Chiral polyamines from reduction of polypeptides: asymmetric pyridoxamine-mediated transaminations

BH3.THF can reduce polypeptides to polyamines with retention of chirality. The resulting polyamines are intriguing general platforms for asymmetric catalysis, given the diverse structures available and their relative ease of synthesis. We have constructed a number of chiral pyridoxamine catalysts based on reduced peptides. These compounds transaminate alpha-ketoacids with moderate to good enantioselectivity, while their peptidyl counterparts show almost no chiral induction.     

RNA: versatility in form and function

RNA performs a remarkable range of functions in all cells. In addition to its central role in information transfer from DNA to protein, it is essential for functions as diverse as RNA processing, chromosome end-maintenance and dosage compensation. The versatility of RNA derives from its unique ability to use direct readout via base-pairing for sequence specific targeting (or templating) in combination with its capacity to form elaborate three dimensional structures. Such structures can perform catalysis or serve as protein recognition surfaces. In this short review, we attempt to give a flavor for the diversity of functional RNAs in the cell and highlight, using selected examples, two quite distinct activities, catalysis and sequence specific targeting. Within each section, we discuss how the lessons we have learned from these systems may apply to other, less well understood, RNAs.     

Origins of tmRNA: the missing link in the birth of protein synthesis?

The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis.     

Exploring new methods and materials for enantioselective separations and catalysis

In this article, we will review and highlight some recent computational work on enantioselective adsorption and catalysis in zeolites and metal–organic frameworks. The design, development and understanding of chiral structures will help expand the utility of nanoporous materials into chiral technology. The highlighted works are examples of how molecular simulations can provide a fundamental understanding of chirality in nanoporous materials. This understanding is essential to help in the design and development of next-generation enantioselective separation devices and catalysts.     

A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage

Oligoribonucleotides containing a 5'-phosphorothiolate linkage have provided effective tools to study the mechanisms of RNA catalysis, allowing resolution of kinetic ambiguity associated with mechanistic dissection and providing a strategy to establish linkage between catalysis and specific functional groups. However, challenges associated with their synthesis have limited wider application of these modified nucleic acids. Here, we describe a general semisynthetic strategy to obtain these oligoribonucleotides reliably and relatively efficiently. The approach begins with the chemical synthesis of an RNA dinucleotide containing the 5'-phosphorothiolate linkage, with the adjacent 2'-hydroxyl group protected as the photolabile 2'-O-o-nitrobenzyl or 2'-O-α-methyl-o-nitrobenzyl derivative. Enzymatic ligation of the 2'-protected dinucleotide to transcribed or chemically synthesized 5' and 3' flanking RNAs yields the full-length oligoribonucleotide. The photolabile protecting group increases the chemical stability of these highly activated oligoribonucleotides during synthesis and long-term storage but is easily removed with UV irradiation under neutral conditions, allowing immediate use of the modified RNA in biochemical experiments.     

The model structure of the hammerhead ribozyme formed by RNAs of reciprocal chirality

RNA-based tools are frequently used to modulate gene expression in living cells. However, the stability and effectiveness of such RNA-based tools is limited by cellular nuclease activity. One way to increase RNA’s resistance to nucleases is to replace its D-ribose backbone with L-ribose isomers. This modification changes chirality of an entire RNA molecule to L-form giving it more chance of survival when introduced into cells. Recently, we have described the activity of left-handed hammerhead ribozyme (L-Rz, L-HH) that can specifically hydrolyse RNA with the opposite chirality at a predetermined location. To understand the structural background of the RNA specific cleavage in a heterochiral complex, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as performed molecular modelling and dynamics simulations of homo- and heterochiral RNA complexes. The active ribozyme-target heterochiral complex showed a mixed chirality as well as low field imino proton NMR signals. We modelled the 3D structures of the oligoribonucleotides with their ribozyme counterparts of reciprocal chirality. L- or D-ribozyme formed a stable, homochiral helix 2, and two short double heterochiral helixes 1 and 3 of D- or L-RNA strand thorough irregular Watson–Crick base pairs. The formation of the heterochiral complexes is supported by the result of simulation molecular dynamics. These new observations suggest that L-catalytic nucleic acids can be used as tools in translational biology and diagnostics.     

