Executive Summary : | Aromatic Nitration with high regio and chemoselectivity is the most soughtout reaction in chemistry, pharmaceuticals and other fine chemical industry. There are plethora of drug molecules, explosives, dyes and other fine chemicals such as pesticides, insecticides and many others who have aromatic ring structure containing Nitro group as functional group. However, these reactions are very tedious to carry out at industrial scale due to involvement of very harsh acids like Nitric and Sulphuric acid and also many unstable compounds. Thus, a green biocatatylic process which can give access to this functionality at high regio and chemoselectivity and good yield will be a boon for these industries. Recently, a new enzyme called RufO has been discovered from the rufomycin biosynthetic pathway which has been shown to catalyze regio and chemoselective nitration on tyrosine (a phenol containing amino acid) to generate 3-Nitrotyrosine. However, this enzyme has not been studied in detail beyond the one report of its discovery because of poor activity of the enzyme. Given the importance of phenol ring as a template for direct nitrations, my research group picked up this interesting enzyme for further exploration. Interestingly, in our lab, we managed to study and characterize this enzyme by optimizing the assays for its production, increasing its native activity by optimizing multiple parameters with various redox factors, coenzymes, time, pH, temperature optimization and several fine tuning techniques to upto 50-60% conversion with tyrosine (figure 2) and also developed a colorimetric method for measuring its activity (earlier, the activity has been measured using LC-MS). However, to further evolve the enzyme using rational engineering, we need to study its mechanism of action and structure activity relationship (SAR) with various substrates. Thus, this study is being proposed for conducting the thorough analysis of enzymatic active site including the study of both first coordination sphere (Objective 1) and second coordination sphere (Objective 2) along with further optimization of enzymatic activity of new mutants with various redox factors and coenzymes (Objective 3). |