Biochemical impact of the host adaptation-associated PB2 E627K mutation on the temperature-dependent RNA synthesis kinetics of influenza A virus polymerase complex

Most avian influenza A viruses, which preferentially replicate at the high temperatures found in the digestive tract of birds, have a glutamic acid at residue 627 of the viral RNA polymerase PB2 subunit (Glu-627), whereas the human viruses, which optimally replicate at the low temperatures observed in the human respiratory tract, have a lysine (Lys-627). The mechanism of action for this mutation is still not understood, although interaction with host factors has been proposed to play a major role. In this study, we explored an alternative, yet related, hypothesis that this PB2 mutation may alter the temperature-dependent enzymatic polymerase activity of the viral polymerase. First, the avian polymerase protein, which was purified from baculovirus expression system, indeed remained significantly active at higher temperatures (i.e. 37 and 42 °C), whereas the human E627K mutant drastically lost activity at these high temperatures. Second, our steady-state kinetics data revealed that the human E627K mutant polymerase is catalytically more active than the avian Glu-627 polymerase at 34 °C. Importantly, the E627K mutation elevates apparent K(cat) at low temperatures with little effect on K(m), suggesting that the E627K mutation alters the biochemical steps involved in enzyme catalysis rather than the interaction with the incoming NTP. Third, this temperature-dependent kinetic impact of the human E627K mutation was also observed with different RNA templates, with different primers and also in the presence of nucleoprotein. In conclusion, our study suggests that the amino acid sequence variations at residue 627 of PB2 subunit can directly alter the enzyme kinetics of influenza polymerase.     

Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V

The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements. Structural and functional data indicate that the mutations induce the preferential population of a state that resembles the ADP-bound state. Moreover, the Gly-680 mutants display greatly reduced dynamic properties, which appear to be related to the recovery of myosin motor function at elevated temperatures.     

Some like it cold: biocatalysis at low temperatures

In the last few years, increased attention has been focused on a class of organisms called psychrophiles. These organisms, hosts of permanently cold habitats, often display metabolic fluxes more or less comparable to those exhibited by mesophilic organisms at moderate temperatures. Psychrophiles have evolved by producing, among other peculiarities, "cold-adapted" enzymes which have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency associated, however, with a low thermal stability. Thanks to recent advances provided by X-ray crystallography, structure modelling, protein engineering and biophysical studies, the adaptation strategies are beginning to be understood. The emerging picture suggests that psychrophilic enzymes are characterized by an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. Due to their attractive properties, i.e., a high specific activity and a low thermal stability, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications.     

On the biological role of a proteolytic-enzyme inhibitor from the ectoparasitic tick Boophilus microplus.

The tick Boophilus microplus contains a protein that inhibits a range of proteolytic enzymes. Variations in the concentration of this protein throughtout the life cycle were followed by measuring simultaneously the inhibition of trypsin and chymotrypsin and reaction with an antiserum to the purified inhibitor. The protein is present in large amounts in eggs and in unfed larvae, but its concentration falls very rapidly after the start of the parasitic stage of the life cycle. This, together with previous evidence, suggests that the inhibitor is important both in eggs and in the initial establishment of the parasite on its host. The activity of the protein towards several enzymes has been measured as an indication of its possible function. Bovine trypsin, chymotrypsin and plasmin and pig pancreatic kallikrein are all inhibited. The protein also affects the blood-coagulation system at several points, since it prolongs both activated-partial-thromboplastin time and prothrombin time. It inhibits the complement-dependent lysis of erythrocytes, but is without significant effect on mitogen-induced lymphocyte stimulation. Thus the inhibitor could have several effects on the host that would be beneficial to the parasite.     

The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection

Mutation and polymorphism detection is of increasing importance in the field of molecular genetics. This is reflected by the plethora of chemical, enzymatic, and physically based methods of mutation detection. The ideal method would detect mutations in large fragments of DNA and position them to single base-pair (bp) accuracy. Few methods are able to quickly screen kilobase lengths of DNA and position the mutation at the same time. The Enzyme Mismatch Cleavage (EMC) method of mutation detection is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp. This method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and more recently, a second bacteriophage resolvase, T7 endonuclease I. The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and mutant DNA. Digestion fragments indicate the presence, and the position, of any mutations. The method is robust and reliable and much faster and cheaper than sequencing. These attributes have resulted in its increasing use in the field of mutation detection.     