Structure of ribonuclease P--a universal ribozyme

Ribonuclease P (RNase P) is one of only two known universal ribozymes and was one of the first ribozymes to be discovered. It is involved in RNA processing, in particular the 5' maturation of tRNA. Unlike most other natural ribozymes, it recognizes and cleaves its substrate in trans. RNase P is a ribonucleoprotein complex containing one RNA subunit and as few as one protein subunit. It has been shown that, in bacteria and in some archaea, the RNA subunit alone can support catalysis. The structure and function of bacterial RNase P RNA have been studied extensively, but the detailed catalytic mechanism is not yet fully understood. Recently, structures of one of the structural domains and of the entire RNA component of RNase P from two different bacteria have been described. These structures provide the first atomic-level information on the structural assembly of the RNA component, and the regions involved in substrate recognition and catalysis. Comparison of these structures reveals a highly conserved core that comprises two universally conserved structural modules. Interestingly, the same structural core can be found in the context of different scaffolds.     

Stable RNA structures can repress RNA synthesis in vitro by the brome mosaic virus replicase

A 15-nucleotide (nt) unstructured RNA with an initiation site but lacking a promoter could direct the initiation of RNA synthesis by the brome mosaic virus (BMV) replicase in vitro. However, BMV RNA with a functional initiation site but a mutated promoter could not initiate RNA synthesis either in vitro or in vivo. To explain these two observations, we hypothesize that RNA structures that cannot function as promoters could prevent RNA synthesis by the BMV RNA replicase. We documented that four different nonpromoter stem-loops can inhibit RNA synthesis from an initiation-competent RNA sequence in vitro. Destabilizing these structures increased RNA synthesis. However, RNA synthesis was restored in full only when a BMV RNA promoter element was added in cis. Competition assays to examine replicase-RNA interactions showed that the structured RNAs have a lower affinity for the replicase than do RNAs lacking stable structures or containing a promoter element. The results characterize another potential mechanism whereby the BMV replicase can specifically recognize BMV RNAs.     

Generation and selection of ribozyme variants with potential application in protein engineering and synthetic biology

Over the past two decades, RNA catalysis has become a major topic of research. On the one hand, naturally occurring ribozymes have been extensively investigated concerning their structure and functional mechanisms. On the other hand, the knowledge gained from these studies has been used to engineer ribozyme variants with novel properties. In addition to RNA engineering by means of rational design, powerful techniques for selection of ribozymes from large pools of random sequences were developed and have been widely used for the generation of functional nucleic acids. RNA as catalyst has been accompanied by DNA, and nowadays a large number of ribozymes and deoxyribozymes are available. The field of ribozyme generation and selection has been extensively reviewed. With respect to the field of biotechnology, RNA and DNA catalysts working on peptides or proteins, or which are designed to control protein synthesis, are of utmost importance and interest. Therefore, in this review, we will focus on engineered nucleic acid catalysts for peptide synthesis and modification as well as for intracellular control of gene expression.     

'In-line attack' conformational effect plays a modest role in an enzyme-catalyzed RNA cleavage: a free energy simulation study

Since the proposal of ‘in-line attack’ conformation as a possibly important intermediate in RNA cleavage, its structure has been captured in various protein and RNA enzymes; these structures strengthen the belief that this conformation plays an essential role in the catalysis of RNA cleavage. As generally discussed, this intermediate structure can be involved in energy barrier reduction in two possible ways, e.g. through either conformational effect or electrostatic effect. In order to quantitatively elucidate the contribution of conformational effect in this type of enzyme catalysis, free energy simulations were performed on the RNA structures both in a splicing endonuclease complex and in the aqueous solution. Our free energy simulation results revealed that the ‘in-line attack’ conformational effect plays a modest role in facilitating the reaction rate enhancement (~12-fold) compared with the overall 10¹²-fold rate increase. The close agreement between the present computational estimation and an experimental measurement on the spontaneous RNA cleavage in an in vitro evolved ATP aptamer motives us to realize that the conformation distribution of an enzyme substrate prior to rather than after its binding determines the upper bound of the rate enhancement ability through the conformational strategy.     

