Abstract
The effect of non-synonymous single nucleotide polymorphisms (SNPs) on cytochrome P450 (CYP450) drug metabolism is currently poorly understood due to the large number of polymorphisms, the diversity of potential substrates and the complexity of CYP450 function. Previously we carried out in silico studies to explore the effect of SNPs on CYP450 function, using in silico calculations to predict the effect of mutations on protein stability. Here we have determined the effect of eight CYP3A4 and seven CYP2C9 SNPs on the thermostability of proteins in solution to test these predictions. Thermostability assays revealed distinct CYP450 sub-populations with only 65–70% of wild-type CYP3A4 and CYP2C9 susceptible to rapid heat-induced P450 to P420 conversion. CYP3A4 mutations G56D, P218R, S222P, I223R, L373F and M445T and CYP2C9 mutations V76M, I359L and I359T were destabilising, increasing the proportion of protein sensitive to the rapid heat-induced P450 to P420 conversion and/or reducing the half-life of this conversion. CYP2C9 Q214L was the only stabilising mutation. These results corresponded well with the in silico protein stability calculations, confirming the value of these predictions and together suggest that the changes in thermostability result from destabilisation/stabilisation of the protein fold, changes in the haem-binding environment or effects on oligomer formation/conformation.
Highlights
Cytochrome P450 (CYP450) enzymes, arguably nature’s most versatile catalysts, are a superfamily of haem-thiolate proteins found across all lineages of life[1]
Recombinant wild-type (WT) and variant His-tagged CYP3A4 and CYP2C9 proteins were expressed in E. coli and purified using immobilized metal affinity chromatography
The reduced ferrous form of the haem ion binds carbon monoxide (CO), yielding an absorption peak at 450 nm, provided the haem group is correctly incorporated into the cytochrome P450 (CYP450), providing a measure of the active protein in the sample (Fig. S1)
Summary
Cytochrome P450 (CYP450) enzymes, arguably nature’s most versatile catalysts, are a superfamily of haem-thiolate proteins found across all lineages of life[1]. To account for direct effects of mutations on protein function, a cytochrome P450 SNP map was created which combined information from previous functional studies to delineate regions important for substrate recognition[27,28], haem binding, interactions with CPR29–34 and residues implicated in the gating of substrate and product access/egress tunnels[35,36,37]. The combination of this information provides a useful tool for predicting the effect of mutations on protein function and for prioritising variants for in vitro testing[25]. In our previous study we found that SNPs predicted to alter protein stability or located in regions important for catalytic function are more likely to have effects on CYP450 function, based on available in vitro activity data[25]
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