Structural and Dynamic Analyses of Cytochromes P450
Creators: name: Wong, Nathan Ross
Publisher: name: Brandeis University
Year: 2022
Subjects: CYP106A2
Cytochrome
Isonitrile
NMR
P450
Steroid
Chemistry

description: Cytochromes P450 are a superfamily of heme-containing mono-oxygenases that typically catalyze the insertion of an oxygen atom from O2 into a carbon-hydrogen bond or carbon-carbon double bond, often with regiospecific and stereospecific precision. These enzymes are primordial, likely having evolved as long as 3.5 billion years ago, and are thought to have initially evolved as a reaction to the increase of oxygen in the atmosphere during the Archean eon. The early evolution and continued prevalence of these enzymes in all three domains of life is no mistake, as their uses in life vary from detoxification and catabolism of soil terpenes in bacteria to functionalization of prodrugs into active forms in humans. Because P450s can perform these oxidations with greater precision, higher yield, and less waste than what is possible with organic synthesis techniques, the potential value that P450-catalyzed reactions have for pharmaceutical and industrial applications is limitless. However, to harness the potential that P450s have, it is necessary to overcome certain limitations. A desired oxidation reaction for a substrate may not have a known P450 that catalyzes that reaction, or even if it does, that P450 may not produce the desired compound as major product. So, if a P450 is to be reengineered to increase efficiency and yield a more pure product, target residues for mutagenesis must be identified. Our work in the Pochapsky group has revealed significant commonalities between P450s using NMR structural and dynamic characterization, identifying regions responsive to and influential on substrate recognition and binding.
description: In this work, the methodology used to characterize a P450 with NMR techniques is applied to CYP106A2, a P450 from Bacillus megaterium that hydroxylates a variety of steroids and terpenoids. The orientation of substrates of CYP106A2 in its solution state are investigated by measuring the paramagnetic relaxation enhancement induced by the heme iron, allowing comparison of CYP106A2’s binding behavior in solution to its crystal structure and turnover data.
description: Beyond that, an exciting new technology developed by our group is reported, in which analogues of substrates presenting isonitrile moieties are synthesized and bound to P450s our lab studies. Spectral data collected support my hypothesis that P450s are sensitive and selective for substrate isonitrile derivatives that present isonitrile moieties at or near where that P450 normally oxidizes. These molecules bear great potential as inhibitors for P450s in a pharmaceutical context, but also are able to stabilize the diamagnetic form (S = 0) of P450s for detailed NMR studies.
description: Finally, a detailed methodology for assigning backbone resonances for CYP106A2 is described. With the benefit of the isonitrile-derivatives, ~80% of CYP106A2’s backbone was assigned, which permitted many observations about the solution-state structure of the enzyme. The results corroborate previous findings with other P450s that there is conservation in the regions involved in conformational changes induced by substrate binding, observations that could not be made with the crystal structure alone.