In vitro selection of deoxyribozymes with DNA capping activity.

In vitro selection was used to isolate a series of deoxyribozymes from a pool of random-sequence DNAs that catalyze an ATP-dependent self-capping reaction. Each deoxyribozyme catalyzes the transfer of the nucleoside and alpha-phosphate moieties of ATP to the phosphate group located at its 5' terminus, thereby creating a 5',5'-pyrophosphate cap. This same pyrophosphate cap structure is formed by T4 DNA ligase during the classical process of DNA ligation. These DNA capping enzymes representative of a collection of self-processing deoxyribozymes that can be used for the directed modification of DNA.     

Capping DNA with DNA

Twelve classes of deoxyribozymes that promote an ATP-dependent "self-capping" reaction were isolated by in vitro selection from a random-sequence pool of DNA. Each deoxyribozyme catalyzes the transfer of the AMP moiety of ATP to its 5'-terminal phosphate group, thereby forming a 5',5'-pyrophosphate linkage. An identical DNA adenylate structure is generated by the T4 DNA ligase during enzymatic DNA ligation. A 41-nucleotide class 1 deoxyribozyme requires Cu(2+) as a cofactor and adopts a structure that recognizes both the adenine and triphosphate moieties of ATP or dATP. The catalytic efficiency for this DNA, measured at 10(4) M(-1) x min(-1) using either ATP or dATP as substrate, is similar to other catalytic nucleic acids that use small substrates. Chemical probing and site-directed mutagenesis implicate the formation of guanine quartets as critical components of the active structure. The observation of ATP-dependent "self-charging" by DNA suggests that DNA could be made to perform the reactions typically associated with DNA cloning, but without the assistance of protein enzymes.     

Selection of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage

The chemical ligation of DNA molecules can be mediated by terminal phosphorothioate displacement of a 5' iodine. We have selected deoxyribozymes that can catalyze the formation of such phosphorothioester internucleotide linkages. The selected deoxyribozymes enhance the rate of ligation in part through the provision of a template that aligns the ligation junction and do not appear to require metal ions for catalysis.     

Metagenomics: Is it a powerful tool to obtain lipases for application in biocatalysis?

In recent years, metagenomic strategies have been widely used to isolate and identify new enzymes from uncultivable components of microbial communities. Among these enzymes, various lipases have been obtained from metagenomic libraries from different environments and characterized. Although many of these lipases have characteristics that could make them interesting for application in biocatalysis, relatively little work has been done to evaluate their potential to catalyze industrially important reactions. In the present article, we highlight the latest research on lipases obtained through metagenomic tools, focusing on studies of activity and stability and investigations of application in biocatalysis. We also discuss the challenges of metagenomic approaches for the bioprospecting of new lipases.     

Deoxyribozymes: new players in the ancient game of biocatalysis.

The repetitive and extraordinarily stable polynucleotide chains of DNA serve as an ideal storage system for genetic information. Although it is best known for its helical structure and relatively inert character, in vitro selection can be used to compel DNA to perform a surprising variety of chemical reactions. These artificial DNA enzymes or 'deoxyribozymes' generate large chemical rate enhancements and demonstrate precise substrate recognition, much like their protein and RNA counterparts. Recent studies with these prototypic deoxyribozymes indicate that DNA has a substantial untapped potential for intricate structure formation that could be exploited in novel chemical and biological catalysis.     

The α-thio and/or β-γ-hypophosphate analogs of ATP as cofactors of T4 DNA ligase

T4 DNA ligase is one of the most commonly used enzymes for in vitro molecular research and a useful model for testing the ligation mechanism of ATP-dependent DNA ligation. To better understand the influence of phosphate group modifications in the ligation process, a series of ATP analogs were tested as cofactors. P-diastereomers of newly developed β,γ-hypo-ATPαS (thio) and β,γ-hypo-ATP (oxo) were synthesized and their activity was compared to ATPαS and their natural precursors. The evaluation of presented ATP analogs revealed the importance of the α-phosphate stereogenic center in ATPαS for the T4 DNA ligase activity and sheds new light on the interaction between ATP-dependent DNA ligases and cofactors.     

