Abstracts Biological Sciences

Metabolic Adaptation For Utilization Of Short-Chain Fatty Acids In Salmonella Typhimurium : Structural And Functional Studies On 2-methylcitrate Synthase, Acetate And Propionate Kinases

by Sagar Chittori

Three-dimensional structures of proteins provide insights into the mechanisms of macromolecular assembly, enzyme catalysis and mode of activation, substrate-specificity, ligand-binding properties, stability and dynamical features. X-ray crystallography has become the method of choice in structural biology due to the remarkable methodological advances made in the generation of intense X-ray beams with very low divergence, cryocooling methods to prolong useful life of irradiated crystals, sensitive methods of Xray diffraction data collection, automated and fast methods for data processing, advances and automation in methods of computational crystallography, comparative analysis of macromolecular structures along with parallel advances in biochemical and molecular biology methods that allow production of the desired biomolecule in quantities sufficient for X-ray diffraction studies. Advances in molecular biology techniques and genomic data have helped in identifying metabolic pathways responsible for metabolism of short-chain fatty acids (SCFAs). The primary objective of this thesis is application of crystallographic techniques for understanding the structure and function of enzymes involved in the metabolism of SCFAs in S. typhimurium. Pathways chosen for the present study are (i) propionate degradation to pyruvate and succinate by 2-methylcitrate pathway involving gene products of the prp operon, (ii) acetate activation to acetyl-CoA by AckA-Pta pathway involving gene products of the ack-pta operon, (iii) threonine degradation to propionate involving gene products of the tdc operon, (iv) 1,2-propanediol (1,2-PD) degradation to propionate involving gene products of the pdu operon. These metabolic pathways utilize a large number of enzymes with diverse catalytic mechanisms. The main objectives of the work include structural and functional studies on 2-methycitrate synthase (PrpC), acetate kinase (AckA), propionate kinase isoforms (PduW and TdcD) and propanol dehydrogenase (PduQ) from S. typhimurium. In the present work, these proteins were cloned, expressed, purified and characterized. The purified proteins were crystallized using standard methods. The crystals were placed in an X-ray beam and diffraction data were collected and used for the elucidation of structure of the proteins. The structures were subjected to rigorous comparative analysis and the results were complemented with suitable biochemical and biophysical experiments. The thesis begins with a review of the current literature on SCFAs metabolism in bacteria, emphasizing studies carried out on S. typhimurium and the closely related E. coli as well as organisms for which the structure of a homologue has been determined (Chapter 1). Metabolic pathways involving acetate utilization by activation to acetyl- CoA, propionate degradation to pyruvate and succinate, anaerobic degradation of Lthreonine to propionate and, 1,2-PD degradation to propionate are described in this chapter. Common experimental and computational methods used during the course of…