Cytochrome P450(cin) (CYP176A1) D241N: investigating the role of the conserved acid in the active site of cytochrome P450s

Stok, Jeanette E., Yamada, Sean, Farlow, Anthony J., Slessor, Kate E. and De Voss, James J. (2013) Cytochrome P450(cin) (CYP176A1) D241N: investigating the role of the conserved acid in the active site of cytochrome P450s. Biochimica et Biophysica Acta: Proteins and Proteomics, 1834 3: 688-696. doi:10.1016/j.bbapap.2012.12.022


Author Stok, Jeanette E.
Yamada, Sean
Farlow, Anthony J.
Slessor, Kate E.
De Voss, James J.
Title Cytochrome P450(cin) (CYP176A1) D241N: investigating the role of the conserved acid in the active site of cytochrome P450s
Journal name Biochimica et Biophysica Acta: Proteins and Proteomics   Check publisher's open access policy
ISSN 1570-9639
Publication date 2013-03-01
Sub-type Article (original research)
DOI 10.1016/j.bbapap.2012.12.022
Open Access Status Not Open Access
Volume 1834
Issue 3
Start page 688
End page 696
Total pages 9
Place of publication Amsterdam, Netherlands
Publisher Elsevier
Language eng
Subject 1602 Analytical Chemistry
1304 Biophysics
1303 Biochemistry
1312 Molecular Biology
Abstract P450cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450cin D241N mutant has been produced and found to be analogous to the P450cam D251N mutant. P450 cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31 ± 3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.
Keyword Cytochrome P450
Cineole
Citrobacter braakii
Enzyme mechanism
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2014 Collection
School of Chemistry and Molecular Biosciences
 
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Created: Thu, 28 Mar 2013, 20:17:42 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences