After 4 years of work by our team, a final paper of the OPUS project is released as a pre-print version in ChemRxiv (https://lnkd.in/drpv8_6E). In the paper, we provide a new structure of ketosteroid dehydrogenase, which contains a previously uncharacterized domain. This domain binds to and orients the enzyme toward the cellular membrane where its substrates are dissolved (as well as a natural electron acceptor).
With a combination of kinetics, directed mutagenesis, QM:MM MD modeling we have described the role of this domain in substrate binding and enhancement of enzymes specificity toward steroids with extended substituents - this fact is very important for designing new enzymes which can dehydrogenate novel steroid drugs with extended structure beyond D ring of steroid core. Here we show one of such compounds - diosgenon - a saponin with a range of biological activities that can be catalytically modified with the enzyme.
We also analyzed by directed mutagenesis catalytic roles of tyrosine residues in the active site. The effects of mutations were analyzed with kinetics and explained with QM:MM MD modeling. So we can see now how a particular mutation is influencing the free energy of particular barriers or intermediates. This brings us to a better understanding of the catalytically important residues and proton-relay system involved in the transportation of abstracted protons to the solvent.
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