Abstract: Cytochrome P450 (CYP450) enzymes are the most versatile and ubiquitous heme proteins that catalyze a plethora of chemical reactions such as C-H activation, C-H amination, epoxidation, and oxidative transformations of various exogenous and endogenous substrates into potentially less toxic and hydrophilic molecules. Due to its chemical versatility and biological importance, it is one of the most prolific topics for biochemists and bioengineers. Although in the last few decades many seminal studies have been reported on the structure and functions of CYP450s, several mechanistic aspects of CYP450 chemistry are yet to be elucidated. Therefore, the present doctoral thesis provides a comprehensive study of several such mechanistic dilemmas and various fundamental problems related to the functions of different CYP450 enzymes using highly efficient computational chemistry tools such as Molecular Dynamics (MD) simulations, Density Functional Theory (DFT), and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) methods. In our work, we first attempted to investigate the fundamental aspect of CYP450 catalysis focusing on its catalytic cycle and then we gradually progressed to study the mechanistic issue of more recent non-natural reaction catalysis. The works shown in chapters two and three are devoted to unfolding the enigmatic mechanistic issues associated with the dioxygen diffusion and protonation machinery at the catalytic cycle. Thereafter, we moved on to study a long-standing mechanistic puzzle of C-C bond cleavage reaction facilitated by a natural CYP450 enzyme i.e. CYP51 using active oxidant Cpd I. Finally, with the wave of recent trends, we reveal the mechanistic conundrum of a bioengineered CYP450 enzyme, CYP411 that catalyzes an unorthodox C-H amination reaction selectively and efficiently. In summary, the present thesis rectifies a previous consensus of the CYP450 mechanism and provides several new mechanistic insights that may be helpful to bioengineer the CYP450 enzyme for new functions.
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Ph.D. dissertation, Shiv Nadar University 2022 " Mechanistic Study of Cytochrome P450 Enzymes: A Computational Insight "
List of Publications:
(Included in the thesis)
1) Kalita, S.; Shaik, S.; Kisan, H. K.; Dubey, K. D. A Paradigm Shift in the Catalytic Cycle of P450: The Preparatory Choreography during O 2 Binding and Origins of the Necessity for Two Protonation Pathways. ACS Catalysis. 2020, 10, 11481-11492.
2) Kalita, S.; Shaik, S.; Dubey, K. D. MD Simulations and QM/MM Calculations Reveal the Key Mechanistic Elements which are Responsible for the Efficient C–H Amination Reaction Performed by a Bioengineered P450 Enzyme. Chemical Science. 2021, 12, 14507-14518.
3) Kalita, S.; Shaik, S.; Dubey, K. D. Mechanistic Conundrum of C–C Bond Cleavage by CYP51. ACS Catalysis. 2022, 12, 5673-5683.
(Not included in the thesis)
4) Dubey, K. D.; Stuyver, T.; Kalita, S.; Shaik, S. Solvent Organization and Rate Regulation of a Menshutkin Reaction by Oriented External Electric Fields are Revealed by Combined MD and QM/MM Calculations. J. Am. Chem. Soc. 2020, 142, 9955-9965.
5) Yadav, S.; Shaik, S.; Siddiqui, S. A.; Kalita, S.; Dubey, K. D. Local Electric Fields Dictate Function: The Different Product Selectivities Observed for Fatty Acid Oxidation by Two Deceptively Very Similar P450-Peroxygenases OleT and BSβ. J. Chem. Inf. Model. 2022, 62,1025-1035.
6) Kardam, V.; Kalita, S.; Dubey, K. D. Computations Reveal a Crucial Role of F121 and F363 Aromatic Dyad in the Catalytic Function of CYP71D of Mint Superfamily, J. Inorg. Biochem, 2022 (Revision requested).
7) Kalita, S. † ; Yadav, S. † ; Dubey, K. D. MD Simulations and QM/MM Calculations Dictate Catalytic Machinery of Novel Metal-Free Carbonic Anhydrase Enzyme. (Manuscript is under communication).
8) Kalita, S.; Shaik, S.; Dubey, K. D. Harnessing the Oriented External Electric Field for disruption of β-Amyloid Aggregation. (Manuscript is under preparation).
9) Siddiqui, S. A.; Kalita, S.; Shaik; S.; Dubey, K. D. Designing a Highly Selective and Efficient Porphyrin-Based Molecular Cage Guided by Designed Local Electric Field. (Manuscript is under preparation).
Research Advisor: Dr. Kshatresh Dutta Dubey
Research Co-Advisor: Prof. Parthapratim Munshi