Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes

Abstract

Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.

The Multienzyme Complex Nature of Dehydroepiandrosterone Sulfate Biosynthesis

“…Some microsomal CYPs are capable of forming oligomers with the involvement of the N-terminal domain of a protein in the oligomerization process [42]. CYP17A1 is predominantly in an oligomeric state, but it can exist in a monomeric state [43]. CYP17A1full is more likely to form oligomers in an aqueous solution because of its transmembrane domain.…”

Heterologous Expression of Recombinant Human Cytochrome P450 (CYP) in Escherichia coli: N-Terminal Modification, Expression, Isolation, Purification, and Reconstitution

“…The current results show that, although not an exclusive requirement for maximal production of all human CYP proteins in E. coli , the supplementation of δ-ALA could enhance the expression dramatically [ 65 ]. δ-ALA is readily taken up by E. coli cells, followed by heme synthesis catalysed by bacterial enzymes, which is subsequently inserted into the recombinant CYP polypeptide to form an enzymatically active protein [ 89 ]. The most commonly used final concentration of δ-ALA added before induction is 0.5 mM, with exceptions such as 1 mM for CYP3A5 [ 72 ] and 1.5 mM for CYP1A2 [ 26 ].…”

Metalloporphyrins Reduce Proteinuria in Podocyte Immune Injury: The Role of Metal and Porphyrin Moieties

“…and assessment of this substitution on enzyme function. This approach was further supported by the demonstration that exogenous administration of not only FePPIX but also non-iron PPs, such as cobalt (Co) PPIX, can be incorporated into human membrane cytochrome P450 enzymes without significant perturbation in the overall protein structure [ 2 ]. These observations lead to the engineering of new types of metalloporphyrin-based biocatalysts that can perform desired reactivity and effect.…”