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

Flavins play a central role in cellular metabolism as molecules that catalyze a wide range of oxidation-reduction reactions in living organisms. Several interesting variations in flavin biosynthesis exist among the domains of life, and the analysis of enzymes on this pathway have put forth many unique structural and mechanistic insights till date. The CTP-dependent riboflavin kinase in archaea is one such example - unlike most kinase enzymes that use adenosine triphosphate to conduct phosphorylation reactions, riboflavin kinases from archaea utilizes cytidine triphosphate (CTP) to phosphorylate riboflavin to produce flavin mononucleotide (FMN). In this study, we present the characterization of a new mesophilic archaeal riboflavin kinase homolog from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic homolog from Methanocaldococcus jannaschii (MjRibK). We reconstitute the activity of the CTP-dependent MmpRibK, determine its kinetic parameters, and analyse the molecular factors that contribute to the uncommon properties of this class of enzymes. Specifically, we probe the flexibility of MmpRibK and MjRibK under varying temperatures and the role of a metal ion for substrate binding and catalysis using molecular dynamics simulation and a series of experiments. Furthermore, based on the high degree of sequence similarity between the mesophilic MmpRibK and the thermophilic MjRibK, we use comparative analysis and site-directed mutagenesis to 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.