Rajni Verma

• In the last decades, enzymatic catalysis emerges as a convenient and environmentally friendly substitute for the traditional chemical processes range from the synthesis of many pharmaceutical and agrochemical building blocks to fine and bulk chemicals, and more recently, the components of biofuel. The combination of experimental and computational methods holds particular promise in the field of enzymatic catalysis to tailor enzymes for the tasks not yet exploited by natural selection. Therefore, it is important to develop computational tools that help to exploit this goal. The scope of this thesis is to propose novel bioinformatics tools and to explore computational methods aimed to support and guide protein evolution experiments. The thesis is divided into two parts. First part of the thesis (Part I, Chapter 1 and Chapter 2) is focused on extending the benchmarking system of random mutagenesis methods (MAP: Mutagenesis Assistant Program) towards the sequence/structure and structure/function analysis and to evaluate this approach on commonly used enzymes as biocatalysts. Chapter 1 offers the comprehensive information about the computational methods used to assist protein engineering experiments. Chapter 2 describes a completely renewed and improved version of MAP server, named as MAP2.03D server that correlates the generated amino acid substitution patterns to the structural information of the target protein. Therefore, the latter helps to identify in advance the random mutagenesis method that can introduce mutations having less deleterious effect and to improve protein fitness towards an expected property, e.g. charged amino acid substitutions to increase solubility of protein in water. The capability of the server was illustrated by in-silico screening of different enzymes and the predicted results were in agreement with the experimental findings. The atomic level understanding of the subtle intertwining among structure, dynamics and function of enzymes plays an important role to rationally design new or improved functions. Second part of the thesis (Part II, Chapter 3 – 6) is based on molecular modeling approach to gain insight into the structural and dynamic properties of P450BM-3 (CYP102) complex in water and in the presence of cobalt(II)sepulchrate (CoSep) as an electron transfer (ET) mediator. P450BM-3, isolated from Bacillus megaterium is an attractive target and model system for biochemical (catalyzes the wide variety of industrially attractive substrates) and biomedical (being a bacterial model for microsomal P450s system) applications. The comprehensive theoretical aspects of MD simulation are provided in Chapter 3 with the overview about the system preparation for MD simulation and the analysis of protein conformation and dynamics in the generated trajectory. In Chapter 4, the structural and dynamic properties of P450BM-3 FMN (Flavin mononucleotide) domain as holo-protein, with the cofactor in oxidized and reduced states and as apo-protein are investigated. The results illustrate the effect of FMN cofactor and its protonation state on the conformation and dynamics of the FMN domain that can be related to ET pathway from FMN to HEME cofactor. The study is further extended to garner insight into the binding modes and the structural determinant of inter-domain ET in HEME/FMN complex of P450BM-3. MD simulations were performed on both FMN and HEME domains, isolated and in their crystallographic complex and results are reported in Chapter 5. HEME/FMN complex undergoes the rearrangement process to decrease the distance between their redox centers to promote favorable ET rate under physiological condition. In Chapter 6, MD simulation of P450BM-3 domains (isolated HEME domain and HEME/FMN complex) were performed in the presence of CoSep, as ET mediator. The results illustrate the preferential binding modes of CoSep in P450BM-3 domains and the putative ET pathways from CoSep to the iron center of HEME cofactor and are in agreement with the experimental findings.