p450 - publications

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1. Bioconjug Chem. 2012 Mar 21. [Epub ahead of print]

Site-specific fluorescent labelling and oriented immobilization of a triple
mutant of CYP3A4 via C64.

Menard A, Huang Y, Karam P, Cosa G, Auclair K.

The generation of site-specific bioconjugates of proteins is highly desired for a
number of biophysical and nanotechnological applications. To this end, many
strategies have been developed that allow the specific modification of certain
canonical amino acids and, more recently, non-canonical functional groups. P450
enzymes are heme-dependent monooxygenases involved in xenobiotic metabolism and
in the biosynthesis of a variety of secondary metabolites. We became interested
in the site-specific modification of these enzymes, CYP3A4 in particular, through
our studies of their in vitro biocatalytic properties and our desire to exploit
their remarkable ability to oxidize unactivated C-H bonds in a regio- and
stereospecific manner. Obtained via a partial cysteine-depletion approach, a
functional triple mutant of CYP3A4 (C98S/C239S/C468G) is reported here which is
singly modified at C64 by maleimide-containing groups. While cysteine-labelling
of the wild-type enzyme abolished >90% of its enzymatic activity, this mutant
retained ≥75% of the activity of the unmodified wild-type enzyme with 9 of the 18
maleimides that were tested. These included both fluorescent and solid-supported
maleimides. The loss of activity observed after labelling with some maleimides is
attributed to direct enzyme inhibition rather than to steric effects. We also
demonstrate the functional immobilization of this mutant on
maleimide-functionalized agarose resins and silica microspheres.

PMID: 22433037 [PubMed - as supplied by publisher]