Ribozymes and Cofactors

One of the acknowledged benefits of using proteins as catalysts is that the available chemical diversity of the amino acid side chains is much greater than that of the four canonical ribonucleotides. Amino acids enable reactions such as proton exchange near neutral pH (histidine), thiol chemistry (cysteine) and nucleophilic attack (serine). Amino acids also have divergent physicochemical properties and include positively charged moieties (histidine, lysine, arginine) and hydrophobic groups (isoleucine, leucine, valine and so forth). These features are largely absent in natural nucleic acids. Nonetheless, even with this seemingly vast advantage many protein catalysts still require cofactors. Interestingly, many ofthese cofactors are nucleotide-based (Fig. 11.1). As suggested above, this correlation strongly suggests that these cofactors were established in and have been carried over from the RNA World.

The early existence of nucleotide-based cofactors in the RNA World is plausible as both pantetheine (a CoA precursor) and nicotinamide (a NAD precursor) would have been synthesized from materials that should have been prebiotically available.18,19 There are different models for how cofactors could have been utilized by early ribozymes, but one can imagine that nucleotide-based cofactors could have been covalently appended to nucleic acid catalysts as a consequence of nonspecific ligation reactions. Indeed the Group I intron ribozyme has been shown to be capable of covalently attaching itself to cofactors (NAD+) and cofactor analogs (de-phosphorylated CoA) using the same mechanism it normally uses to insert guanosine into RNA during splicing initiation.20 Similar mechanisms may have also played a role in the establishment of amino acids and translation, as we will discuss below.

As an example of how covalently-attached cofactors would have augmented ribozyme functionality, directed evolution experiments have shown that redox active, alcohol oxidase ribozymes that utilize NAD+ can be selected from random sequence pools.21 The same ribozyme was also shown to catalyze the reverse reaction—a reduction of benzyl aldehyde back to benzyl alcohol.22 Two other interesting facts emerged from these studies: first, the alcohol dehydrogenase ribozymes selected had an absolute requirement for Zn2+, which is also a requirement of the protein enzyme. Second, oxidation of the alcohol could occur by generating NAD+ via uncatalyzed hydride transfer from NADH to FAD, indicating that coupled redox reactions similar to those commonly observed in modern biochemistry could have also arisen in the earliest metabolic pathways.

Directed evolution has also been used to generate ribozymes that can utilize noncovalently attached cofactors. An adenosine-binding RNA domain (the RNA equivalent of the Rossman fold found in proteins) was appended to a random sequence pool and kinase ri-bozymes that could catalyze the transfer ofthe thiophosphate from soluble ATP-yS to the 5'-hydroxyl of the ribozyme were selected by capturing self-kinases on a thiol column.23 One of the selected ribozymes was able to catalyze multiple turnover phosphorylation of an oligonucleotide identical to the 5' end of the ribozyme, a reaction akin to the one catalyzed by the protein enzyme poly-nucleotide kinase.

The availability of small amounts of premade cofactors in the prebiotic soup might have led not only to the evolution of cofac-tor-dependent ribozymes, but also to the evolution of biosynthetic pathways to reproduce the cofactors. Satisfyingly, directed evolution experiments have demonstrated that ribozymes can catalyze the formation of cofactors from precursors. A pyrophosphate transferase ('cappase') ribozyme originally evolved by Yarus and coworkers has been shown to have very loose substrate specificity.24 Virtually any molecule that contained a phosphate can add itselfto the 5' triphosphate ofthe ribozyme, displacing pyrophosphate. This ribozyme was initially shown to be able to attach cofactors to itself, similar to the aforementioned experiments with the Group I intron. Other ribozyme variants joined precursors and the 5' adenosine of the ribozyme to form common cofactors containing canonical disphosphate linkages25 (Fig. 11.3). For example, the ribozyme could synthesize CoA, NAD and FAD from their 4'-phosphopantetheine, nicotinamide mononucleotide (NMN) and flavin mononucleotide (FMN) precursors, respectively. These experiments again support the hypothesis that cofactors could have been covalently attached to early RNA catalysts, possibly at their termini. As metabolism expanded and the demand for diffusible cofactors increased the covalently-linked cofactors may have become detached and the ribozymes would evolve to synthesize the cofactors using ATP as a substrate in trans. Whether cofactors began as tethered appendages

Ribozyme Prebiotic

Figure 11.3. Ribozyme mechanism for cofactor synthesis. Virtually any chemical moiety (R) that contains a phosphate can react with the terminal ATP on the 'cappase' ribozyme to attach itself to the ribozyme's 5' terminus. The R-group could be any of the several cofactor moieties shown in Figure 11.1.

Figure 11.3. Ribozyme mechanism for cofactor synthesis. Virtually any chemical moiety (R) that contains a phosphate can react with the terminal ATP on the 'cappase' ribozyme to attach itself to the ribozyme's 5' terminus. The R-group could be any of the several cofactor moieties shown in Figure 11.1.

or as diffusible moieties with nucleotide handles is debatable, but what is clear is that the ability to synthesize and utilize cofactors could have been present in the early RNA World.26

Directed evolution experiments have been used to extensively explore ribozyme reactions relevant to primordial biosynthesis, many of which require cofactors. For a more detailed review of ribozyme chemistry and cofactors the reader is encouraged to read the references listed in the Further Reading Section.

0 0

Post a comment