Introducing CYPs

The newest area in the lab is Michael's joint project with Mark Paine's group at the Liverpool School of Tropical Medicine. The cytochrome P450 class of enzymes (CYPs for short) interested me when I first heard about them in a seminar some years ago. The most interesting aspect from my own perspective was the combination of a variable substrate recognition domain with a common domain for redox chemistry. CYPs therefore share some similarities with C5 MTases, where a common methyl transfer domain is coupled to a diversity of sequence specific base flipping domains in each specific enzyme. The most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction, e.g., insertion of one atom of oxygen into the aliphatic position of an organic substrate (RH) while the other oxygen atom is reduced to water.

My own interest lies not in the specific properties of CYPs; that's very much Mark Paine's interest and underpins his group's interest in understanding detoxification mechanisms in insect vectors and the mechanisms underlying insecticide resistance. No, I am interested in the primary structure determinants of enzyme specificity and catalysis, in particular those that are not easily identified through structural methods. 

I am particularly interested in the application of random mutagenesis combined with simple genetic screening in E.coli, or if necessary in conjunction with high throughput biochemical assay, for  building libraries of mutants with altered catalytic properties, and comparing the mutational data with sequence alignments from genomics, in order to inform strategic biophysical experiments aimed at fully understanding structure and function.Of course there others, notably Frances Arnold's group at Caltech in the US have published widely on CYPs as targets for directed evolution. Take a look at the home page for some insight into methods and targets. The work of Arnold has demonstrated that random methods as part of a programme of directed evolution, reveal unexpected roles for amino acids that were not anticipated from structural studies. 

Michael is working with CYP6Z2, and is currently optimising expression, purification, solubility etc. in order to support the first crystal structure of an insect CYP. So far so good, but a number of site directed mutants are proving challenging. One of our aims is to address specificity, possibly by coupling random mutagenesis with selective screening for new substrate specificities. By using internal indicators of general protein integrity (such as sensitive spectral analysis), we hope to identify mutants with novel specificities.