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In response to the stress of infection, Mycobacterium tuberculosis (Mtb) reprograms its metabolism to accommodate nutrient and energetic demands in a changing environment. Pyruvate kinase (PYK) is an essential glycolytic enzyme in the phosphoenolpyruvate-pyruvate-oxaloacetate node that is a central switch point for carbon flux distribution. Here we show that the competitive binding of pentose monophosphate inhibitors or the activator glucose 6-phosphate (G6P) to MtbPYK tightly regulates the metabolic flux. Intriguingly, pentose monophosphates were found to share the same binding site with G6P. The determination of a crystal structure of MtbPYK with bound ribose 5-phosphate (R5P), combined with biochemical analyses and molecular dynamic simulations, revealed that the allosteric inhibitor pentose monophosphate increases PYK structural dynamics, weakens the structural network communication, and impairs substrate binding. G6P, on the other hand, primes and activates the tetramer by decreasing protein flexibility and strengthening allosteric coupling. Therefore, we propose that MtbPYK uses these differences in conformational dynamics to up- and down-regulate enzymic activity. Importantly, metabolome profiling in mycobacteria reveals a significant increase in the levels of pentose monophosphate during hypoxia, which provides insights into how PYK uses dynamics of the tetramer as a competitive allosteric mechanism to retard glycolysis and facilitate metabolic reprogramming toward the pentose-phosphate pathway for achieving redox balance and an anticipatory metabolic response in Mtb. Ministry of Education (MOE) National Medical Research Council (NMRC) National Research Foundation (NRF) Accepted version This research was supported by the National Research Foundation of Singapore through the Singapore–MIT Alliance for Research and Technology Antimicrobial Resistance research program, and a Singapore–MIT Alliance for Research and Technology Postdoctoral Fellowship (W.Z.). During the course of this study, the J.L. laboratory was supported by grant NMRC/CBRG/ 0073/2014. The Y. M. laboratory was supported by the grant of MOE Tier 1 RG146/17 from Ministry of Education Singapore.

DRH-3 is critically involved in germline development and RNA interference (RNAi) facilitated chromosome segregation via the 22G-siRNA pathway in Caenorhabditis elegans. DRH-3 has similar domain architecture to RIG-I-like receptors (RLRs) and belongs to the RIG-I-like RNA helicase family. The molecular understanding of DRH-3 and its function in endogenous RNAi pathways remains elusive. In this study, we solved the crystal structures of the DRH-3 N-terminal domain (NTD) and the C-terminal domains (CTDs) in complex with 5'-triphosphorylated RNAs. The NTD of DRH-3 adopts a distinct fold of tandem caspase activation and recruitment domains (CARDs) structurally similar to the CARDs of RIG-I and MDA5, suggesting a signaling function in the endogenous RNAi biogenesis. The CTD preferentially recognizes 5'-triphosphorylated double-stranded RNAs bearing the typical features of secondary siRNA transcripts. The full-length DRH-3 displays unique structural dynamics upon binding to RNA duplexes that differ from RIG-I or MDA5. These features of DRH-3 showcase the evolutionary divergence of the Dicer and RLR family of helicases. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) Published version Singapore Ministry of Health’s National Medical Re- search Council, Individual Research Grant (OF-IRG) [NMRC/OFIRG/0075/2018]; Singapore Ministry of Edu- cation, Education Academic Research Fund Tier 1 [2018- T1-002-010]; Howard Hughes Medical Institute. Funding for open access charge: Singapore Ministry of Health’s National Medical Research Council, Individual Research Grant (OF-IRG) [NMRC/OFIRG/0075/2018].

RNA interference (RNAi) is a major antiviral defense mechanism in nematodes. In Caenorhabditis elegans (C. elegans), Dicer-related helicase 1 (DRH-1) acts to enhance the production of primary virus-derived small interfering RNA (siRNA) in antiviral RNAi pathway. However, Dicer-related helicase 3 (DRH-3), is involved in the biogenesis of secondary siRNA in both endogenous and exogenous RNAi pathways. DRH-1 and DRH-3 are orthologs to RIG-I like receptors (RLRs), based on their structural and sequential conservation of the helicase domain (HEL) and the C-terminal RNA binding domain (CTD). The structure and function of worm-specific N-terminal domains (NTDs) of DRH-1 and DRH-3 are currently unknown. This thesis describes biochemical, biophysical and structural studies of DRH-1 and DRH-3 (DRHs) to provide new insights into the RNAi mechanism at the molecular level. We first established the methods for expressing and purifying the full-length and various domains of DRHs. The purified recombinant proteins were then fully characterized to understand their functions in RNA recognition. For DRH-3, we applied X-ray crystallography and solved the crystal structure of DRH-3 NTD, as well as two crystal structures of DRH-3 CTD in complex with 5′-triphosphate (5′-ppp) RNAs. The NTD of DRH-3 adopts a novel fold of tandem CARDs that is different from the CARDs of RIG-I. This suggests DRH-3 may recruit an unknown protein partner in the endogenous RNAi pathway. Crystal structures and RNA binding assay confirmed that CTD preferentially recognizes RNAs with 5′-ppp, which is the typical feature of secondary siRNA transcript. Furthermore, the full-length DRH-3 displays unique structural dynamics and RNA differentiation patterns that are different from RIG-I and MDA5, as revealed by the hydrogen/deuterium exchange coupled to mass spectrometry experiment (HDX-MS). For DRH-1, we provided the first evidence that the Hel-CTD of DRH-1 has a dsRNA-dependent ATPase function. Moreover, we demonstrated that DRH-1 preferred short dsRNAs, especially those with 5'-ppp and 3' overhang for its ATPase hydrolysis activity and the protein stability. Collectively, our findings reveal unique molecular features of DRHs in RNAi and provide new perspectives for advancing our understanding of small RNA processing and antiviral defense mechanism in worms. Doctor of Philosophy