Oxygen activation by P450(cin): Protein and substrate mutagenesis

Slessor, KE, Farlow, AJ, Cavaignac, SM, Stok, JE and De Voss, JJ (2011) Oxygen activation by P450(cin): Protein and substrate mutagenesis. Archives of Biochemistry And Biophysics, 507 1: 154-162. doi:10.1016/j.abb.2010.09.009

Author Slessor, KE
Farlow, AJ
Cavaignac, SM
Stok, JE
De Voss, JJ
Title Oxygen activation by P450(cin): Protein and substrate mutagenesis
Journal name Archives of Biochemistry And Biophysics   Check publisher's open access policy
ISSN 0003-9861
Publication date 2011-03
Year available 2010
Sub-type Article (original research)
DOI 10.1016/j.abb.2010.09.009
Volume 507
Issue 1
Start page 154
End page 162
Total pages 9
Place of publication Maryland Heights MO, United States
Publisher Academic Press
Collection year 2012
Language eng
Formatted abstract
A conserved threonine found in the majority of cytochromes P450 (P450s) has been implicated in the activation of dioxygen during the catalytic cycle. P450cin (CYP176A) has been found to be an exception to this paradigm, where the conserved threonine has been replaced with an asparagine. Prior studies with a P450cin N242A mutant established that the Asn-242 was not a functional replacement for the conserved threonine but was essential for the regio- and stereocontrol of the oxidation of cineole. To explore further how P450cin controls the activation of the dioxygen in the absence of the conserved threonine, two concurrent lines of investigation were followed. Modification of P450cin indicated that the Thr-243 was not involved in controlling the protonation of the hydroperoxy species. In addition, the N242T mutant did not enhance the rate and/or efficiency of catalytic turnover of cineole by P450cin. In parallel experiments, the substrate cineole was modified by removing the ethereal oxygen to produce camphane or 2,2-dimethylbicyclo[2.2.2]octane (cinane). An analogous experiment with P450EryF showed that a hydroxyl group on the substrate was vital, and in its absence catalytic turnover was effectively abolished. Catalytic turnover of P450cin with either of these alternative substrates (camphane or cinane) revealed that in the absence of the ethereal oxygen there was still a significant amount of coupling of the NADPH-reducing equivalents to the formation of oxidised product. Again the substrate itself was not found to be important in controlling oxygen activation, in contrast to P450EryF, but was shown to be essential for regio- and stereoselective substrate oxidation. Thus, it still remains unclear how dioxygen activation in the catalytic turnover of cineole by P450cin is controlled.

Research highlights

 Asn-242 in P450cin does not act as a functional replacement for the conserved threonine of P450s.  The ethereal oxygen of cineole is not important in controlling molecular oxygen activation.  Asn-242 and the ethereal oxygen of cineole are important in controlling the regio- and stereochemistry of hydroxylation.

Keyword Cytochrome P450
Citrobacter braakii
Enzyme mechanism
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Available online 17 September 2010

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2012 Collection
School of Chemistry and Molecular Biosciences
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Citation counts: TR Web of Science Citation Count  Cited 12 times in Thomson Reuters Web of Science Article | Citations
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Created: Sun, 13 Mar 2011, 00:13:55 EST