SET domain protein lysine methyltransferases: Structure, specificity and catalysis

Site- and state-specific lysine methylation of histones is catalyzed by a family of proteins that contain the evolutionarily conserved SET domain and plays a fundamental role in epigenetic regulation of gene activation and silencing in all eukaryotes. The recently determined three-dimensional structures of the SET domains from chromosomal proteins reveal that the core SET domain structure contains a two-domain architecture, consisting of a conserved anti-parallel β-barrel and a structurally variable insert that surround a unusual knot-like structure that comprises the enzyme active site. These structures of the SET domains, either in the free state or when bound to cofactor S-adenosyl-L-homocysteine and/or histone peptide, mimicking an enzyme/cofactor/substrate complex, further yield the structural insights into the molecular basis of the substrate specificity, methylation multiplicity and the catalytic mechanism of histone lysine methylation. Received 10 June 2006; accepted 22 August 2006     

Medium- and short-chain dehydrogenase/reductase gene and protein families

Zinc plays an important role in the structure and function of many enzymes, including alcohol dehydrogenases (ADHs) of the MDR type (mediumchain dehydrogenases/reductases). Active site zinc participates in catalytic events, and structural site zinc maintains structural stability. MDR-types of ADHs have both of these zinc sites but with some variation in ligands and spacing. The catalytic zinc sites involve three residues with different spacings from two separate protein segments, while the structural zinc sites involve four residues and cover a local segment of the protein chain (Cys97-Cys111 in horse liver class I ADH). This review summarizes properties of both ADH zinc sites, and relates them to zinc sites of proteins in general. In addition, it highlights a separate study of zinc binding peptide variants of the horse liver ADH structural zinc site. The results show that zinc coordination of the free peptide differs markedly from that of the enzyme (one His / three Cys versus four Cys), suggesting that the protein zinc site is in an energetically strained conformation relative to that of the peptide. This finding is a characteristic of an entatic state, implying a functional nature for this zinc site.     

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.     

Hammerhead ribozyme design and application

The emerging knowledge about RNA-based enzymes has already had great impact on our concept of evolutionary history, making the ‘RNA world’ more likely. It may well have an equally important impact on the diagnostic and therapeutic practices of human and veterinary medicine in the next decade. We are not quite there yet. This review addresses the design and application of hammerhead ribozymes, two aspects of a conserved and most commonly studied and used enzymatically active entity among the RNA enzymes. The emerging picture is one of great diversity. There is at this stage no general cell model nor a clearly preferable ribozyme structure. Each and every cell line (and tissue) may be unique in that they vary with respect to structural requirements for optimal uptake, activity and stability of ribozymes. We may have seen only the tip of the iceberg when it comes to RNA-based enzymes and their roles in biology and medicine. Received 3 June 1998; received after revision 28 July 1998; accepted 28 July 1998     

Molecular mechanisms of thrombin function

The discovery of thrombin as a Na⁺-dependent allosteric enzyme has revealed a novel strategy for regulating protease activity and specificity. The allosteric nature of this enzyme influences all its physiologically important interactions and rationalizes a large body of structural and functional information. For the first time, a coherent mechanistic framework is available for understanding how thrombin interacts with fibrinogen, thrombomodulin and protein C, and how Na⁺ binding influences the specificity sites of the enzyme. This information can be used for engineering thrombin mutants with selective specificity towards protein C and for the rational design of potent active site inhibitors. Thrombin also serves as a paradigm for allosteric proteases. Elucidation of the molecular basis of the Na⁺-dependent allosteric regulation of catalytic activity, based on the residue present at position 225, provides unprecedented insights into the function and evolution of serine proteases. This mechanism represents one of the simplest and most important structure-function correlations ever reported for enzymes in general. All vitamin K-dependent proteases and some complement factors are subject to the Na⁺-dependent regulation discovered for thrombin. Na⁺ is therefore a key factor in the activation of zymogens in the coagulation and complement systems.     

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway. Received 28 May 1998; received after revision 6 July 1998; accepted 6 July 1998     

Elastase digested urokinase (ED-UK)

Summary Elastase digested urokinase (ED-UK) was prepared from human high mol. wt urokinase (HMW-UK). It resembled low mol. wt urokinase (LMW-UK) in its mol. wt, specific activity, and active sites. The steady-state kinetic parameters of each enzyme for the activation of human Glu-plasminogen also resembled each other, as did their amidase parameters (with pyro-Glu-Gly-Arg-pNA).     

