Deoxyribozymes: new activities and new applications

DNA in its single-stranded form has the ability to fold into complex three-dimensional structures that serve as highly specific receptors or catalysts. Only protein enzymes and ribozymes are known to be responsible for biological catalysis, but deoxyribozymes with kinetic parameters that rival ribozymes can be created in the laboratory. Some of these engineered DNA catalysts are showing surprising potential as therapeutic agents, which makes them biologically relevant if not biologically derived. If DNA's natural role is strictly genomic, how significant is its innate catalytic prowess? New examples of engineered deoxyribozymes serve as empirical examples of the potential for catalysis by DNA. These results indicate that the true catalytic power of DNA is limited by discovery and not by chemistry.     

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p₁ ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p₂ and p₀ have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p₂ , and Lys-66 in p₀ ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p₂ are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p₀ electrostatic interaction contributes only to binding of the RNA.     

The pleiotropic functions of aspirin: mechanisms of action

Recent studies have suggested that aspirin and aspirin-like compounds have a variety of actions in addition to their well-studied ability to inhibit cyclooxygenases. These actions include inhibition of the uncoupling of oxidative phosphorylation, decreases in adenosine triphosphate stores, increases in extracellular adenosine, downregulation of the expression and activity of inducible nitric oxide synthetase, inhibition and/or stimulation of various mitogen-activated protein kinase activities and inhibition of nuclear factor binding κB site (NF-κB) activation. Moreover, aspirin-like compounds have recently been shown to have previously unappreciated clinical and biological effects, some apparently independent of cyclooxygenase. In this review we discuss the various mechanisms of action of aspirin-like compounds and their relevance to clinical disease and therapy. Received 1 February 1999; received after revision 1 April 1999; accepted 7 May 1999     

Review¶Psychrophilic enzymes: molecular basis of cold adaptation

Psychrophilic organisms have successfully colonized polar and alpine regions and are able to grow efficiently at sub-zero temperatures. At the enzymatic level, such organisms have to cope with the reduction of chemical reaction rates induced by low temperatures in order to maintain adequate metabolic fluxes. Thermal compensation in cold-adapted enzymes is reached through improved turnover number and catalytic efficiency. This optimization of the catalytic parameters can originate from a highly flexible structure which provides enhanced abilities to undergo conformational changes during catalysis. Thermal instability of cold-adapted enzymes is therefore regarded as a consequence of their conformational flexibility. A survey of the psychrophilic enzymes studied so far reveals only minor alterations of the primary structure when compared to mesophilic or thermophilic homologues. However, all known structural factors and weak interactions involved in protein stability are either reduced in number or modified in order to increase their flexibility.     

Insights into acylphosphatase structure and catalytic mechanism

Acylphosphatase is one of the smallest enzymes known (about 98 amino acid residues). It is present in organs and tissues of vertebrate species as two isoenzymes sharing over 55% of sequence homology; these appear highly conserved in differing species. The two isoenzymes can be involved in a number of physiological processes, though their effective biological function is not still certain. The solution and crystal structures of different isoenzymes are known, revealing a close packed protein with a fold similar to that shown by other phosphate-bind ing proteins. The structural data, together with an extended site-directed mutagenesis investigation, led to the identification of the residues involved in enzyme catalysis. However, it appears unlikely that these residues are able to perform the full catalytic cycle: a substrate-assisted catalytic mechanism has therefore been proposed, in which the phosphate moiety of the substrate could act as a nucleophile activating the catalytic water molecule. Received 12 November 1996; accepted 27 November 1996     

Water in enzyme reactions: biophysical aspects of hydration-dehydration processes

Water has been recognized as one of the major structuring factors in biological macromolecules. Indeed, water clusters influence many aspects of biological function, and the water-protein interaction has long been recognized as a major determinant of chain folding, conformational stability, internal dynamics, binding specificity and catalysis. I discuss here several themes arising from recent progress in understanding structural aspects of ‘direct’ and ‘indirect’ ligands in terms of enzyme-substrate interactions, and the role of water bridges in enzyme catalysis. The review also attempts to illuminate issues relating to efficiency, through solvent interactions associated with enzymic specificity, and versatility. Over the years, carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) has played a significant role in the continuing delineation of principles underlying the role of water in enzyme reactions. As a result of its pronounced catalytic power and robust constitution CA was transformed into a veritable ‘laboratory’ in which active site mechanisms were rigorously tested and explored.     

