Biochemical characterization of mRNA capping enzyme from Faustovirus.

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Methods to Unravel the Roles of ATPases in Fe-S Cluster Biosynthesis.

Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. Each of the four cluster biogenesis systems that have been discovered requires at least one ATPase. Generally, the function of nucleotide hydrolysis in Fe-S cluster biogenesis is understudied. For example, the cytosolic Fe-S cluster assembly (CIA) pathway is proposed to begin with a scaffold, which assembles nascent Fe-S clusters destined for cytosolic and nuclear enzymes. This scaffold, comprised of Nbp35 and Cfd1 in yeast, possesses an ATPase site that is necessary for CIA function, but the role of nucleotide hydrolysis is poorly understood. Herein, we describe the in vitro methods that have been developed to uncover how the ATPase site of the scaffold regulates interaction with one of its partner proteins, Dre2. We describe a qualitative affinity copurification assay and a quantitative assay for evaluating the dissociation constant for the scaffold-partner protein complex. Finally, we describe kinetic methods to measure the kcat and KM values for ATP hydrolysis by the scaffold-partner protein complex and the execution of the ATPase assays in an anaerobic environment. These methods could be applied to study other ATPases to advance our mechanistic understanding of nucleotide hydrolases involved in metallocluster biogenesis.