Improvement of pectinase,xylanase and cellulase activities by ultrasound: Effects on enzymes and substrates,kinetics and thermodynamic parameters

Ultrasound effects were investigated on pectinase (PE), xylanase (XLN) and cellulase (CE) activities at different pH, temperatures, and by sonication pre-treatment, comparing the reaction at ultrasound bath (US) and at a mechanical stirring (MS). In general, US increased the activity of the enzymes by 5% for PE, 30 % for XLN and 25% for CE compared to MS. US provided a higher activity at extremes pH (pH 3 and 7), mainly for XLN and CE. The substrate and enzyme pre-sonication enhanced the activities. The previous sonication of xylan increased the xylanase activity in almost 30% under US and almost 20% under MS. On the other hand, cellulase pre-sonication increased the activity in 50% under US and 40% under MS. The catalytic efficiency (V_max/K_M) increase 25% for PE and 17% for higher XLN and CE under US. US affected the PE activity at low temperature improving 10% the PE activity, while its effect was more representative at high temperatures, where the enzymatic activities of XLN and CE were 33% and 15% higher. Our results demonstrated that ultrasound can affect enzymes and substrates, making it a powerful tool for enzymatic-catalyzed reactions.     

A Gene, ALCA, Affecting the Life Cycle Form Expressed in PHYSARUM POLYCEPHALUM

The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-plasmodium. So-called "amoebaless life cycle" or alc mutants of this Myxomycete undergo a simplified plasmodium-spore-plasmodium life cycle. We have analyzed three independently isolated alc mutants and found in each case that the failure of the spores to give rise to amoebae is due to a recessive Mendelian allele. The three mutations are tightly linked to one another and belong to a single complementation group, alcA. The mutations are pleiotropic, not only interfering with the establishment of the amoebal form at spore germination, but also affecting the phenotype of alc amoebae, which occasionally arise from alc spores. The alc amoebae (1) grow more slowly than wild type, particularly at elevated temperatures; (2) tend to transform directly into plasmodia, circumventing the sexual fusion of amoebae that usually accompanies plasmodium formation; and (3) form plasmodia by the sexual mechanism less efficiently than wild-type amoebae. The various effects of an alc mutation seem to derive from mutation of a single gene, since reversion for one effect is always accompanied by reversion for the other effects. Moreover, a mutation, aptA1, that blocks direct plasmodium formation by alcA amoebae, also increases their growth rate to near normal. The manner of plasmodium formation in alcA strains differs significantly from that in another class of mutants, the gad mutants. Unlike gad amoebae, alcA amoebae need not reach a critical density in order to differentiate directly into plasmodia and do not respond to the extracellular inducer of differentiation. In addition, alcA differentiation is not prevented by a mutation, npfA1, that blocks direct differentiation by most gad amoebae.     

Structural and Biochemical Characterization of an Active Arylamine N-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad

Arylamine N-acetyltransferases (NATs), a class of xenobiotic-metabolizing enzymes, catalyze the acetylation of aromatic amine compounds through a strictly conserved Cys-His-Asp catalytic triad. Each residue is essential for catalysis in both prokaryotic and eukaryotic NATs. Indeed, in (HUMAN)NAT2 variants, mutation of the Asp residue to Asn, Gln, or Glu dramatically impairs enzyme activity. However, a putative atypical NAT harboring a catalytic triad Glu residue was recently identified in Bacillus cereus ((BACCR)NAT3) but has not yet been characterized. We report here the crystal structure and functional characterization of this atypical NAT. The overall fold of (BACCR)NAT3 and the geometry of its Cys-His-Glu catalytic triad are similar to those present in functional NATs. Importantly, the enzyme was found to be active and to acetylate prototypic arylamine NAT substrates. In contrast to (HUMAN) NAT2, the presence of a Glu or Asp in the triad of (BACCR)NAT3 did not significantly affect enzyme structure or function. Computational analysis identified differences in residue packing and steric constraints in the active site of (BACCR)NAT3 that allow it to accommodate a Cys-His-Glu triad. These findings overturn the conventional view, demonstrating that the catalytic triad of this family of acetyltransferases is plastic. Moreover, they highlight the need for further study of the evolutionary history of NATs and the functional significance of the predominant Cys-His-Asp triad in both prokaryotic and eukaryotic forms.     