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus

In the hepatitis delta virus, ribozymes are encoded in both the genomic strand RNA and its complement, the antigenomic strand. The two ribozymes are similar in sequence and structure, are most active in the presence of divalent cation and catalyze RNA cleavage reactions which generate a 5′-hydroxyl group and a 2′,3′-cyclic phosphate group. Recent progress has been made in understanding the catalytic mechanism. One key was a crystal structure of the genomic ribozyme that revealed a specific cytosine positioned to act as a general acid-base catalyst. The folding of the ribozyme in the context of the longer viral RNA is another area of interest. The biology requires that each ribozyme act only once, and mechanisms proposed for regulation of ribozyme activity sometimes invoke alternative RNA structures. Likewise, interference of ribozyme function by polyadenylation of the antigenomic RNA strand could be controlled through alternative structures, and a model for such control is proposed. Received 21 June 2001; received after revision 18 July 2001; accepted 20 July 2001     

Homing endonucleases: structure, function and evolution

‘Homing’ is the lateral transfer of an intervening genetic sequence, either an intron or an intein, to a cognate allele that lacks that element. The end result of homing is the duplication of the intervening sequence. The process is initiated by site-specific endonucleases that are encoded by open reading frames within the mobile elements. Several features of these proteins make them attractive subjects for structural and functional studies. First, these endonucleases, while unique, may be contrasted with a variety of enzymes involved in nucleic acid strand breakage and rearrangement, particularly restriction endonucleases. Second, because they are encoded within the intervening sequence, there are interesting limitations on the position and length of their open reading frames, and therefore on their structures. Third, these enzymes display a unique strategy of flexible recognition of very long DNA target sites. This strategy allows these sequences to minimize nonspecific cleavage within the host genome, while maximizing the ability of the endonuclease to cleave closely related variants of the homing site. Recent studies explain a great deal about the biochemical and genetic mechanisms of homing, and also about the structure and function of several representative members of the homing endonuclease families. Received 6 January 1999; received after revision 24 February 1999; accepted 24 February 1999     

Deoxyribozymes: useful DNA catalysts in vitro and in vivo

Deoxyribozymes (DNA enzymes; DNAzymes) are catalytic DNA sequences. Using the technique of in vitro selection, individual deoxyribozymes have been identified that catalyze RNA cleavage, RNA ligation, and a growing range of other chemical reactions. DNA enzymes have been used in vitro for applications such as biochemical RNA manipulation and analytical assays for metal ions, small organic compounds, oligonucleotides, and proteins. Deoxyribozymes have also been utilized as in vivo therapeutic agents to destroy specific mRNA targets. Although many conceptual and practical challenges remain to be addressed, deoxyribozymes have substantial promise to contribute meaningfully for applications both in vitro and in vivo.     

The right neuron at the wrong place: biology of heterotopic neurons in cortical neuronal migration disorders, with special reference to associated pathologies

During the development of the neocortex, neurogenesis and neuronal differentiation occur in two separate locations. Thus neurons have to migrate through the future white matter. Arrested or excessive migration leads neurons to differentiate in a heterotopic position. Such neuronal migration disorders (NMDs) occur sporadically in normal development but are markedly increased as a consequence of genetic defects or after exposure to toxic drugs during the period of migration. Anatomofunctional studies in rodents with NMDs have revealed that heterotopic neurons form essentially normal afferent and efferentconnections, which has been interpreted as evidence that the connectionpattern of cortical neurons is specified prior to migration. In addition, recent data show that heterotopic neurons can be contacted by environmental, that is local, fibres that normally never innervate the neocortex. This dual connectivity leads heterotopias to form bridges between their environmental and original network. Such an abnormal pattern of connectivity could contribute to the pathophysiology of disorders associated with NMDs such as epilepsy. Received 16 December 1998; received after revision 5 February 1999; accepted 9 February 1999     

Short-range linkage relationships, genomic organisation and sequence comparisons of a cluster of five HSP70 genes in Fugu rubripes

