Molecular determinants of stability and nucleobase specificity in flavin biosynthesis enzymes

Abstract

Nucleoside triphosphates (NTP) are the building blocks of RNA, and also play a central role in various metabolic pathways. Even though the NTPs - adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP) - are largely similar in structure, the nucleobase moiety differs among them and appears to be the determining factor for their roles in cellular processes. For instance, ATP is used as the energy currency in cellular metabolism, GTP is typically utilized in signal transduction, and CTP and UTP are involved in phospholipid and glycogen biosynthesis, respectively. However, the molecular mechanism by which enzymes distinguish between the various NTPs is not well explored in the literature. Even though the binding motifs as well as mode of binding of NTPs in some classes of enzymes has been established, these rules do not apply across the vast variety of NTP-utilizing enzymes. In this thesis, we undertake the exploration of the molecular determinants of stability and nucleobase specificity of the NTP-utilizing enzymes involved in flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) biosynthesis, with a goal of expanding the range of NTPs that these enzymes use. Our studies done with homologs of riboflavin kinase, FMN adenylyltransferase and FAD synthetase from various organisms have culminated in understanding some aspects of the NTP choice of these enzymes which has allowed us to produce a series of enzymes that show diverse nucleotide preferences. Investigations conducted on the CTP-dependent riboflavin kinase from the thermophilic archaea Methanocaldococcus jannaschii (MjRibK) suggest that a loop containing a small one-turn helix in the middle section of the enzyme is important for the recognition of the cytosine nucleobase. The perturbation of this one-turn helix via mutations has led to altered NTP specificity. Further, comparative sequence and structure analysis with a mesophilic homolog of MjRibK led us to identify the molecular interactions responsible for its thermostability. Next, we analyzed homologs of FMN adenylyltransferases and FAD synthetases for their promiscuity in NTP utilization, which helped us establish a robust enzymatic route for the synthesis of FAD cofactor analogs such as FGD and dFAD. Our studies lay the basis for understanding nucleotide specificity in flavin biosynthesis enzymes. Furthermore, our studies on engineering these enzymes to exhibit altered nucleotide specificity lay the foundation for the enzymatic synthesis of functional FAD analogs. The molecular insights that we derive from our studies inform the broader question of how a specific nucleobase is selected by nucleotide-utilizing enzymes for their cellular function. This exploration can be extended to the fields of synthetic biology, metabolic circuits, bioremediation, and the development of potent inhibitors for flavin biosynthesis enzymes and flavoenzymes.