Bacterial cell surfaces are decorated with glycoconjugates, including polysaccharides such as O-antigens, enterobacterial common antigens, glycogen, and glycoproteins that contribute to bacterial interactions with the environment. These glycoconjugates play essential roles in virulence, immune evasion, host-pathogen interactions, and biofilm formation-processes crucial for bacterial growth and survival. Remarkably, bacterial glycoconjugates contain some of the over 700 “prokaryote-specific” or “rare” sugars that are absent in mammals, and represent unique motifs for differentiating bacterial and mammalian cells. The biosynthesis of these rare sugars offers new avenues for disabling bacterial virulence and pathogenicity; however, their exact role in mediating host-pathogen interactions remains unclear. Nucleotidyltransferases, a prominent class of enzymes, catalyze the activation of sugar-1- phosphates (S-1-Ps) with nucleoside triphosphates (NTPs) to produce nucleoside diphosphate sugars (NDP-sugars), which serve as activated precursors for rare sugar incorporation in glycoconjugate assembly. The biosynthesis of these glycoconjugate precursors is tightly regulated via feedback inhibition of nucleotidyltransferases to control substrate flux and mediate glycan assembly. Among these nucleotidyltransferases, a well-characterized enzyme called RmlA, a glucose-1-phosphate thymidylyltransferases, facilitates the activation of glucose-1-phosphate (Glc-1-P) by coupling it with deoxythymidine triphosphate (dTTP) to form dTDP-glucose (dTDPGlc). dTDP-Glc is a key precursor for the biosynthesis of several NDP-rare sugar, including dTDPβ-L-rhamnose (dTDP-β-L-Rha), which also serves as an allosteric inhibitor of RmlA, regulating viii the pathway through feedback inhibition. RmlA has been exploited for substrate flexibility, enabling the chemoenzymatic synthesis of diverse NDP-sugars, though some reactions suffer from poor catalytic efficiency. While most studies have focused on active site engineering to enhance substrate promiscuity for non-canonical substrates, a single enzyme capable of catalyzing the production of NDP-rare sugars has yet to be found. These NDP-rare sugars remain difficult to access using traditional synthetic methods, and engineering nucleotidyltransferases for one-step activation of rare sugars remains underexplored, collectively limiting advances in bacterial glycan assembly and host-pathogen interaction studies. In this thesis, we expand on rational mutation studies of RmlA, initially focusing on mitigating allosteric regulation by NDP-sugars. Previous studies have shown that RmlA activity is tightly controlled through product inhibition by dTDP-Glc and feedback inhibition by dTDP-βL-Rha. We hypothesized that the key limitation to improving the catalytic efficiency of RmlA with non-canonical substrates is NDP-rare sugar binding to the allosteric site, which suppresses enzyme activity. Thus, we postulated that modulation of RmlA regulation would improve nucleotidyltransferase activity in vitro. As predicted, rational mutations of RmlA in both the active and allosteric sites substantially expand substrate tolerance while simultaneously improving catalytic activity. We identified a highly promiscuous triple mutant, designated as RmlA*, which contains mutations in the active site (Y146F) and allosteric site (E256D), along with a stabilizing mutation (D104N). This variant showed the greatest promiscuity across all (d)NTPs and noncanonical α-D-S-1-P substrates. Kinetic and structural analyses revealed that these mutations ix induce subtle conformational changes in the quaternary structure of RmlA, leading to enhanced flexibility in the active site and modulating ligand interactions and enzyme catalytic efficiency. RmlA initiates the biosynthesis of several NDP-rare sugars, including dTDP-β-L-Rha, dTDP-β-L-6-deoxy-talose (dTDP-β-L-6dTal), and dTDP-α-D-N-acetylfucosamine (dTDP-α-DFucNAc), which play a crucial role in regulating downstream glycan assembly through feedback inhibition of RmlA. To better understand this regulatory mechanism, we next investigated the impact of allosteric regulation of RmlA from various species using synthetic NDP-rare sugars. We identified critical structural features required for feedback inhibition across diverse bacterial RmlA homologs and uncovered species-specific differences in regulatory mechanisms. We also observed that different RmlA species display varying degrees of cooperativity in response to tested NDPrare sugars, with P. aeruginosa RmlA exhibiting the highest sensitivity to NDP-rare sugar inhibition. Notably, we found that eliminating allosteric binding of NDP-rare sugars relieved feedback inhibition, thereby enabling efficient utilization of noncanonical rare sugar-1-phosphate substrates. This is the first reported one-step chemoenzymatic reaction of rare sugar precursors using engineered RmlA to produce NDP-rare sugars. This discovery not only enhances our understanding of nucleotidyltransferase regulation by metabolic intermediates but also provides a novel strategy for generating rare sugar substrates to investigate bacteria-specific glycan biosynthetic pathways. Moreover, the differing sensitivities of RmlA from different species to NDP-rare sugar inhibition set the foundation for the design of species-selective inhibitors for Gram-negative bacteria. Overall, our biochemical findings provide a framework for engineering x RmlA and related enzymes, advancing the biosynthesis of NDP-sugars with promising applications in glycoengineering and synthetic biology. Upon obtaining a promiscuous mutant with abolished feedback regulation, we sought to systematically evaluate the cellular effects of disrupting the feedback inhibition and expanding the substrate scope of RmlA in E. coli. Additionally, we aimed to leverage RmlA as a probe to understand how flux of various NDP-sugar biosynthetic pathways impact overall cellular metabolism. Initially, we hypothesized that RmlA* expression might alter glycan sequences due to the production of non-native NDP-sugars. Surprisingly, we found that E. coli cells expressing RmlA* showed impaired O-Ag production, leading to increased bacterial lysis in host serum. Furthermore, RmlA* expression induced a filamentation phenotype, likely due to excess usage of the DNA precursor dTTP, resulting in increased sensitivity to cell division inhibitors, and aberrant FtsZ ring formation. In addition, we found that non-regulated nucleotidyltransferases disrupt cellular homeostasis by altering intracellular concentrations of various key NDP-sugars involved in metabolism and cell wall biosynthesis. Collectively, this thesis provides a blueprint for engineering nucleotidyltransferases to enable chemoenzymatic activation of NDP-rare sugars and offers a structural scaffold for designing species-selective RmlA inhibitors. Although nucleotidyltransferases are not individually essential in bacteria, modulation of nucleotidyltransferases that utilize essential precursors like dT may lead to new avenues of targeting pathogenic bacteria.