DNA and RNA ligases: structural variations and shared mechanisms

DNA and RNA ligases join 3' OH and 5' PO4 ends in polynucleotide substrates using a three-step reaction mechanism that involves covalent modification of both the ligase enzyme and the polynucleotide substrate with AMP. In the past three years, several polynucleotide ligases have been crystallized in complex with nucleic acid, providing the introductory views of ligase enzymes engaging their substrates. Crystal structures for two ATP-dependent DNA ligases, an NAD+-dependent DNA ligase, and an ATP-dependent RNA ligase demonstrate how ligases utilize the AMP group and their multi-domain architectures to manipulate nucleic acid structure and catalyze the end-joining reaction. Together with unliganded crystal structures of DNA and RNA ligases, a more comprehensive and dynamic understanding of the multi-step ligation reaction mechanism has emerged.     

Recent advances in DNA catalysis

Studies of catalytically active DNA sequences have expanded considerably since the first artificial deoxyribozyme was identified in 1994. Nevertheless, the field is still quite young, and advances in both fundamental understanding and practical applications of deoxyribozymes are still developing. Deoxyribozymes that either cleave or ligate two RNA substrates have been most widely investigated, and this review describes recent advances in the fundamental studies and applications of these DNA enzymes. Deoxyribozymes with catalytic activities other than RNA ligation and cleavage are also increasingly pursued, and this review covers several key examples.     

原核生物细胞色素P450酶系及其应用

细胞色素P450酶系广泛分布于各种生物中,它们通常由一组基因超家族编码并含有血红素,能够催化一系列化学反应,具有多种生物学功能。特别是原核生物P450酶在催化内源性和外源性化合物的反应中具有重要的工业生产应用价值,成为近年来P450酶系研究的热点。本文对近年来原核生物P450酶系的重组表达和生物催化领域的研究进展进行综述。     

In vitro selection, characterization, and application of deoxyribozymes that cleave RNA

Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg²⁺ to catalyze attack by a specific RNA 2′-hydroxyl group on the adjacent phosphodiester linkage, forming a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.     

Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action.

ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.     

Exploitation of carbohydrate processing enzymes in biocatalysis

Exploitation of enzymes in biocatalytic processes provides scope both in the synthesis and degradation of molecules. Enzymes have power not only in their catalytic efficiency, but their chemoselectivity, regioselectivity, and stereoselectivity means the reactions they catalyze are precise and reproducible. Focusing on carbohydrate processing enzymes, this review covers advances in biocatalysis involving carbohydrates over the last 2–3 years. Given the notorious difficulties in the chemical synthesis of carbohydrates, the use of enzymes for synthesis has potential for significant impact in the future. The use of catabolic enzymes in the degradation of biomass, which can be exploited in the production of biofuels to provide a sustainable and greener source of energy, and the synthesis of molecules that have a range of applications including in the pharmaceutical and food industries will be explored.     

DNA ligase and the pyridine nucleotide cycle in Salmonella typhimurium.

Bacterial DNA ligases use NAD as an energy source. In this study we addressed two questions about these enzymes. First, what is the physiological consequence of completely removing the NAD-dependent enzyme and replacing it with an ATP-dependent DNA ligase? We constructed Salmonella typhimurium strains in which the endogenous NAD-dependent DNA ligase activity was inactivated by an insertion mutation and the ATP-dependent enzyme from bacteriophage T4 was provided by a cloned phage gene. Such strains were physiologically indistinguishable from the wild type, even under conditions of UV irradiation or treatment with alkylating agents. These results suggest that specific functional interactions between DNA ligase and other replication and repair enzymes may be unimportant under the conditions tested. Second, the importance of DNA ligation as the initiating event of the bacterial pyridine nucleotide cycle was critically assessed in these mutant strains. Surprisingly, our results indicate that DNA ligation makes a minimal contribution to the pyridine nucleotide cycle; the Salmonella strains with only an ATP-dependent ligase had the same NAD turnover rates as the wild-type strain with an NAD-dependent ligase. However, we found that NAD turnover was significantly decreased under anaerobic conditions. We suggest that most intracellular pyridine nucleotide breakdown occurs in a process that protects the cell against oxygen damage but involves a biochemical mechanism other than DNA ligation.     

