Cytochrome P450cin

Hawkes, David B. (2003). Cytochrome P450cin PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author Hawkes, David B.
Thesis Title Cytochrome P450cin
Formatted title
Cytochrome P450CIN
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2003
Thesis type PhD Thesis
Supervisor Dr James De Voss
Total pages 277
Collection year 2003
Language eng
Subjects L
250399 Organic Chemistry not elsewhere classified
780103 Chemical sciences
Formatted abstract
Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here the isolation and characterization a novel cytochrome P450 (P450cin, CYP176A1) and an associated flavodoxin redox partner purified from a strain of Citrobacter braakii which is capable of using cineole as its sole source of carbon and energy.

The complete CIN operon involved in this oxidation has been isolated by first purifying the P450 from the natural host and using peptide sequencing information to develop a probe. Southern hybridization, and colony hybridisation allowed the isolation of a 4kb DNA fragment, and subsequently further 5.5kb and 2.9kb fragments. Sequencing of these fragments revealed an 8kb contig containing three open reading frames identified on the basis of sequence homology as a cytochrome P450 (P450cin), an NADPH dependent flavodoxin/ferrodoxin reductase (cindoxin reductase) and a flavodoxin (cindoxin). This arrangement suggests that P450cin may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved catalytically important, active site threonine with an asparagine residue.

The P450 gene (cinA) was subcloned and heterologously expressed in E. coli at approximately 2000 nmol/l of original culture and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450cin, it was expressed in E. coli in the presence of cineole. A product was formed in vivo that was tentatively identified by GCMS as 2-hydroxycineoIe. Examination of P450cin by UV/visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole (KD = 0.7 µM), and a large spin state change of the heme iron associated with binding of cineole. These facts support the hypothesis that cineole is the natural substrate for this enzyme and that P450cin catalyses the initial monooxygenation of cineole biodegradation. This constitutes the first characterisation of an enzyme involved in this pathway.

Expression of the NADPH dependent flavodoxin/ferrodoxin reductase (cinB) identified in the CIN operon failed to produce fully folded protein, however the cindoxin gene (cinC) was successfully subcloned expressed in E.coli at approximately 4000nmol/l of original culture, purified and characterized by UV/Visible spectroscopy and mass spectrometry. Characterisation confirmed the protein as a flavodoxin yet its genetic arrangement suggested a novel role as a redox partner for P450cin.

Investigation of this role was achieved by in vitro 1,8-cineole turnover experiments in which the unexpressed NADPH dependent flavodoxin/ferrodoxin reductase was substituted with E. coli reductase and used in conjunction with cindoxin and P450cin. This system displays high turnover numbers of 388µM/min/µM P450 (using 0.5µM P450, 1µM reductase 4µM flavodoxin). The product of 1,8-cineole hydroxylation was found to be (lR,2S,4S)-2-hydroxy-1,8- cineole by P450cin.

Development of tricistronic (P450, E. coli reductase, cindoxin) and bicistronic (P450, cindoxin) expression systems allowed for large scale (bioreactor) oxidation in vivo of 1,8-cineole to the hydroxy product. As an attempt at improving the efficiency of the in vivo bioreactor an E. coli reductase/cindoxin fusion protein was constructed. Turnover using the fusion protein however dramatically lowered the production of hydroxycineole.
Keyword Cytochrome P-450

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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Created: Fri, 24 Aug 2007, 18:18:24 EST