Comparative analysis of hairpin ribozyme structures and interference data

Great strides in understanding the molecular underpinnings of RNA catalysis have been achieved with advances in RNA structure determination by NMR spectroscopy and X-ray crystallography. Despite these successes the functional relevance of a given structure can only be assessed upon comparison with biochemical studies performed on functioning RNA molecules. The hairpin ribozyme presents an excellent case study for such a comparison. The active site is comprised of two stems each with an internal loop that forms a series of non-canonical base pairs. These loops dock into each other to create an active site for catalysis. Recently, three independent structures have been determined for this catalytic RNA, including two NMR structures of the isolated loop A and loop B stems and a high-resolution crystal structure of both loops in a docked conformation. These structures differ significantly both in their tertiary fold and the nature of the non-canonical base pairs formed within each loop. Several of the chemical groups required to achieve a functioning hairpin ribozyme have been determined by nucleotide analog interference mapping (NAIM). Here we compare the three hairpin structures with previously published NAIM data to assess the convergence between the structural and functional data. While there is significant disparity between the interference data and the individual NMR loop structures, there is almost complete congruity with the X-ray structure. The only significant differences cluster around an occluded pocket adjacent to the scissile phosphate. These local differences may suggest a role for these atoms in the transition state, either directly in chemistry or via a local structural rearrangement.     

The hairpin ribozyme

The hairpin ribozyme is a naturally occurring RNA that catalyzes sequence-specific cleavage and ligation of RNA. It has been the subject of extensive biochemical and structural studies, perhaps the most detailed for any catalytic RNA to date. Comparison of the structures of its constituent domains free and fully assembled demonstrates that the RNA undergoes extensive structural rearrangement. This rearrangement results in a distortion of the substrate RNA that primes it for cleavage. This ribozyme is known to achieve catalysis employing exclusively RNA functional groups. Metal ions or other catalytic cofactors are not used. Current experimental evidence points to a combination of at least four mechanistic strategies by this RNA: (1) precise substrate orientation, (2) preferential transition state binding, (3) electrostatic catalysis, and (4) general acid base catalysis.     

Enzymatic and chemoenzymatic approaches to polymer synthesis.

The function of many specialized polymers calls for properties such as chirality and biodegradability. The stereo, -positional-and chemo-selectivities characteristic of enzymatic catalysis are highly desirable attributes for incorporation into strategies for synthesizing such polymers. Enzymes alone, or in combination with chemical synthesis (i.e. chemoenzymatic methodologies), are finding increased use in the synthesis of novel materials. Potential applications include water-absorbents, hydrogels, biodegradable materials, chiral adsorbents, liquid crystals and permselective membranes.     

Metal-ion-dependent catalysis and specificity of CCA-adding enzymes: a comparison of two classes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases] catalyze synthesis of the conserved and essential CCA sequence to the tRNA 3' end. These enzymes are divided into two classes of distinct structures that differ in the overall orientation of the head to tail domains. However, the catalytic core of the two classes is conserved and contains three carboxylates in a geometry commonly found in DNA and RNA polymerases that use the two-metal-ion mechanism for phosphoryl transfer. Two important aspects of the two-metal-ion mechanism are tested here for CCA enzymes: the dependence on metal ions for catalysis and for specificity of nucleotide addition. Using the archaeal Sulfolobus shibabae enzyme as an example of the class I, and the bacterial Escherichia coli enzyme as an example of the class II, we show that both enzymes depend on metal ions for catalysis, and that both use primarily Mg2+ and Mn2+ as the "productive" metal ions, but several other metal ions such as Ca2+ as the "nonproductive" metal ions. Of the two productive metal ions, Mg2+ specifically promotes synthesis of the correct CCA, whereas Mn2+ preferentially accelerates synthesis of the noncognate CCC and poly(C). Thus, despite evolution of structural diversity of two classes, both classes use metal ions to determine catalysis and specificity. These results provide critical insights into the catalytic mechanism of CCA synthesis to allow the two classes to be related to each other, and to members of the larger family of DNA and RNA polymerases.     

Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P

In bacteria, the tRNA-processing endonuclease ribonuclease P is composed of a large ( approximately 400 nucleotide) catalytic RNA and a smaller ( approximately 100 amino acid) protein subunit that is essential for substrate recognition. Current biochemical and biophysical investigations are providing fresh insights into the modular architecture of the ribozyme, the mechanisms of substrate specificity and the role of essential metal ions in catalysis. Together with recent high-resolution structures of portions of the ribozyme, these findings are beginning to reveal how the functions of RNA and protein are coordinated in this ribonucleoprotein enzyme.     

Understanding the transition states of phosphodiester bond cleavage: insights from heavy atom isotope effects

The nucleotides of DNA and RNA are joined by phosphodiester linkages whose synthesis and hydrolysis are catalyzed by numerous essential enzymes. Two prominent mechanisms have been proposed for RNA and protein enzyme catalyzed cleavage of phosphodiester bonds in RNA: (a) intramolecular nucleophilic attack by the 2'-hydroxyl group adjacent to the reactive phosphate; and (b) intermolecular nucleophilic attack by hydroxide, or other oxyanion. The general features of these two mechanisms have been established by physical organic chemical analyses; however, a more detailed understanding of the transition states of these reactions is emerging from recent kinetic isotope effect (KIE) studies. The recent data show interesting differences between the chemical mechanisms and transition state structures of the inter- and intramolecular reactions, as well as provide information on the impact of metal ion, acid, and base catalysis on these mechanisms. Importantly, recent nonenzymatic model studies show that interactions with divalent metal ions, an important feature of many phosphodiesterase active sites, can influence both the mechanism and transition state structure of nonenzymatic phosphodiester cleavage. Such detailed investigations are important because they mimic catalytic strategies employed by both RNA and protein phosphodiesterases, and so set the stage for explorations of enzyme-catalyzed transition states. Application of KIE analyses for this class of enzymes is just beginning, and several important technical challenges remain to be overcome. Nonetheless, such studies hold great promise since they will provide novel insights into the role of metal ions and other active site interactions.     

手性技术与生物催化

简要介绍了手性,手性技术与生物催化的基本概念。手性,是指一个有机分子具有不对称性,形成两种空间排布方式不同的对映异构体。手性技术即生产手性化合物的技术,手性化合物的制备方法主要有手性源、外消旋体拆分、不对称合成等几种。生物催化,即利用酶或微生物等生物材料催化进行某种化学反应,被认为是手性化合物生产取得突破的关健技术。文章还介绍了生物催化外消旋体拆分、生物催化不对称合成等几种生产手性化合物的应用实例。     

3 Origins and evolution of the translation machinery

The protein synthesis machinery largely evolved prior to the last common ancestor and hence its study can provide insight to early events in the origin of life, including the transition from the hypothetical RNA world to living systems as we know them. By utilizing information from primary sequences, atomic resolution structures, and functional properties of the various components, it is possible to identify timing relationships (Hsiao et al., 2009; Fox, 2010). Taken together, these timing events are used to develop a preliminary time line for major evolutionary events leading to the modern protein synthesis machinery. It has been argued that a key initial event was the hybridization of two or more RNAs that created the peptidyl transferase center, (PTC), of the ribosome (Agmon et al. 2005). The PTC, left side of figure, contains a characteristic cavity/pore that serves as the entrance to the exit tunnel and is thought to be essential to the catalysis (Fox et al., 2012). This cavity is distinct from typical RNA pores (right side of figure) in that the nitrogenous bases face towards the lumen of the pore and thus are available for hydrogen bonding interactions. In typical RNA pores, the bases carefully avoid the lumen region. In support of Agmon et al. 2005), it is argued that this key difference reflects the fact the pore was created by an early hybridization event rather than normal RNA folding.