Abstract: |
RNA 3′-phosphate cyclase (RtcA) synthesizes RNA 2′,3′ cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-(histidinyl-Nε)-AMP intermediate; transfer of adenylate to an RNA 3′-phosphate to form RNA(3′)pp(5′)A; and attack of the vicinal O2′ on the 3′-phosphorus to form a 2′,3′ cyclic phosphate and release AMP. Here we report the crystal structures of RtcA·ATP, RtcA·ATP·Mn 2+, and RtcA·ATP·Co 2+ substrate complexes and an RtcA·AMP product complex. Together with the structures of RtcA apoenzyme and the covalent RtcA-AMP intermediate, they illuminate the mechanism of nucleotidyl transfer, especially the stereochemical transitions at the AMP phosphate, the critical role of the metal in orienting the PP i leaving group of ATP during step 1, and the protein conformational switches that accompany substrate binding and product release. The octahedral metal complex of RtcA·ATP·Mn 2+ includes nonbridging oxygens from each of the ATP phosphates, two waters, and Glu14 as the sole RtcA component. Whereas the RtcA adenylylation step is metal-catalyzed, the subsequent steps in the cyclization pathway are metal-independent. |