPBA, borneol was the major product. To further demonstrate the origin of Hexo in borneol, we monitored the bioconversion of camphor in deuterated buffer (pD = 7.4) under shunt conditions using m-CPBA as the oxidant and mono-deuterated borneol at C-2 was detected. We demonstrate that the source of electrons for this reduction reaction is water and not the nicotinamide cofactor. When 17O labeled buffer was used in the reaction mixture, labeled hydrogen peroxide (H217O2) formed. We propose a novel reduction mechanism for P450cam, discuss its generality and also the ecological implications of this reaction for P. putida and E. coli. To accommodate unnatural substrates in the active site, a mutant library of P450cam was constructed by Sequence Saturation Mutagenesis (SeSaM). With an objective to identify mutants from SeSaM library that would dehalogenate the chlorinated pesticide endosulfan, the library was screened with 3-chloroindole as a substrate and the active clone(s) were identified by isatin/indigo formation. The mutant (E156G/V247F/V253G/F256S) was the most active in the conversion of 3-chloroindole to isatin, (KM = 250 µM) compared to the WT enzyme (which did not accept 3-chloroindole). The mutant also degrades endosulfan and endosulfan diol to pthalaldialdehyde under shunt conditions using m-CPBA. We propose a mechanism for the dechlorination of endosulfan and the formation of pthalaldialdehyde with mutant (E156G/V247F/V253G/F256S) of P450cam.
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Thesis advisor: Plettner, Erika
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