| dc.description.abstract | Microorganisms, mainly bacteria and fungi, are the producers of structurally diverse, complex organic compounds called as secondary metabolites. These metabolites include polyketides (PKs), non-ribosomal peptides (NRPs), and hybrid PKs/NRPs. The final products from these three classes display different characteristics like antibiotics, antiparasitic agents, antifungals, anticancer drugs, and immunosuppressants. As these products have wide range of potential application in pharmaceuticals, a number of biochemical studies have been carried out to elucidate of their biosynthetic pathways. The biosynthesis of these products is catalysed by large, multi-modular proteins including the NRPSs (non-ribosomal peptide synthases), the PKSs (polyketide synthases) and hybrid NRPS/PKS. Knowledge of the quaternary structure of PKS, NRPS and hybrid NRPS/PKS assembly line enzymes have been topic of interest since it helps not only in elucidating the crosstalk between different domains but also useful in modification of products. It has been demonstrated in the literature that FAS and PKS are homodimeric enzyme complexes, whereas the majority of NRPS are monomeric in nature. Whether NRPS is monomeric or homodimeric in the case of hybrid NRPS/PKS of a modular enzymatic manufacturing line has been questioned. To gain structural insights into the hybrid multidomain NRPS, we focused module-2 (Cy1-Cy2-A-PCP-Ox) of hybrid NRPS/PKS of leinamycin biosynthetic pathway. We performed cryo-EM of module 2 (Cy1-Cy2-A-PCP-Ox) which resulted in a low resolution cryo-EM map of this NRPS perhaps as a result of the linkers present in between the domains. However, to elucidate crucial interdomain interfaces and interactions that occur during different steps of the NRPS catalytic cycle, we undertook truncation studies including the domains (Cy1-Cy2) and (A-PCP-Ox) of the module2 of NRPS. We determined the structure of multidomain constructs (PCP-Cy1-Cy2) and (A-PCP-Ox) at overall resolutions of 5.2 Å and 7 Å respectively. The unravelling of architecture, organization, and mechanism of NRPS module 2 of leinamycin biosynthesis by cryo-EM will help design bioengineering approaches to understand the mechanistic insight into this novel pathway (swapping modules and domains).
DNA-binding protein under starvation (Dps), is a miniature ferritin complex, which plays a vital role in protecting bacterial DNA during starvation for maintaining the integrity of bacteria from hostile conditions. Mycobacterium smegmatis is one such bacteria that express MsDps2, which binds DNA to protect it under oxidative and nutritional stress conditions. Several approaches, including cryo-electron tomography (Cryo-ET), were implemented to identify the structure of the Dps protein that is bound to DNA. However, none of the structures of the Dps-DNA complex was resolved to high resolution to be able to identify the DNA binding residues. In this study, we implemented various biochemical and biophysical studies to characterize the DNA protein interactions of Dps protein. We employed single-particle cryo-EM-based structural analysis of MsDps2-DNA and identify that the region close to N-terminal confers the DNA binding property. Based on cryo-EM data, we performed mutations of several arginine residues proximal to the DNA binding region, which dramatically reduced the MsDps2-DNA interaction. In addition, we propose a model for DNA compaction during lattice formation. We performed single-molecule imaging experiments of MsDps2-DNA interactions that corroborate well with our structural studies. Single molecule imaging also deciphers the mechanism of compaction required for DNA protection. | en_US |
