Expanding the chemical range of RNA-mediated catalysis : structure and mechanism

在活细胞中发生的所有化学反应都是由酶催化的，其中绝大多数是由蛋白质组成的。然而，有一小部分酶是由RNA组成的，称为核酶。这些都非常重要，原因有很多。首先，它们催化一些非常重要的生物反应，如蛋白质合成。其次，它们可能在地球上生命的早期发展中发挥了关键作用，当时RNA可能既是遗传信息的载体，也是新陈代谢反应的催化剂。第三，理解化学上简单的(与蛋白质的组合复杂性相比)RNA如何起催化剂的作用对生物化学家来说是一个挑战，并提供了对生物催化机制的一般洞察。最后，RNA催化剂有可能为有机化学、生物技术和医学提供有用和新颖的试剂和工具。已知的大多数天然核酶催化生成或断裂磷-氧键的反应。如何利用有限的RNA化学资源来催化这些磷酸基转移反应在一定程度上已经被理解，但重要的问题仍然存在。在自然界中不断发现新的核酶，例如出现在人类转座元件中的Line-1核酶。这些为现有的核酶机制分类提供了一个新的视角。原始的RNA催化的新陈代谢需要更大范围的反应来加速，包括诸如碳-碳键和碳-氮键的形成等困难的反应。体外选择提供了催化不同化学反应的核酶来源。我们最近解决了一种选定的RNA的晶体结构，该RNA催化从O6-甲基鸟嘌呤或O6-烷基鸟嘌呤到RNA腺嘌呤核苷酸上的特定氮原子的甲基或烷基转移。甲基转移酶核酶目前在生物化学领域引起了人们的极大兴趣，因为RNA的甲基化程度及其功能仍在阐明中。我们现在正在使用结构和机制相结合的方法来充分阐明这种核酶的机制，我们目前的信息已经表明，核酶使用复杂的化学机制来加速反应。我们建议将这种烷基转移酶核酶发展成一种工具来设计RNA的定点修饰，包括荧光探针和用于交联的位点，首先是在体外，然后是在体内。尽管RNA可以进行非凡的催化壮举，速度可以加快一百万倍或更多，但可能的化学范围将受到RNA相对化学简单的限制。然而，辅酶的招募可能会极大地扩大RNA的催化谱系。这些是与酶结合并参与催化反应的小分子。RNA是一种很好的结合小分子的受体，这一点在生物学上被RNA中的核糖开关所利用，这些核糖开关可以结合代谢产物来控制邻近的基因。许多核糖开关结合了强大的辅酶，如S-腺苷蛋氨酸、硫胺素焦磷酸和烟酰胺腺嘌呤二核苷酸，这些都可以极大地扩展核酶的催化谱，我们假设一些辅酶结合的核糖开关已经从核酶进化而来。我们建议通过使用体外选择将这种核糖开关反向工程成核酶来检验这一想法。这种核酶可以潜在地催化多种化学转化，包括C-C键的形成和氧化还原反应。这种新型催化剂可能在化学合成和诊断方面有应用。

All chemical reactions that take place in living cells are catalysed by enzymes, the great majority of which are made of protein. However, a small subset of enzymes are made of RNA, called ribozymes. These are very important for a number of reasons. First, they catalyse some very important biological reactions, such as protein synthesis. Second, they probably played a key role in the early development of life on the planet, when RNA likely served as both the carrier of genetic information and the catalyst for metabolic reactions. Third, understanding how the chemically-simple (compared to combinatorial complexity of protein) RNA can act as a catalyst is a challenge to the biological chemist, and offers general insight into the mechanisms of biocatalysis. Lastly, RNA catalysts could potentially provide a source of useful and novel reagents and tools for organic chemistry, biotechnology and medicine. The great majority of known, natural ribozymes catalyse reactions making or breaking phosphorus-oxygen bonds. How the limited chemical resources of RNA are exploited to catalyse these phosphoryl transfer reactions is understood to some degree, but important questions remain. New ribozymes continue to be found in nature, such as the LINE-1 ribozyme that occurs in a human transposable element. These offer a new perspective on the existing mechanistic classification of ribozymes.A primitive RNA-catalysed metabolism would have required a much greater range of reactions to be accelerated, including 'difficult' reactions such as the formation of carbon-carbon and carbon-nitrogen bonds. In vitro selection provides a source of ribozymes that catalyse different chemical reactions. We have recently solved the crystal structure of a selected RNA that catalyses the transfer of a methyl or alkyl group from O6-methyl guanine or O6-alkyl guanine to a specific nitrogen atom on an adenine nucleotide of the RNA. Methyl transferase ribozymes are currently creating a lot of interest in biological chemistry as the full extent of methylation of RNA and its functions are still being elucidated. We are now using a combination of structural and mechanistic approaches to elucidate fully the mechanism of this ribozyme, and our present information already indicates that the ribozyme uses a sophisticated chemical mechanism to accelerate the reaction. We propose to develop this alkyl transferase ribozyme into a tool to engineer site-specific modification of RNA, including fluorescent probes and sites for crosslinking, first in vitro and subsequently in vivo. Although RNA can carry out remarkable feats of catalysis, with rate acceleration of a million-fold or more, the range of chemistry that is possible will be limited by the relative chemical simplicity of RNA. However the catalytic repertoire of RNA might be greatly expanded by the recruitment of co-enzymes. These are small molecules that bind to enzymes and participate in the reaction that is catalysed. RNA is an excellent receptor for binding small molecules, and this is exploited in biology by riboswitches in mRNA that bind metabolites to control adjacent genes. Many riboswitches bind powerful co-enzymes like S-adenosyl methionine, thiamine pyrophosphate and nicotinamide adenine dinucleotide that could greatly expand the catalytic repertoire of ribozymes, and we have hypothesised that some coenzyme-binding riboswitches have evolved from RNA world ribozymes. We propose to test this idea by reverse-engineering such riboswitches into ribozymes using in vitro selection. Such ribozymes could potentially catalyse a wide variety of chemical conversions including the formation of C-C bonds and oxidation-reduction reactions. Such novel catalysts could have applications in chemical synthesis and diagnostics.