January 7, 2019 at 4.00 pm in AG-69
Title :
Structural Biochemistry: A versatile tool to study biological reactions
Abstract :
Structural biochemistry employs structure in conjunction with biochemical and biophysical tools to understand molecular mechanism in biological system. Here, we study two major problems of importance to the Indian scenario; first towards understanding and devising strategies towards combating antibiotic resistance and second in development of biosensors for water quality monitoring. The first problem pertains towards combating the problem of antibiotic resistance. Here, we take a two-fold approach, first, towards discovery of novel enzymes that are divergent between human and pathogens as potential new targets and second focuses on understanding why do pathogens become resistant to existing drugs? Towards the first approach we have selected nucleobase deaminases as model systems to search for alternative therapies. These deaminases are essential enzymes and are structurally very different between humans and bacteria. This difference has already lead them to be used as prodrug-enzyme systems for cancer therapy and now we are exploiting them towards developing therapies for antimicrobial resistance. The problem pertaining to origins of antibiotic resistance involves unearthing molecular mechanisms that promote it. Two prominent systems have been undertaken, the tetracycline efflux pump regulators that activate efflux pumps that deplete antibiotic concentrations in the cells and ribosomal modifying enzymes with focus on methyltransferases that cause a steric clash with certain antibiotics thus result in evading their action. By solving a series of crystal structures of antibiotic efflux pump regulators as well as ribosomal methytransferases with and without DNA and complimenting these studies with biochemical and fluorescence spectroscopy we have delineated strategies to combat antibiotic resistance.
Towards developing biosensors,NtrC transcription regulators that activate sigma 54 class of polymerases and facilitate transcription of stress and virulence combating genes were selected for study. This is because these proteins have a self contained sensor and readout domain on a single polypeptide and thus pose as an efficient sensing-readout system. In this regards the class of sensors that we focused pertain to aromatic xenobiotics like phenol, benzene etc which are prominent pollutants from petroleum, dye and petrochemical industries. The crystal structure of the phenol sensing domain solved by us for this class of enzymes opened the doors towards design of a battery of specific sensors for both phenol as well as benzene group of compounds. The detection of these compounds was further optimized to low ppb levels and the shelf life, stability and sensitivity optimized to yield sensors that are potentially compatible in a commercial setting. Their xenobiotic sensing potential was further exploited by employing a combination of structure based design as well as mutagenesis created biosensors for not only phenol but xyenols, benzene derivatives and other aromatic pollutants. The sensor design has been translated to a chip based design where, the protein has been immobilized onto mesoporous silica nanoparticles. The aim is to create cheap and effective biosensor units that can detect these pollutants insitu.
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