Twelve cosmids containing sequences resembling genes encoding members of the 70-kDa heat-shock protein family, HSP70, have been isolated from Fugu rubripes. They can be broadly divided into three groups of overlapping cosmids. Restriction analysis and sequencing of one set of five cosmids have revealed five intronless Fugu HSP70 genes spanning 42 kb, arranged in a combined head-to-head, tail-to-tail and head-to-tail orientation. The levels of DNA and amino acid identity are very high with respect to one another, and are most similar to HSP70 sequences linked to the major histocompatibility complex (MHC) region in other species. Putative heat-shock consensus elements are identified. Non-HSP70 sequences with homology to known genes have been found physically linked to this Fugu HSP70 cluster: the Drosophila melanogaster SOL gene, the Drosophila melanogaster nemo gene, the Caenorhabditis elegans T17E9.1 gene and the sequence encoding the serine protease domain. The linkage relationships described here so far bear no resemblance to those of HSP70 in other organisms. Convergence of mammalian HSP70 and MHC class I and II loci probably occurred after fish had diverged. Received 17 November 1998; received after revision 25 February 1999; accepted 26 February 1999     

Specific localization of the catalytic subunits of protein kinase CK2 at the centrosomes

The protein kinase CK2 holoenzyme is composed of two regulatory β subunits and two catalytic α or α' subunits. Although experimental evidence for involvement of the enzyme in the regulation of cell proliferation is accumulating, the exact mechanism of its action is still unclear. The subcellular localization of the enzyme may be a key to its function. We have recently shown that the CK2 holoenzyme is tightly associated with the Golgi complex and the endoplasmic reticulum. Centrosomes, which organize spindle formation during the cell cycle and microtubule cytoskeleton formation and, thereby, the location and orientation of different organelles in the cell, are in close vicinity to the Golgi complex. Because several kinases and phosphatases have been described to regulate the functions of the centrosome, we analysed the association of CK2 with these organelles. Using biochemical cell fractionation and coimmunoprecipitation, we never found the holoenzyme but only the catalytic asubunits associated with the centrosome. These data were confirmed by immunoelectron microscopy. Thus, the present data point to a particular role of the catalytic α and α' subunit of protein kinase CK2, which may be different from their roles in the holoenzyme. Received 2 August 2002; received after revision 2 October 2002; accepted 22 October 2002 RID="*" ID="*"Corresponding author.     

Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin

Depending on the availability of suitable reducing agents, alloxan can be either a prooxidant or an antioxidant. Alloxan and its reduced derivative, dialuric acid, act as a redox couple, driven by reduced glutathione (GSH) or L-cysteine, generating in vitro in the presence of oxygen, both superoxide radical and hydrogen peroxide. The production of superoxide radicals was shown by the appearance of lucigenin chemiluminescence (CL) as well as by the generation of formazan from nitroblue tetrazolium (NBT). The lucigenin CL as well as the NBT reduction was inhibited by superoxide dismutase and partially by catalase. Melatonin inhibited alloxan-mediated CL. In contrast, in the absence of reducing agents, alloxan is a scavenger of superoxide radicals formed by other reactions. Because of the high content of reducing compounds in the cell (e.g. glutathione), it is suggested that alloxan acts in vivo mainly as a generator of reactive oxygen species. Received 9 November 1998; received after revision 15 January 1999; accepted 15 January 1999     

A molecular recognition hypothesis for nonpeptides: Na+ K+ ATPase and endogenous digitalis-like peptides

