RNA-Catalyzed CoA, NAD, and FAD Synthesis from Phosphopantetheine, NMN, and FMN ² Faqing Huang,* ,‡ Charles Walter Bugg, ‡ and Michael Yarus § Department of Chemistry and Biochemistry, UniVersity of Southern Mississippi, Hattiesburg, Mississippi 39406-5043, and Department of Molecular, Cellular, and DeVelopmental Biology, UniVersity of Colorado at Boulder, Boulder, Colorado 80309-0347 ReceiVed August 31, 2000; ReVised Manuscript ReceiVed October 17, 2000 ABSTRACT: A novel in vitro selection method was developed to isolate RNA sequences with coenzyme- synthesizing activities. We used size-heterogeneous libraries containing randomized ribonucleotide sequences of four different lengths (30N, 60N, 100N, and 140N), all with 5′-ATP initiation. Two RNAs, CoES7 (30N) and CoES21 (60N), are able to catalyze the synthesis of three common coenzymes, CoA, NAD, and FAD, from their precursors, 4′-phosphopantetheine, NMN, and FMN, respectively. Both ribozymes require divalent manganese for activities. The results support the availability of these coenzymes in an RNA world, and point to a chemical explanation for the complex bipartite structures of many coenzymes. Coenzymes are densely functionalized small molecules. They play essential roles in metabolism by performing chemistry that is inefficient or impossible for typical amino acids. Three common coenzymes, coenzyme A (CoA), nicotinamide adenine dinucleotide (NAD), and flavin adenine dinucleotide (FAD), carry out a variety of acyl group and electron/hydride transfer reactions. Structurally, CoA, NAD, and FAD are complex (Figure 1), consisting of a ribonucleo- side adenosine, a pyrophosphate linkage, and a functional group (pantetheine, nicotinamide, or riboflavin). However, the chemically functional elements within these coenzymes are the simpler moieties: sulfhydryl, nicotinamide, and isoalloxazine, respectively. It has not been clear why these coenzymes have their complex conserved structures. While adenosine and pyro- phosphate serve to anchor these coenzymes to host proteins, it seems unlikely that coenzyme availability and reactivity are uniquely optimized by these groups. Therefore, the universality of adenosine pyrophosphate may be the result of evolutionary descent instead of functional requirement. In fact, the existence of these coenzymes in all kingdoms suggests their persistence since the last common ancestor of life on earth (1). Furthermore, the inclusion of ribonucleotide adenosine in these coenzymes may imply the utilization of CoA, NAD, and FAD in a more ancient RNA world (2) as parts of a prior generation of RNA enzymes, as suggested by White (3, 4). To gain insight into the complex nature of coenzyme structures relative to their functions, we have isolated relatively small RNA molecules that synthesize RNA-linked CoA, NAD, and FAD from their corresponding precursors, 4′-phosphopantetheine (pan-p), nicotinamide mononucleotide (NMN), and flavin mononucleotide (FMN). The finding suggests a plausible mechanism of coenzyme synthesis and utilization in the RNA world, and may provide clues about coenzyme origin and evolution. MATERIALS AND METHODS Substrate Preparation. Two terminal phosphate-containing molecules (Figure 2) were prepared for use as the substrates in the isolation of active ribozymes. The first compound, biotin-p, was prepared from the reaction of sulfo-NHS-SS- biotin (Pierce) with phosphocolamine (Fluka) as follows. Phosphocolamine (110 mg) and sulfo-NHS-SS-biotin (90 mg) were dissolved in 8 mL of water, and the pH of the solution was adjusted to 8.3 with 1.0 N NaOH. The sample was allowed to react with constant stirring for 4 h at room temperature. Two volumes of EtOH and 2 volumes of acetone were then added to precipitate biotin-p at -20 °C. A collected solid sample was dissolved in 8 mL of water, and the pH was adjusted to 3.7 with acetic acid. The solution was loaded onto a C18 Sep-Pak column (Waters), washed with water, and eluted with methanol. The sample was dried under vacuum and dissolved in 8 mL of solution. The solution pH was adjusted to 6.0 with 1.0 M NaOH. Amino group analysis by ninhydrin gave a negative result, indicating that phosphocolamine was absent. The sample was analyzed by mass spectrometry. The expected molecular weight (MW) is 530. Peaks found by ESI-MS (Mass Consortium, San Diego, CA) were as follows: positive ion at 575 (M - H + + 2Na + ) and negative ion at 529 (M - H + ). No free biotin peak was observed. The concentration of biotin-p was determined by alkaline phosphatase digestion followed by quantitative inorganic phosphate analysis (5). ² This work was supported by NSF Grant MCB9974487 to F.H. and NIH Grants GM30881 and GM48080 to M.Y. * To whom correspondence should be addressed. Phone: (601) 266- 4371. Fax: (601) 266-6075. E-mail: faqing.h.huang@usm.edu. ‡ University of Southern Mississippi. § University of Colorado at Boulder. 15548 Biochemistry 2000, 39, 15548-15555 10.1021/bi002061f CCC: $19.00 © 2000 American Chemical Society Published on Web 11/23/2000