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.

  

Lab updates and formats

I have been thinking over the way in which progress is monitored and documented, now that you have most of your methodology in place for your experimental work. I suggest that you all write up your work in the form of a manuscript submission to Nucleic Acids Research (it doesn't matter about the nature of the data, it is the style of presentation that counts). This will enable you to progress to thesis submission much more efficiently. I suggest you submit the document to me by email and I will then send back comments. You should make sure you follow the instructions to authors to the letter and that means the annotation of Figures and their judicious use.

Instructions to Authors

I think this might help us all identify problems that need fixing and more importantly any new trends or unexpected observations will emerge more clearly. I look forward to your submissions! Many thanks

Trying to reconcile the PC-PG phenotype

I recall one of the first experiments that Paul Hurd carried out on mutants at the Pro-Cys element (motif IV) in order to get to the bottom of the pyrimidinone data that we had been obtaining, thanks to some lovely chemical synthesis by Bernard Connolly. Briefly, it was difficult to explain whether the oligos containing pyrimidinone in place of the target C in CCGG sites, was eliciting covalent adduct formation with M.MspI. We wanted to demonstrate that this was Cys dependent (with an outside possibility that the pyrimidinone could be sufficiently reactive to surrender to an attack by Ser). Anyway, for completeness, we decided to make the PC-PG mutant as well as several others, since Rich Roberts had shown anomalies with the Gly substitution, and Ashok Bhagwat had seen similar behaviour with M.EcoRII. Probably the most surprising result turned out to be the viability of the M.MspI C81G mutant (using M.HhaI numbering). Or that's what we thought.....

What we hadn't appreciated was the impact of the cloning vector, or rather the consequences of fusing our M.MspI ORF in frame with GST (we were routinely using pGEX vectors), since fusions to GST seemed to stabilise the expressed methylase enzymes, made them easy to purify the GST seemed to be tolerated well, so we generally didn't bother removing it in gel shift assays. However, GST is a homodimer (left). When Paul sequenced the apparently viable clones, we realised that something was amiss. In fact, we took a while to realise what had happened, since SDS PAGE analysis of the expressed ProGly mutant ran in its expected molecular weight position. The sequence analysis revealed that the first set of clones had an in frame 30bp deletion which took out ten amino acids at the active site, including the Pro-Gly sequence! Over the next few years, we investigated different methylase genes and used different host strains. In all cases, the induction of plasmid mutations turned out to be dependent on the presence of a dimerisation domain and led to insertions, deletions or mobilisation of transposable elements etc. All mutants eliminated the function of the ProGly mutant.

Having tried to rationalise the observation for a number of years and looked into copy number issues etc., it occurred to me that these observations have some resonance with chromothripsis. This relatively recent phenomenon, in which it has been suggested that a catastrophic replication associated event leads to massive genetic mutation (insertions, deletions etc), could provide some insight. If we sequence the genomes of several Pro-Gly transformants and compare them with standard plasmid transformants, we should see whether the error prone repair is plasmid directed or a generalized error prone mechanism/s. The results will enable us to plan our next set of experiments more strategically, if nothing else! The questions I ask, is, have we developed a bacterial model for chromothripsis? Could it be applicable as a general mutagenesis strategy?

How do enzymes work?

Maybe I am an optimist, but by now, you would think this question would have been answered definitively? I recall as an undergraduate being shown a Cambridge University question set by the famous geneticist and enzymologist JBS Haldane, in which undergraduates were asked to consider the possibilities and challenges surrounding the de novo design of a protein that could fulfill a Biochemical function. I imagine that question was set around 1930! The challenges that remain in delivering this objective are significant, despite the deposition of thousands of protein structures in the PDB and the determination of the sequences of many genomes. In fact the uncertainty that exists in defining gene function remains a significant barrier to the effective "translation" of Molecular Cell Biology into Biotechnological success: the "Holy Grail" of Synthetic Biology.

Perhaps one of the problems that exists is that proteins often have multiple

functions, and we tend to be (naturally) drawn to the earliest description of function. Thus, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, shown right) is well known to Biochemists as the sixth step in Glycolysis, thanks to Embden and Myerhof and Parnas. But it is also implicated in transcriptional regulation, apoptosis and vesicle transport. The point being, that technology (as Sydney Brenner has pointed out) often limits knowledge, and it was possible (over 90 years ago) to identify the GAPDH metabolic reaction, but only 15 years ago to identify its other functions. So GAPDH related sequences in genome analyses are primarily considered glycolysis related. How many other unknown functions of seemingly known genes exist?

This brings me on to the scope of our mutational work on C5 MTases and the forthcoming work from Michael in collaboration with Mark Paine's lab on P450s (and hopefully with Richard Pleass on immune molecules). Since we have a limited (at best) understanding of structure function relationships in protein chemistry, I am interested in using model enzymes to identify those residues that are essential for function (in as unbiased a way as possible). Thereafter, attempt to rationalise these observations using the most appropriate technology and subsequently use this knowledge in a synthetic biology programme. We are at step one at the moment.

The current state of our knowledge in respect of C5 DNA MTases has been

reviewed extensively and therefore we have a good knowledge base. More importantly, by using mcrBC positive and negative strains, we can select for inactivation by mutagenesis. The data from Laila and Sam using our Phusion based error prone PCR system, confirm the essential nature of the catalytic Cys as expected (and this is our best internal control). However, I remain puzzled by the 20 or so point mutations in the TRD of M.HhaI that abolish methylation activity (producing colonies on mcrBC+). I think Laila's subsequent analysis of mutants that are base flipping proficient, but do not complete methyl transfer (as identified by our indirect base flipping screen) are very interesting candidates for structural analysis (we should certainly look carefully at Norbert Reich's NMR data). I shall draw up an infographic to try and simplify the data from Laila's thesis, which are now ready for publication. For me this has demonstrated that we need to accumulate a large data set for M.HhaI. Remember that the mutants that we recover from mcrBC+ plates will be inactive and there is value in establishing which classes of amino acid substitutions inactivate as well as those that do not. I think we should dig in for a comprehensive analysis, since the methodology is now well established. Asma, Michael and Mohammed, you should get together and discuss these concepts: I shall write up Laila's data. Next an update on my take on our historic Pro-Cys Pro-Gly data and the genome analysis that Asma carried out.