Cold-hardiness of the arctic beetle, Pytho americanus Kirby Coleoptera,Pythidae (Salpingidae)

The arctic beetle, Pytho americanus Kirby, is frost tolerant in both larval and adult stages. This is the first demonstration that an insect can tolerate freezing in more than one life stage, a situation which would be congruous with its northern distribution and allow it to spread its life cycle over a number of growing seasons. The main biochemical correlates during the cold hardening process of low temperature acclimation are increasing glycerol and decreasing glycogen concentrations. Glycerol is the only polyol to be synthesized during acclimation, and it accumulates to a maximum of 8.2 and 12.2% of the fresh body weight in larvae and adults respectively. This coincides with the peak of frost tolerance. In addition to its normally assumed roles in cryoprotection it is suggested that glycerol may further serve to minimize dehydration in the overwintering insect by increasing the level of ‘bound’ water. Evidence is presented that indicates that glycerol is synthesized mainly from carbohydrate reserves, especially glycogen, but it does not rule out the possibility that a proportion of free glycerol comes from glyceride sources.P. americanus larvae and adults have low supercooling potential and maintain their supercooling points in the region of ?4° to ?8°C. It is hypothesized that these elevated supercooling points are a result of the presence in the haemolymph of nucleating agents which ensure ice formation at high sub-zero temperatures. It is believed that this beetle overwinters in a frozen state within its microhabitat, which is under bark of fallen spruce which is, in turn, covered by an insulating blanket of snow. The advantages of this overwintering strategy are discussed.     

Mechanistic studies of the autoactivation of PAK2: a two-step model of cis initiation followed by trans amplification

Protein kinase activation, via autophosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation-dependent signaling cascades. Despite the prevalence of this reaction and its importance in biological regulation, the molecular mechanisms of autophosphorylation are poorly understood. In this study, we developed a kinetic approach to distinguish quantitatively between cis- and trans-pathways in an autocatalytic reaction. Using this method, we have undertaken a detailed kinetic analysis for the autoactivation mechanism of p21-activated protein kinase 2 (PAK2). PAK2 is regulated in vivo and in vitro by small GTP-binding proteins, Cdc42 and Rac. Full activation of PAK2 requires autophosphorylation of the conserved threonine, Thr(402), in the activation loop of its catalytic kinase domain. Analyses of the time courses of substrate reaction during PAK2 autoactivation suggest that autophosphorylation of Thr(402) in PAK2 obeys a two-step mechanism of cis initiation, followed by trans amplification. The unphosphorylated PAK2 undergoes an intramolecular (cis) autophosphorylation on Thr(402) to produce phosphorylated PAK2, and this newly formed active PAK2 then phosphorylates other PAK2 molecules at Thr(402) in an intermolecular (trans) manner. Based on the kinetic equation derived, all microscopic kinetic constants for the cis and trans autophosphorylation have been estimated quantitatively. The advantage of the new method is not only its usefulness in the study of fast activation reactions, but its convenience in the study of substrate effects on modification reaction. It would be particularly useful when the regulatory mechanism of the autophosphorylation reaction toward certain enzymes is being assessed.     

A mathematical model of breast cancer development, local treatment and recurrence

Cancer development is a stepwise process through which normal somatic cells acquire mutations which enable them to escape their normal function in the tissue and become self-sufficient in survival. The number of mutations depends on the patient's age, genetic susceptibility and on the exposure of the patient to carcinogens throughout their life. It is believed that in every malignancy 4-6 crucial similar mutations have to occur on cancer-related genes. These genes are classified as oncogenes and tumour suppressor genes (TSGs) which gain or lose their function respectively, after they have received one mutative hit or both of their alleles have been knocked out. With the acquisition of each of the necessary mutations the transformed cell gains a selective advantage over normal cells, and the mutation will spread throughout the tissue via clonal expansion. We present a simplified model of this mutation and expansion process, in which we assume that the loss of two TSGs is sufficient to give rise to a cancer. Our mathematical model of the stepwise development of breast cancer verifies the idea that the normal mutation rate in genes is only sufficient to give rise to a tumour within a clinically observable time if a high number of breast stem cells and TSGs exist or genetic instability is involved as a driving force of the mutation pathway. Furthermore, our model shows that if a mutation occurred in stem cells pre-puberty, and formed a field of cells with this mutation through clonal formation of the breast, it is most likely that a tumour will arise from within this area. We then apply different treatment strategies, namely surgery and adjuvant external beam radiotherapy and targeted intraoperative radiotherapy (TARGIT) and use the model to identify different sources of local recurrence and analyse their prevention.     

