Site-Specific Fluorescent Labeling and Oriented Immobilization of a Triple Mutant of CYP3A4 via C64

Abstract

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, noncanonical 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-labeling 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 labeling 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 resin and silica microspheres.

Studying Direct Electron Transfer by Site‐Directed Immobilization of Cellobiose Dehydrogenase

“…This approach has been used by Ménard et al . for the oriented immobilization of a human cytochrome P450 enzyme (CYP3 A4) to commercial resin or silica microspheres . The reaction between the thiol and maleimide occurs spontaneously, without the need for added reagents, and with high specificity to give a stable linkage at pH 6.5 to 7.5 making this a very efficient way to immobilize the enzyme variants.…”

High-Level Production and Properties of the Cysteine-Depleted Cytochrome P450 3A4

“…A similar trend was observed for the C98S/C239S/C468G mutant of Δ3–12 CYP3A4, reported to possess a 1.5-fold higher activity than WT. 17 …”

“…9 Menard and co-workers reported that the C98S/C239S/C468G mutant is 1.5-fold more active than the WT and undergoes maleimide modification primarily at the Cys64 site without a significant loss of the enzymatic activity. 17 Based on the latter mutant, a poorly expressed C58T/C64A/C98S/C239S/C468G variant was built for mechanistic studies of the allostery and binding cooperativity in CYP3A4. 18 Another group investigated C64S/C98S, C98S/C377S/C468G, C98S/C239S/C468G, C98S/C239S/C377S/C468A, and C58T/C64A/C98S/C239S/C377S/C468A variants 6,7,9,10,12 and noticed that elimination of more than four native cysteines markedly lowers the level of CYP3A4 expression.…”

Steroid bioconjugation to a CYP3A4 allosteric site and its effect on substrate binding and coupling efficiency

“…Wild type CYP3A4 and the C215 mutant were expressed and purified as previously described, with minor modifications [34,36]. Wild type refers to the N-terminus-truncated Δ2–12 CYP3A4, which was used as a template for preparation of the C215 mutant containing the following mutations: C58T/C64A/C98S/F215C/C239S/C468G.…”