The molecular recognition hypothesis for peptides is that binding sites of ligands and their receptors are encoded by short, complementary segments of DNA. A corollary hypothesis for nonpeptide ligands posited here is that peptide replicas may be encoded by the DNA segment complementary to the receptor binding sites for nonpeptides. This corollary was tested for digitalis, a family of cardiotonic and natriuretic steroids including ouabain. A hexapeptide (ouabain-like peptide, OLP) complementary to a ouabain binding site on sodium/potassium dependent adenosine triphosphatase (Na⁺ K⁺ ATPase) exhibited activity in a digitalis bioassay. Antisera to the complementary peptide (OLP) stained the neurohypophysis in an immunocytochemical procedure. The complementary peptide was found to share an identical 4-amino acid region with the 39-amino acid glycopeptide moiety of the vasopressin-neurophysin precursor. This glycopeptide was isolated from pituitary extracts; it exhibited digitalis-like activity in the submicromolar range and cross-reacted with complementary peptide antibodies. Another digitalis-like substance with high activity also was detected in the extracts. These results demonstrate that the vasopressin-neurophysin glycopeptide has digitalis-like activity. Moreover, the findings are consistent with the hypothesis that peptide mimetics of nonpeptides are encoded in the genome. Received 23 November 1998; received after revision 18 January 1999; accepted 19 February 1999     

Radical catalysis of B12 enzymes: structure,mechanism, inactivation,and reactivation of diol and glycerol dehydratases

Enzymatic radical catalysis is defined as a mechanism of catalysis by which enzymes catalyze chemically difficult reactions by utilizing the high reactivity of free radicals. Adenosylcobalamin (coenzyme B12) serves as a cofactor for enzymatic radical reactions. The recent structural analysis of adenosylcobalamin-dependent diol dehydratase revealed that the substrate 1,2-propanediol and an essential potassium ion are located inside a (beta/alpha)8 barrel. Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity. Cobalamin bound in the base-on mode covers the cavity to isolate the active site from solvent. Based on the three-dimensional structure and theoretical calculations, a new mechanism for diol dehydratase is proposed in which the potassium ion plays a direct role in the catalysis. The mechanisms for generation of a catalytic radical by homolysis of the coenzyme Co-C bond and for protection of radical intermediates from undesired side reactions during catalysis are discussed based on the structure. The reactivating factors for diol and glycerol dehydratases have been identified. These factors are a new type of molecular chaperone which participate in reactivation of the inactivated holoenzymes by mediating ATP-dependent exchange of the modified coenzyme for free intact coenzyme.     

Human sulfatases: A structural perspective to catalysis

The sulfatase family of enzymes catalyzes hydrolysis of sulfate ester bonds of a wide variety of substrates. Seventeen genes have been identified in this class of sulfatases, many of which are associated with genetic disorders leading to reduction or loss of function of the corresponding enzymes. Amino acid sequence homology suggests that the enzymes have similar overall folds, mechanisms of action, and bivalent metal ion-binding sites. A catalytic cysteine residue, strictly conserved in prokaryotic and eukaryotic sulfatases, is post-translationally modified into a formylglycine. Hydroxylation of the formylglycine residue by a water molecule forming the activated hydroxylformylglycine (a formylglycine hydrate or a gem-diol) is a necessary step for the enzyme's sulfatase activity. Crystal structures of three human sulfatases, arylsulfatases A and B(ARSA and ARSB), and estrone/dehydroepiandrosterone sulfatase or steroid sulfatase (STS), also known as arylsulfatase C, have been determined. While ARSA and ARSB are water-soluble enzymes, STS has a hydrophobic domain and is an integral membrane protein of the endoplasmic reticulum. In this article, we compare and contrast sulfatase structures and revisit the proposed catalytic mechanism in light of available structural and functional data. Examination of the STS active site reveals substrate-specific interactions previously identified as the estrogen-recognition motif. Because of the proximity of the catalytic cleft of STS to the membrane surface, the lipid bilayer has a critical role in the constitution of the active site, unlike other sulfatases.     

α-Endosulfine, a new entity in the control of insulin secretion

ATP-dependent potassium (K_ATP) channels occupy a key position in the control of insulin release from the pancreatic β cell since they couple cell polarity to metabolism. These channels close when more ATP is produced via glucose metabolism. They are also controlled by sulfonylureas, a class of drugs used in type 2 diabetic patients for triggering insulin secretion from β cells that have lost part of their sensitivity to glucose. We have demonstrated the existence of endogenous counterparts to sulfonylureas which we have called ‘endosulfines.’ In this review, we describe the discovery, isolation, cloning, and biological features of the high-molecular-mass form, α-endosulfine, and discuss its possible role in the physiology of the β cell as well as in pathology. Received 1 February 1999; received after revision 26 March 1999; accepted 26 March 1999