Distinct Contributions of Enzymic Functional Groups to the 2',3'-Cyclic Phosphodiesterase, 3'-Phosphate Guanylylation, and 3'-ppG/5'-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway.

UNLABELLED: Escherichia coli RtcB is a founding member of a family of manganese-dependent RNA repair enzymes that join RNA 2′,3′-cyclic phosphate (RNA>p) or RNA 3′-phosphate (RNAp) ends to 5′-OH RNA (HORNA) ends in a multistep pathway whereby RtcB (i) hydrolyzes RNA>p to RNAp, (ii) transfers GMP from GTP to RNAp to form to RNAppG, and (iii) directs the attack of 5′-OH on RNAppG to form a 3′-5′ phosphodiester splice junction. The crystal structure of the homologous archaeal RtcB enzyme revealed an active site with two closely spaced manganese ions, Mn1 and Mn2, that interact with the GTP phosphates. By studying the reactions of wild-type E. coli RtcB and RtcB alanine mutants with 3′-phosphate-, 2′,3′-cyclic phosphate-, and 3′-ppG-terminated substrates, we found that enzymic constituents of the two metal coordination complexes (Cys78, His185, and His281 for Mn1 and Asp75, Cys78, and His168 for Mn2 in E. coli RtcB) play distinct catalytic roles. For example, whereas the C78A mutation abolished all steps assayed, the D75A mutation allowed cyclic phosphodiester hydrolysis but crippled 3′-phosphate guanylylation, and the H281A mutant was impaired in overall HORNAp and HORNA>p ligation but was able to seal a preguanylylated substrate. The archaeal counterpart of E. coli RtcB Arg189 coordinates a sulfate anion construed to mimic the position of an RNA phosphate. We propose that Arg189 coordinates a phosphodiester at the 5′-OH end, based on our findings that the R189A mutation slowed the step of RNAppG/HORNA sealing by a factor of 200 compared to that with wild-type RtcB while decreasing the rate of RNAppG formation by only 3-fold. IMPORTANCE: RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that ligate 2′,3′-cyclic phosphate ends to 5′-OH ends via RNA 3′-phosphate and RNA(3′)pp(5′)G intermediates. The RtcB active site includes two adjacent manganese ions that engage the GTP phosphates. Alanine scanning of Escherichia coli RtcB reveals distinct contributions of metal-binding residues Cys78, Asp75, and His281 at different steps of the RtcB pathway. The RNA contacts of RtcB are uncharted. Mutagenesis implicates Arg189 in engaging the 5′-OH RNA end.