Engineering P450s by the ‘Molecular Lego’

The human P450s are difficult to work with because;

they are membrane bound and hard to solubilise
the enzymes are large and easy to inactivate, and
they require a multi-component redox chain.

In the human hepatocytes, the necessary reductase is attached to the endoplasmic reticulum membrane together with the P450s and the electrons are supplied by NADPH. The key idea in the NBD technology is to link the P450 covalently (using genetic engineering) to the reductase to form an assembly that is then attached to an electrode. The electrode will supply the reducing units and the rate at which the P450 turns over the substrate can then be measured directly by measuring the current on the electrode (typically µA to nA).

The Molecular Lego idea, protected by the IP of NBD, looks at proteins and enzymes as “modules” that can be fused with a genetic/protein engineering approach to generate multi-component systems, where an electron-transfer modules interacts favourably with the electrode and the catalytic (P450) module turns over the substrate.

With this approach, the gene of the various human P450 isoforms can be fused to a number of different reductases, such as a small electron transfer protein like flavodoxin or the redox partner cytochrome P450 reductase to achieve optimal interaction with the electrode surface.

The solubility of the enzymes, and therefore theirs amenability to laboratory experiments, is achieved by elimination of the N-terminal portion that anchors the protein to the membranes and by linkage of a large bacterial reductase that is per se highly soluble. The NBD technology allows covalent linkage of the soluble and engineered P450 onto the surface of gold electrodes and then measures the electrochemical signal of the enzyme via a three electrode system.

Molecular Lego
The soluble human P450 module is indicated in red, the electron transfer module in blue, the engineered connecting loop in green. The covalent linkage of the reductase to the gold electrode is achieved via a unique cysteine indicated in yellow through a spacer enlarged between the dotted lines. A drug R-H gives a product R-OH by drawing electrons (vertical arrows) from the electrode.