Riboflavinator: a molecular machine for efficient riboflavin biosynthesis

University Of Texas At San Antonio, San Antonio TX

Investigators

Abstract

With support from the Chemistry of Life Processes (CLP) Program in the Chemistry Division, Professor Audrey Lamb from the University of Texas at San Antonio (UTSA) will investigate the chemical mechanism of the biosynthesis of riboflavin (vitamin B2) with a special focus on the last enzymes in the pathway. Riboflavin is required by all living organisms and is synthesized from one guanosine 5’-triphosphate (GTP) and two ribulose 5-phosphate molecules using five unique enzymes. Despite unparalleled chemistry and the ubiquitous requirement for riboflavin, compelling evidence for the chemical mechanisms of the enzymes in this biosynthetic pathway has not been presented. We will investigate the hypothesis that the enzymes work in concert as a molecular machine for efficient production of this critical vitamin. This project will allow graduate students and postdoctoral fellows to acquire specialized training in enzyme kinetics and high-resolution X-ray crystallography. Portions of this project are structured such that undergraduate students will obtain meaningful research experiences to inspire careers in chemical research. This project also includes professional development outside of the laboratory for graduate students and postdoctoral fellows and pedagogical training for faculty at UTSA. Under this grant, the Lamb research team aims to establish how riboflavin is formed enzymatically from GTP and ribulose-5-phosphate, including structural identification of intermediates that are detected kinetically. This proposal focuses on the two final enzymes of the biosynthetic pathway that perform curious reactions. RibE (lumazine synthase) adds 4 carbons derived from the sugar ribulose 5-phosphate to a pyrimidinedione derived from GTP, generating lumazine. RibC performs an unusual dismutation reaction in which those same 4 carbons of the RibE reaction are transferred from one lumazine to another, generating riboflavin. The team will apply nuclear magnetic resonance methodology that they have developed to observe the formation and disappearance of intermediates for enzymes earlier in the biosynthetic pathway to substantiate or refute the largely hypothetical chemical mechanisms of RibE and RibC. Earlier work on this system has led the Lamb team to propose that efficient catalysis occurs when a molecular machine is formed, in which RibE forms as capsid that encapsulates the other 4 enzymes of the pathway. They have labelled this system the riboflavinator. The outcomes of this project are expected to advance the understanding of how this essential cofactor is biosynthesized. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.