The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases

ATP- and NAD(+)-dependent DNA ligases, ATP-dependent RNA ligases and GTP-dependent mRNA capping enzymes comprise a superfamily of proteins that catalyze nucleotidyl transfer to polynucleotide 5' ends via covalent enzyme-(lysyl-N)-NMP intermediates. The superfamily is defined by five peptide motifs that line the nucleotide-binding pocket and contribute amino acid sidechains essential for catalysis. Early crystal structures revealed a shared core tertiary structure for DNA ligases and capping enzymes, which are composed minimally of a nucleotidyltransferase domain fused to a distal OB-fold domain. Recent structures of viral and bacterial DNA ligases, and a fungal mRNA capping enzyme underscore how the substrate-binding and chemical steps of the ligation and capping pathways are coordinated with large rearrangements of the component protein domains and with remodeling of the atomic contacts between the enzyme and the nucleotide at the active site. The first crystal structure of an RNA ligase suggests that contemporary DNA ligases, RNA ligases and RNA capping enzymes evolved by fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.     

Characterization of bacteriophage T3 DNA ligase

DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.     

Relationship between bacteriophage T4 and T6 DNA topoisomerases. T6 39-protein subunit is equivalent to the combined T4 39- and 60-protein subunits

T6 DNA topoisomerase has been purified from bacteriophage T6 infected Escherichia coli. Unlike the T4 DNA topoisomerase which has three subunits, it consists of two subunits of molecular weights 75,000 and 51,000. They are the products of T6 genes 39 and 52, respectively. The purified T6 enzyme can stimulate in vitro T6 DNA replication. It has an ATP-dependent DNA relaxation activity similar to the T4 enzyme. Either ATP or dATP can be used in both reactions. Using a "Western blotting" and radioimmuno-detection methods, we show that T6 39 subunit contains protein sequences specified by both the T4 39 and 60 genes. The 52-proteins of both phages appear to be identical. The T4 and T6 topoisomerase genes represent a naturally occurring example of gene separation or fusion.     

Parallel Selections In Vitro Reveal a Preference for 2′–5′ RNA Ligation upon Deoxyribozyme-Mediated Opening of a 2′,3′-Cyclic Phosphate

We previously used in vitro selection to identify Mg²⁺-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5′-hydroxyl group with a 2′,3′-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2′–5′ phosphodiester bonds rather than native 3′–5′ linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2′–5′ linkages. This includes deoxyribozymes with an arrangement that favors 3′–5′ linkages for a different chemical reaction, that of a 2′,3′-diol plus 5′-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2′–5′ RNA linkages upon opening of a 2′,3′-cyclic phosphate with a 5′-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2′,3′-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.[Reviewing Editor: Niles Lehman]     

Concentration-dependent processivity of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase

Folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-gamma-glutamate derivatives of reduced folates and antifolates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH 4PteGlu 1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high-performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH 4PteGlu 1 catalyzed by FPGS to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to assess folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues to DDAH 4PteGlu 1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells.     

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。     

Bacterial DNA ligases

DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell. All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor. All Bacteria (eubacteria) contain NAD+-dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics. In addition to their NAD+-dependent enzymes, some Bacteria contain genes for putative ATP-dependent DNA ligases. The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism. The putative ATP-dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses. Phylogenetic analysis suggests that all NAD+-dependent DNA ligases are closely related, but the ATP-dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.