Author ORCID Identifier
Year of Publication
Doctor of Philosophy (PhD)
Arts and Sciences
Dr. Edith C. Glazer
Cytochrome P450s (CYPs) are cysteine ligated Fe-heme monooxygenases that are found in all domains of life. In mammals, they have a role in xenobiotic metabolism and steroid synthesis, making them a fundamental requirement for survival. In addition, their ability to perform a variety of chemical reactions on an array of substrates makes CYPs highly sought for biotechnical applications such as wastewater remediation, production of potential drug candidates, and creation of drug metabolites. By mutating specific amino acids, these enzymes can be engineered to change their substrate binding profiles and achieve stereo- and regio-specific chemistry. While these mutations are essential to change CYP activity, the major drawback to using them on an industrial scale is a decrease in stability of the enzyme. This work elaborated how CYP stability is effected by mutations, binding of native and non-native substrates, and changes in iron oxidation state.
Cytochrome P450BM3 (BM3, or CYP102A1), a bacterial enzyme, was used as a model system. In contrast to membrane associated human CYPs, BM3 is soluble and has efficient turnover due to the fusion of the reductase partner the heme domain. BM3 is naturally selective, but mutations can be incorporated to make it promiscuous, similar to CYPs responsible for xenobiotic breakdown. This allowed for the comparison of a selective vs. a promiscuous CYP while conserving the greatest possible sequence identity. An approach was used combining experimental solution phase data, x-ray crystallography, and molecular dynamic simulations. The results showed that mutations resulted in an cumulative decrease in stability as promiscuity increased. This reduction in stability was due to a decrease in the number of salt bridges and disruption of hydrophobic contacts. Regions of P450BM3 were found that could be targeted through mutation to increase the stability of a highly promiscuous and active variant known as the pentuple mutant (PM). Further investigations demonstrated the impact of native and non-native substrate binding. The Gibbs free energy of binding (ΔGb°) was determined for a small library of molecules and was rationalized computationally, concluding that attractive dispersion forces negated the impact of electrostatic and repulsive forces. In addition, the impact of the iron-heme charge state on CYP stability was examined as a function of promiscuity. In general, there was an association between promiscuity and similarities in the stability of the Fe(III) and Fe(II) states. This is consistent with a model where the promiscuous variants of the enzyme are in a more “reduction-ready” state, and can undergo catalysis with greater ease than the wild type enzyme. These findings have implications for the role of CYPs in human health and for biotechnical applications.
Digital Object Identifier (DOI)
Funding for this research was provided by:
- Research Challenge Trust Fund Fellowship (2016 – 2018)
- National Institute of Drug Abuse T32 Fellowship (2014 – 2016) NIH DA016176
- Kentucky National Science Foundation EPSCOR (2014 - 2019) award #1355438
Denning-Jannace, Catherine A., "A BIOPHYSICAL INVESTIGATION OF STABILITY, LIGAND BINDING, AND IRON STATE OF CYP102A1" (2020). Theses and Dissertations--Chemistry. 121.