Characterization of a novel mesophilic CTP-dependent riboflavin kinase and rational engineering to create its thermostable homologs

Abstract

Flavins play a central role in cellular metabolism as cofactors that are engaged in a wide range of oxidation-reduction reactions in living organisms. Several interesting variations exist in the flavin biosynthesis pathway among the domains of life, and the analysis of enzymes on this pathway have resulted in unique structural and mechanistic insights. The CTP-dependent riboflavin kinase in archaea is one such example - unlike most kinase enzymes that use adenosine triphosphate for conducting phosphorylation reactions, riboflavin kinases from tarchaea utilizes cytidine triphosphate (CTP) to phosphorylate riboflavin to produce flavin mononucleotide (FMN). In this study, we present the characterization of a mesophilic archaeal riboflavin kinase homolog from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the thermophilic RFK homolog from Methanocaldococcus jannaschii (MjRibK). We reconstitute the activity of the CTP-dependent MmpRibK and analyse the molecular factors that contribute to the uncommon properties of this class of enzymes. Furthermore, based on the high degree of sequence similarity between the mesophilic MmpRibK and the thermophilic MjRibK, we establish a set of the residues that are responsible for the thermostability of the enzyme without any loss in activity or substrate specificity. Our work contributes to the molecular understanding of flavin biosynthesis in archaea through the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to enzymes, with implications in enzyme engineering and biotechnological applications.