Abstract
RNA-based drugs are an emerging class of therapeutics combining the immense potential of DNA gene-therapy with the absence of genome integration-associated risks. While the synthesis of such molecules is feasible, large scale in vitro production of humanised mRNA remains a biochemical and economical challenge. Human mRNAs possess two post-transcriptional modifications at their 5′ end: an inverted methylated guanosine and a unique 2′O-methylation on the ribose of the penultimate nucleotide. One strategy to precisely methylate the 2′ oxygen is to use viral mRNA methyltransferases that have evolved to escape the host’s cell immunity response following virus infection. However, these enzymes are ill-adapted to industrial processes and suffer from low turnovers. We have investigated the effects of homologous and orthologous active-site mutations on both stability and transferase activity, and identified new functional motifs in the interaction network surrounding the catalytic lysine. Our findings suggest that despite their low catalytic efficiency, the active-sites of viral mRNA methyltransferases have low mutational plasticity, while mutations in a defined third shell around the active site have strong effects on folding, stability and activity in the variant enzymes, mostly via network-mediated effects.
Highlights
Nucleic acid-encoded drugs provide an economical solution to the development and manufacturing of new therapies
The active site in VP39 is centred around K175, which activates the 2′-hydroxyl group of the first transcribed nucleotide, for nucleophilic attack on the methyl group, by a mechanism of orbital steering[18]
The steric mutation D138E was enough to drop the activity of the variant below the detection limit of our methyltransferase assay (Fig. 2), underlining the importance of the precise positioning of the carboxylate group of this residue
Summary
Nucleic acid-encoded drugs provide an economical solution to the development and manufacturing of new therapies. Members of the large double-stranded DNA poxvirus family have evolved the ability to methylate the first transcribed nucleotide of their mRNA, probably as a strategy to escape immunogenic response in the host cell[6,7]. The human 2′O-mRNA methyltransferase CMTr1 is a member of the same superfamily (PFAM Clan 063) whose structure has been determined. CMTr1 differs from VP39 in the mRNA binding mechanism and, unlike VP3914, CMTr1′s activity is not m7G-dependent[15]. For this reason and because VP39 is the most characterised viral mRNA 2′O-methyltransferase to date, it is a potential candidate for the in vitro post-transcriptional enzymatic methylation of therapeutic mRNAs in an industrial context. Density Functional Theory (DFT) studies of the transfer of a methyl group from a sulfur onto a hydroxyl, further confirmed that a proton acceptor significantly lowered the high activation energy barrier of the methyltransfer reaction, eventually matching the experimental kcat[20]
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