miCLIP-MaPseq, a Substrate Identification Approach for Radical SAM RNA Methylating Enzymes

Although present across bacteria, the large family of radical SAM RNA methylating enzymes is largely uncharacterized. <i>Escherichia coli</i> RlmN, the founding member of the family, methylates an adenosine in 23S rRNA and several tRNAs to yield 2-methyladenosine (m²A). However, varied RNA substrate specificity among RlmN enzymes, combined with the ability of certain family members to generate 8-methyladenosine (m⁸A), makes functional predictions across this family challenging. Here, we present a method for unbiased substrate identification that exploits highly efficient, mechanism-based cross-linking between the enzyme and its RNA substrates. Additionally, by determining that the thermostable group II intron reverse transcriptase introduces mismatches at the site of the cross-link, we have identified the precise positions of RNA modification using mismatch profiling. These results illustrate the capability of our method to define enzyme–substrate pairs and determine modification sites of the largely uncharacterized radical SAM RNA methylating enzyme family

Effects of the Donor–Acceptor Distance and Dynamics on Hydride Tunneling in the Dihydrofolate Reductase Catalyzed Reaction

A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling in enhancing enzyme catalyzed reactions. Here, we report a correlation between the donor–acceptor distance (DAD) distribution and intrinsic kinetic isotope effects (KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction. This study compares the nature of the hydride-transfer step for a series of active-site mutants, where the size of a side chain that modulates the DAD (I14 in E. coli DHFR) is systematically reduced (I14V, I14A, and I14G). The contributions of the DAD and its dynamics to the hydride-transfer step were examined by the temperature dependence of intrinsic KIEs, hydride-transfer rates, activation parameters, and classical molecular dynamics (MD) simulations. Results are interpreted within the framework of the Marcus-like model where the increase in the temperature dependence of KIEs arises as a direct consequence of the deviation of the DAD from its distribution in the wild type enzyme. Classical MD simulations suggest new populations with larger average DADs, as well as broader distributions, and a reduction in the population of the reactive conformers correlated with the decrease in the size of the hydrophobic residue. The more flexible active site in the mutants required more substantial thermally activated motions for effective H-tunneling, consistent with the hypothesis that the role of the hydrophobic side chain of I14 is to restrict the distribution and dynamics of the DAD and thus assist the hydride-transfer. These studies establish relationships between the distribution of DADs, the hydride-transfer rates, and the DAD’s rearrangement toward tunneling-ready states. This structure–function correlation shall assist in the interpretation of the temperature dependence of KIEs caused by mutants far from the active site in this and other enzymes, and may apply generally to C–H→C transfer reactions