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

Biochimica et biophysica acta (2013-01-12)
Jeanette E Stok, Sean Yamada, Anthony J Farlow, Kate E Slessor, James J De Voss
ABSTRACT

P450(cin) (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, P450(cin) 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 P450(cin) D241N mutant has been produced and found to be analogous to the P450(cam) D251N mutant. P450(cin) catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450(cin) 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 P450(cin) 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 P450(cam) 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Asparagine, ≥98% (HPLC)
Sigma-Aldrich
Eucalyptol, 99%
Sigma-Aldrich
L-Asparagine, BioReagent, suitable for cell culture, suitable for insect cell culture
Supelco
Eucalyptol, analytical standard
Supelco
1,8-Cineole, primary reference standard
Sigma-Aldrich
Eucalyptol, natural, ≥99%, FCC, FG
Supelco
L-Asparagine, certified reference material, TraceCERT®
Cineole, European Pharmacopoeia (EP) Reference Standard
Supelco
Eucalyptol, certified reference material, TraceCERT®