Re: Cytochrome P450 Enzyme and Xanthobactor Autotropicus

Hi Grant, Thanks for this very interesting question. I'd like to answer it by commenting upon some of the things that we should consider when expressing a foreign protein in a bacterium (heterologous expression).

First off, I'm sure you're aware that the term cytochrome P450 is a rather generic term which refers to a large superfamily of enzymes, responsible for the metabolism of a wide range of compounds. In mammals and other eukaryotic organisms, enzymes that contain cytochrome P450 are localised in different parts of the cell-for instance there are mitochondrial forms, soluble forms and membrane-associated forms of these enzymes. Specific mechanisms exist in cells to target proteins to the correct compartment, so you may find that in a bacterium such as Xanthobacter autotrophicus, an expressed protein from a mammalian source may not assemble or fold properly. This would be your first concern in such an experiment, so you would have to consider carefully the original source of the cytochrome P450 enzyme.

Next, there are a number of issues to consider in a protein expression experiment. First, you would need to know if X. autotrophicus is "genetically amenable", meaning if you cloned a cytochrome P450 gene in a plasmid vector, could you get it into the bacterium and would it replicate? You would also need a strong gene promoter to drive the expression of the gene in the bacterium, either a natural promoter or an artificial one that you could switch on.

Assuming these criteria are met, the amount of protein and its final state can rarely be predicted. The protein may fail to fold and assemble properly, in which case it may form insoluble particles called inclusion bodies, or be degraded by intracellular proteases. In the case of cytochrome P450, you would also need to be sure that the biosynthetic pathways for cofactors such as the heme group are present in your bacterium. Finally, it's almost impossible to predict how much enzyme will be produced and how active it will be (if at all).

You can see that there are many factors to consider in this sort of experiment and really the only way to find out is to do it and see! My gut feeling is that X. autotrophicus might not be the best candidate for expression of cytochrome P450 from mammals, but it certainly sounds like a very interesting project. You might like to pop over to PubMed and search the literature there for cytochrome P450 expression and the genetics of X. autotrophicus, to get an idea of how viable your idea might be.

Good luck!
Neil Saunders