Elastase digested urokinase (ED-UK)

Elastase digested urokinase (ED-UK) was prepared from human high mol. wt urokinase (HMW-UK). It resembled low mol. wt urokinase (LMW-UK) in its mol. wt, specific activity, and active sites. The steady-state kinetic parameters of each enzyme for the activation of human Glu-plasminogen also resembled each other, as did their amidase parameters (with pyro-Glu-Gly-Arg-pNA).     

RNA-splicing endonuclease structure and function

The RNA-splicing endonuclease is an evolutionarily conserved enzyme responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs. Since its first identification from yeast in the late 1970s, significant progress has been made toward understanding the biochemical mechanisms of this enzyme. Four families of the splicing endonucleases possessing the same active sites and overall architecture but with different subunit compositions have been identified. Two related consensus structures of the precursor RNA splice sites and the critical elements required for intron excision have been established. More recently, a glimpse was obtained of the structural mechanism by which the endonuclease recognizes the consensus RNA structures and cleaves at the splice sites. This review summarizes these findings and discusses their implications in the evolution of intron removal processes. Received 24 August 2007; received after revision 24 November 2007; accepted 27 November 2007     

The plasma membrane proton-translocating ATPase

Living cells require membranes and membrane transporters for the maintenance of life. After decades of biochemical scrutiny, the structures and molecular mechanisms by which membrane transporters catalyze transmembrane solute movements are beginning to be understood. The plasma membrane proton-translocating adenosine triphosphatase (ATPase) is an archetype of the P-type ATPase family of membrane transporters, which are important in a wide variety of cellular processes. The H+-ATPase has been crystallized and its structure determined to a resolution of 8 angstrom in the membrane plane. When considered together with the large body of biochemical information that has been accumulated for this transporter, and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by this enzyme.     

2’-5’ Oligoadenylate synthetase shares active site architecture with the archaeal CCA-adding enzyme

The 2'-5' oligoadenylate synthetases (OAS) are interferon-induced antiviral enzymes that recognise virally produced dsRNA and initiate an RNA destabilisation within the infected cell. We compared the structure of OAS to that of poly adenosine polymerase (PAP) and the class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA). This comparison revealed a strong structural homology between the three enzyme families. In particular, the active sites of OAS and CCA class I enzymes are highly conserved. We conducted an extensive mutagenesis of amino acid residues within the putative active site in OAS, thereby identifying enzymatically important residues and confirming the common active site architecture for OAS and the AfCCA. Our findings also have profound implications for our understanding of the evolutionary origin of the OAS enzymes and suggest that the OAS proteins diverged from a common 3'-specific ancestor at the beginning of metazoan evolution.     

Medium- and short-chain dehydrogenase/reductase gene and protein families

In this report we describe the main features of the initially determined alcohol dehydrogenase, that of horse liver, relate this to the human enzyme structures and review recent structural studies on mutants and new complexes of the enzymes. We further review the structure of a bacterial alcohol dehydrogenase to arrive at a coherent picture of medium-chain dehydrogenase/reductase alcohol dehydrogenases in general.     

Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product

Carbamoyl phosphate synthetase (CPS) catalyzes one of the most remarkable reactions ever described in biological chemistry, in which carbamoyl phosphate is produced from one molecule of bicarbonate, two molecules of Mg2+ ATP, and one molecule of either glutamine or ammonia. The carbamoyl phosphate so produced is utilized in the synthesis of arginine and pyrimidine nucleotides. It is also employed in the urea cycle in most terrestrial vertebrates. Due to its large size, its important metabolic role, and the fact that it is highly regulated, CPS has been the focus of intensive investigation for nearly 40 years. Numerous enzymological, biochemical, and biophysical studies by a variety of investigators have led to a quite detailed understanding of CPS. Perhaps one of the most significant advances on this topic within the last 2 years has been the successful X-ray crystallographic analysis of CPS from Escherichia coli. Quite unexpectedly, this structural investigation revealed that the three active sites on the protein are widely separated from one another. Furthermore, these active sites are connected by a molecular tunnel with a total length of approximately 100 A, suggesting that CPS utilizes this channel to facilitate the translocation of reaction intermediates from one site to another. In this review, we highlight the recent biochemical and X-ray crystallographic results that have led to a more complete understanding of this finely tuned instrument of catalysis.     

Assessing heterogeneity in oligomeric AAA+ machines

ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.     

