An enzyme engineering approach to the biosynthesis and utilization of FAD nucleobase analogues

Abstract

The nucleoside triphosphates (NTPs: ATP, GTP, CTP, and UTP) are the building blocks of RNA, and apart from that, they also serve specific functions in cellular metabolism, performing cellular processes such as energy production and signalling. These nucleotides share a common triphosphate and ribose moiety and are only identified based on the nucleobases. Despite the similar structure, a high degree of nucleotide selectivity of enzymes exists for various reactions. For instance, ATP is known as energy currency for all types of cells, and GTP is an intermediate in transmembrane signalling pathways, serving as a substrate for the G-protein family. Additionally, adenosine moiety from ATP serves as a recognition handle in coenzymes like flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), S-adenosyl methionine (SAM), and coenzyme A, while the other nucleobase analogues of these cofactors are not trackable in organisms signifying the specificity of ATP for the enzymes involved in the cofactor biosynthesis. For example, FAD is synthesized from vitamin B 2 (riboflavin), where riboflavin is converted to FMN (flavin mononucleotide), followed by the adenylation of FMN to FAD by the enzymes riboflavin kinase (RFK) and FMN adenylyltransferase (FMNAT), where only ATP is utilized in both the steps among the NTPs. In prokaryotes, both steps are performed by a bifunctional FAD synthetase enzyme possessing two separate modules – RFK and FMNAT module. The best example is the structurally well-explored bifunctional FAD synthetase from Corynebacterium ammoniagenes (CaFADS). This thesis focused on the FAD biosynthesis in Escherichia coli by FAD synthetase (EcFADS), a homolog of CaFADS. We synthesized FAD nucleobase analogues via new chemical and enzymatic approaches requiring no more than three steps and yielding moderate amounts (10-57%). Then, the analogues were subjected to evaluate their binding and functional capability with glutathione reductase flavoprotein and found to be active as reducing agents similar to FAD. The RFK module is ATP-specific, and the FMNAT module shows promiscuity towards each NTP, except GTP, resulting in FAD analogues (FCD, FUD, and dFAD). Despite this, E. coli shows only FAD production. We tuned the efficiency of the FMNAT module with each NTP by using computational and structural homology approaches. Specifically, we swapped the loop around the adenine of ATP within the FMNAT domain with a loop of a cytidylyl transferase enzyme. The LoopSwap mutant is efficiently active with GTP and shows increased activity with each NTP relative to EcFADS. Notably, the expression of the LoopSwap mutant in E. coli resulted in the synthesis of FAD analogous (FGD, FCD and FUD) within the cell. Furthermore, the shortening of the same loop established its key role in the ATP specificity of the FMNAT module, producing FCD and FUD within the cell while showing decreased or diminished in vitro activity with each NTP. Swapping the EcFADS expressing essential ribF gene with the LoopSwap (ribF25) mutant gene resulted in the biosynthesis of FGD, FCD, and FUD. Interestingly, the ribF25 mutant strain shows similar growth as the wild-type strain. Importantly, the mutant strain exhibits antibiotic tolerance against aminoglycosides and β-lactams, outperforming the wild-type strain. Due to low FAD, the mutant strain shows slower growth under anaerobic conditions than the wild-type strain. Yet, the antibiotic tolerance is observable anaerobically. This study proposes that manipulating the structure of vital loops is a potent strategy for fine- tuning enzyme activity without specifically targeting residues within the active site. The biosynthesis of FAD nucleobase analogues has potential applications in understanding FAD's molecular role in cellular metabolism as well as in biotechnology and synthetic biology similar to the applications developed using nucleobase analogue (NCD) of the redox cofactor NAD to make biorthogonal biological systems. Understanding how unnatural molecules, such as FAD analogues, aid antibiotic resistance will help increase antibiotics' efficacy.