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'''The Role of Asn 242 in P450cin''' | '''The Role of Asn 242 in P450cin''' | ||
==Overview== | |||
Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyzes the hydroxylation of cineole to (S)-6beta-hydroxycineole. This initiates the biodegradation of cineole, enabling C. braakii to live on cineole as its sole source of carbon and energy. P450cin lacks the almost universally conserved threonine residue believed to be involved in dioxygen activation and instead contains an asparagine at this position (Asn-242). To investigate the role of Asn-242 in P450cin catalysis, it was converted to alanine, and the resultant mutant was characterized. The characteristic CO-bound spectrum and spectrally determined K(D) for substrate binding were unchanged in the mutant. The x-ray crystal structures of the substrate-free and -bound N242A mutant were determined and show that the only significant change is in a reorientation of the substrate such that (R)-6alpha-hydroxycineole should be a major product. Molecular dynamics simulations of both wild type and mutant are consistent with the change in regio- and stereoselectivity predicted from the crystal structure. The mutation has only a modest effect on enzyme activity and on the diversion of the NADPH-reducing equivalent toward unproductive peroxide formation. Product profile analysis shows that (R)-6alpha-hydroxycineole is the main product, which is consistent with the crystal structure. These results demonstrate that Asn-242 is not a functional replacement for the conserved threonine in other P450s but, rather, is critical in controlling regioselective substrate oxidation. | |||
==About this Structure== | ==About this Structure== | ||
3BE0 is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Citrobacter_braakii Citrobacter braakii]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3BE0 OCA]. | 3BE0 is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Citrobacter_braakii Citrobacter braakii]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3BE0 OCA]. | ||
==Reference== | |||
The critical role of substrate-protein hydrogen bonding in the control of regioselective hydroxylation in p450cin., Meharenna YT, Slessor KE, Cavaignac SM, Poulos TL, De Voss JJ, J Biol Chem. 2008 Apr 18;283(16):10804-12. Epub 2008 Feb 12. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/18270198 18270198] | |||
[[Category: Citrobacter braakii]] | [[Category: Citrobacter braakii]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| Line 20: | Line 26: | ||
[[Category: Protein substrate complex]] | [[Category: Protein substrate complex]] | ||
[[Category: Unknown function]] | [[Category: Unknown function]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jun 11 10:56:56 2008'' | ||
Revision as of 10:56, 11 June 2008
The Role of Asn 242 in P450cin
OverviewOverview
Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyzes the hydroxylation of cineole to (S)-6beta-hydroxycineole. This initiates the biodegradation of cineole, enabling C. braakii to live on cineole as its sole source of carbon and energy. P450cin lacks the almost universally conserved threonine residue believed to be involved in dioxygen activation and instead contains an asparagine at this position (Asn-242). To investigate the role of Asn-242 in P450cin catalysis, it was converted to alanine, and the resultant mutant was characterized. The characteristic CO-bound spectrum and spectrally determined K(D) for substrate binding were unchanged in the mutant. The x-ray crystal structures of the substrate-free and -bound N242A mutant were determined and show that the only significant change is in a reorientation of the substrate such that (R)-6alpha-hydroxycineole should be a major product. Molecular dynamics simulations of both wild type and mutant are consistent with the change in regio- and stereoselectivity predicted from the crystal structure. The mutation has only a modest effect on enzyme activity and on the diversion of the NADPH-reducing equivalent toward unproductive peroxide formation. Product profile analysis shows that (R)-6alpha-hydroxycineole is the main product, which is consistent with the crystal structure. These results demonstrate that Asn-242 is not a functional replacement for the conserved threonine in other P450s but, rather, is critical in controlling regioselective substrate oxidation.
About this StructureAbout this Structure
3BE0 is a Single protein structure of sequence from Citrobacter braakii. Full crystallographic information is available from OCA.
ReferenceReference
The critical role of substrate-protein hydrogen bonding in the control of regioselective hydroxylation in p450cin., Meharenna YT, Slessor KE, Cavaignac SM, Poulos TL, De Voss JJ, J Biol Chem. 2008 Apr 18;283(16):10804-12. Epub 2008 Feb 12. PMID:18270198 Page seeded by OCA on Wed Jun 11 10:56:56 2008