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Sara Thodberg´s studies cytochrome P450 proteins at two very different levels. First with an evolutionary approach, where she tries to unravel the biosynthetic pathway of cyanogenic glucoside (CNG) production in ferns. Secondly she uses Single Molecular Studies on Cytochrome P450 Reductase Mutants to look at enzymatic function and dynamics.
In the project “Hunting for Ancestral Genes” we aim to unravel the biosynthetic pathway of cyanogenic glucoside production in ferns. Ferns are the evolutionary oldest plants known to produce CNGs, however the key gene of the pathway, a cytochrome P450 of the CYP79 family, has not been found in any of the 70 fern transcriptomes sequenced. By hunting down the missing enzymes responsible for making CNGs in ferns, we can determine if there is any evolutionary link between ferns and some of the 2500 modern plant species that make various classes of these compounds.
We perform the largest cyanogenesis screen of fern species to date by i) a colorimetric paper-assay, which enables rapid assessment of cyanogensis of plants in the field, and ii) LC-MS analysis to identify the different classes of CNGs and related compounds present. From highly cyanogenic species we are performing tissue specific radiolabelled feeding assays to identify biosynthetically active tissue from which we can extract RNA for next generation sequencing.
In my second project I use Single Molecular Studies on Cytochrome P450 Reductase Mutants to look at enzymatic function and dynamics. The conformational dynamics are anticipated to be functionally relevant and dictating specificity towards its P450 partners. However, mechanistic details of these highly stochastic and unsynchronized structural dynamics are typically averaged out by ensemble (bulk) computational or NMR techniques. The single molecule (SM) regime harbors the potential to reveal the hidden dynamics of a system by providing a platform to directly observe and quantify the conformational fluctuations. We investigate this by comparing the dynamic of six different mutants of the Human Cytochrome P450 Reductase, which is considered the key of understanding hormonal imbalance in patients with skeletal dysplasia.