Structural redesign of riboflavin kinases from bacteria and archaea for altered nucleobase specificity

Abstract

Riboflavin (also known as vitamin B2) is a water-soluble vitamin that is acquired from dietary sources and converted into FMN and FAD enzymatically. Various flavoenzymes use these two molecules as co-enzymes/co-factors for catalyzing oxidation/reduction reactions. The conversion of riboflavin to FMN is achieved by means of an enzyme called riboflavin kinase that phosphorylates riboflavin using an NTP source. The first section of my thesis dealt with engineering an archaeal CTP-dependent riboflavin kinase from Methanococcus maripaludis (MmpRibK) for the investigation of the strict nucleobase specificity of this enzyme. The basis of this research stems from previous attempts at engineering another archaeal CTP-dependent riboflavin kinase from Methanocaldococcus jannaschii (MjRibK) in order to confer reactivity with ATP. The direction of my thesis moved on to the investigation of a certain conserved part of the nucleobase-surrounding loop in eukaryotic riboflavin kinases. This investigation was performed by characterization of mutants of the human riboflavin kinase (HsRibK) with modifications in this conserved region. The third section of my thesis involved attempts at the characterization of riboflavin kinase and FAD synthetase of Schizosaccharomyces pombe (SpRibK and SpFADS) followed by an analysis of organisms that contain both the monofunctional riboflavin kinase and the bifunctional FAD synthetase.