Engineering the biosynthesis of unnatural vitamin B2 analogs - probing the molecular basis of nucleotide specificity


Amrita Hazra

Associate Professor

Department of Chemistry 

Enzyme mechanisms, Chemical biology of microbes

IISER Pune, Indian Institute of Science, Education and Research at Pune







Amphi Sciences de la Matière, site Monod

Vitamins are essential micronutrients required by organisms in all domains of life. Vitamins assist enzymes in catalysing reactions in the cell, ranging from simple acid-base and oxidation-reduction chemistry to complex radical-based rearrangements. Many organisms produce their own vitamins, however, others including humans are
unable to do so and obtain them from their diet or environment. Important discoveries in vitamin biosynthesis have spanned the fields of biological chemistry, microbiology, and evolution. As these are interesting biomolecules from both the chemistry and biology perspective, my laboratory's research program is built on understanding the molecular mechanisms of vitamin biosynthesis, exchange, and sharing. 

Vitamins such as flavin adenine dinucleotide (FAD), S-adenosyl methionine, and nicotinamide adenine dinucleotide catalyze distinct cellular reactions, yet they contain in common the nucleobase adenine in their structure. The source of adenine for the biosynthesis of these coenzymes is the ribonucleotide adenosine triphosphate (ATP). Nucleotide triphosphates (NTPs) such as ATP, GTP, CTP, and UTP, the building blocks of RNA, differ from one another only on the basis of the nucleobase. In spite of their overall structural similarity, they show high selectivity in being recruited by enzymes to conduct discrete reactions. In this work, we specifically probe the molecular, mechanistic and physiological basis of the choice of NTP in the FAD biosynthesis pathway.

The biosynthesis of FAD typically utilizes two molecules of ATP - the first one activates riboflavin (vitamin B2) to form flavin mononucleotide (FMN), and the second one couples with FMN to form FAD. We interrogate the choice of NTP for the second step in FAD biosynthesis using Escherichia coli FAD synthetase, a bifunctional enzyme that converts riboflavin to FMN using the riboflavin kinase domain followed by adenylation in the FMN:adenylyltransferase (FMNAT) domain resulting in FAD formation. We have engineered the FMNAT domain to produce mutants with altered NTP specificity which synthesize FAD nucleobase analogues under in vitro conditions. Heterologous expression of these mutants in E. coli demonstrates the synthesis of the FAD nucleobase analogue within cells. Finally, on replacing the native FAD synthetase on the bacterial chromosome with the mutant, we observe the biosynthesis of FAD nucleobase analogues and are currently investigating their physiological roles and potential cellular functions. Our study on FAD nucleobase analogues lays the foundation for their use as ‘unnatural vitamins’ to investigate cellular metabolism and for synthetic biology and biotechnology applications such as in vivo imaging and bioremediation.