Gastric lysozyme as a digestive enzyme in the hoatzin (Opisthocomus hoazin), a ruminant-like folivorous bird

The hoatzin is the only bird known to have pregastric fermentation in the crop. This digestive strategy is supported by morphological and microbiological adaptations analogous to those present in ruminants and ruminant-like mammals. The hoatzin expresses a lysozyme-like bacteriolytic activity in its foregut. The enzyme has a high activity, and its low pH optimum, pepsin resistance and localization to the proventriculus allow it to be active for digestion in the stomach. The hoatzin enzyme and the ruminant gastric lysozyme present similar biochemical characteristics. The lysis of bacterial cells may be of significance for the nutrition of the hoatzin. We propose that the hoatzin expresses a lysozyme which has been recruited to function as a digestive enzyme, representing a unique case of evolutionary convergence of digestive adaptations in this bird and foregut fermenter mammals.     

Long‐Term Forecasting of Global Carbon Dioxide Emissions: Reducing Uncertainties Using a Per Capita Approach

Global CO₂ emission forecasts span such a wide range as to yield little guidance for climate policy and analysis. But global per capita emissions appear to be better constrained than total emissions, which we argue has an economic justification. Trend stationarity of per capita emissions may provide a means of characterizing the relative likelihood of global forecasts. On data spanning 1950 to 2009 a unit root hypothesis allowing for endogenous structural breaks is rejected, but adding in the 2010 observation pushes the p‐value slightly over 0.1. Since structural breaks cannot be detected at the end of sample this may simply indicate a power problem. Using Monte Carlo simulations we conclude that the lower half of a well‐known suite of IPCC emission scenarios is more likely to occur than the upper half, and the top quartile is particularly difficult to justify. Copyright © 2013 John Wiley & Sons, Ltd.     

Interleukin-8 is a Cyclosporin A binding protein

Inflammatory immune reactions occur during transplant rejections and autoimmune diseases. Such reactions are mediated by cytokines, including interleukin-8 (IL-8). Cyclosporin A (CsA) exerts immunosuppressive activities^1,2 by binding to immunoregulatory proteins termed cyclophilins³. The anti-inflammatory effects of CsA are still not fully understood. Searching for novel neutrophil-activating proteins, we observed that an antiserum against human recombinant Interleukin-8 (IL-8) cross-reacted with cyclophilins in Western blots. Furthermore, native IL-8 was found to specifically bind CsA, whereas biologically inactive analogs of CsA were not bound by IL-8. Putative binding sites for CsA on IL-8 could be identified on the basis of structural similarities between IL-8 and cyclophilin. However, IL-8 lacks peptidyl-prolyl-isomerase (PPlase) enzyme activity, which is regarded as a characteristic of cyclophilins^4,5,6. We conclude that the specific binding of CsA to IL-8 may explain some of the anti-inflammatory effects of CsA. IL-8 may be a novel member of the cyclophilins lacking PPlase activity.     

Lactoferricin

The peptide lactoferricin (Lfcin) can be released from the multifunctional protein lactoferrin (LF) through proteolysis by pepsin under acidic conditions, a reaction that occurs naturally in the stomach. Lfcin encompasses a large portion of the functional domain of the intact protein, and in many cases it not only retains the activities of LF but is more active. Lfcin possesses strong antimicrobial and weak antiviral activities, and it also has potent antitumor and immunological properties. This review covers the current state of research in this field, focusing on the many beneficial activities of this peptide. Throughout we will discuss the breadth of Lfcin activity as well as the mechanism of action. Many recent studies have drawn attention to the fact that the main site of action for the peptide may be intracellular. In addition the results of structural and dynamic studies of Lfcin are presented, and the relationship between structure and activity is explored.     

Functional, biochemical and genetic diversity of prokaryotic nitrate reductases

Prokaryotic nitrate reduction can serve a number of physiological roles and can be catalysed by a number of biochemically distinct nitrate reductases. Three distinct nitrate reductase classes can be indentified in prokaryotes, NAS, NAR and NAP. NAS is located in the cytoplasmic compartment and participates in nitrogen assimilation. NAR is usually a three-subunit complex anchored to the cytoplasmic face of the membrane with its active site located in the cytoplasmic compartment and is involved in anaerobic nitrate respiration. NAP is a two-subunit complex, located in the periplasmic compartment, that is coupled to quinol oxidation via a membrane anchored tetraheme cytochrome. It shows considerable functional flexibility by participating in anaerobic respiration or redox energy dissipation depending on the organism in which it is found. The members of all three classes of enzymes bind the bis-molybdopterin guanine dinucleotide cofactor at the active site, but they differ markedly in the number and nature of cofactors used to transfer electrons to this site. Analysis of prokaryotic genome sequences available at the time of writing reveals that the different nitrate reductases are phylogenetically widespread.