Bleaching in coral reef anthozoans: effects of irradiance,ultraviolet radiation,and temperature on the activities of protective enzymes against active oxygen

Recent widespread bleaching of coral reef anthozoans has been observed on the Great Barrier Reef, the Pacific coast of Panama, and in the Caribbean Sea. Bleaching events have been correlated with anomalously high sea surface temperatures which are presumed to cause the expulsion of zooxanthellae from their hosts. Our experimental results show that increases in temperature significantly reduce the total number of zooxanthellae per polyp. At the same time temperature, irradiance (photosynthetically active radiation=PAR), and ultraviolet radiation (UV) independently increase the activities of the enzymes superoxide dismutase, catalase, and ascorbate peroxidase within the zooxanthellae of the zoanthid Palythoa caribaeorum. Enzyme activities within the host are only suggestive of similar changes. These enzymes are responsible for detoxifying active forms of oxygen, and their elevated activities indirectly indicate an increase in the production of active oxygen species by increases in these environmental factors. Historically, bleaching has been attributed to changes in temperature, salinity, and UV. Increases in temperature or highly energetic UV radiation can increase the flux of active forms of oxygen, particularly at the elevated oxygen concentrations that prevail in the tissues during photosynthesis, with oxygen toxicity potentially mediating the bleaching event. Additionally, the concentration of UV absorbing compounds within the symbiosis is inversely related to temperature, potentially increasing exposure of the host and zooxanthellae to the direct effects of UV.     

Fluorescence-based analysis of enzymes at the single-molecule level

Enzymes, and proteins in general, consist of a dynamic ensemble of different conformations, which fluctuate around an average structure. Single-molecule experiments are a powerful tool to obtain information about these conformations and their contributions to the catalytic reaction. In contrast to classical ensemble measurements, which average over the whole population, singlemolecule experiments are able to detect conformational heterogeneities, to identify transient or rare conformations, to follow the time series of conformational changes and to reveal parallel reaction pathways. A number of single-molecule studies with enzymes have proven this potential showing that the activity of individual enzymes varies between different molecules and that the catalytic rate constants fluctuate over time. From a practical point of view this review focuses on fluorescence-based methods that have been used to study enzymes at the single-molecule level. Since the first proof-of-principle experiments a wide range of different methods have been developed over the last 10 years and the methodology now needs to be applied to answer questions of biological relevance, for example about conformational changes induced by allosteric effectors or mutations.     

Pma1, a P-type Proton ATPase,Is a Determinant of Chronological Life Span in Fission Yeast

Chronological life span is defined by how long a cell can survive in a non-dividing state. In yeast, it is measured by viability after entry into stationary phase. To date, some factors affecting chronological life span have been identified; however, the molecular details of how these factors regulate chronological life span have not yet been elucidated clearly. Because life span is a complicated phenomenon and is supposedly regulated by many factors, it is necessary to identify new factors affecting chronological life span to understand life span regulation. To this end, we have screened for long-lived mutants and identified Pma1, an essential P-type proton ATPase, as one of the determinants of chronological life span. We show that partial loss of Pma1 activity not only by mutations but also by treatment with the Pma1 inhibitory chemical vanadate resulted in the long-lived phenotype in Schizosaccharomyces pombe. These findings suggest a novel way to manipulate chronological life span by modulating Pma1 as a molecular target.     

The origin of the biologically coded amino acids

Biology uses essentially 20 amino acids for its coded protein enzymes, representing a very small subset of the structurally possible set. Most models of the origin of life suggest organisms developed from environmentally available organic compounds. A variety of amino acids are easily produced under conditions which were believed to have existed on the primitive Earth or in the early solar nebula. The types of amino acids produced depend on the conditions which prevailed at the time of synthesis, which remain controversial. The selection of the biological set is likely due to chemical and early biological evolution acting on the environmentally available compounds based on their chemical properties. Once life arose, selection would have proceeded based on the functional utility of amino acids coupled with their accessibility by primitive metabolism and their compatibility with other biochemical processes. Some possible mechanisms by which the modern set of 20 amino acids was selected starting from prebiotic chemistry are discussed.     

Sensitivity of pathway rate to activities of substrate-cycle enzymes: application to gluconeogenesis and glycolysis

In a study of metabolic regulation, it is frequently useful to consider the degree to which an enzyme can influence the rate of its pathway. The most productive expression of rate-controlling influence is the fractional change in pathway rate per fractional change in enzyme activity (called control strength or sensitivity coefficient). We have developed a system for considering how a substrate-cycle enzyme's control strength depends on its flux and reaction order and on related features of other enzymes of its pathway. We have applied this system to the gluconeogenic pathway of rat liver and the glycolytic pathway of bovine sperm, where enough fluxes and reaction orders have been published to allow valid estimates of several control strengths. In normal fed animals where gluconeogenesis is slow and unidirectional substrate-to-product and product-to-substrate fluxes are comparable, all substrate-cycle limbs have very high and similar control strengths regardless of their flux rates and positions in the pathway. The activity of a step affects all substrate-cycle control strengths similarly as it affects unidirectional end-to-end fluxes relative to net rate. Control strengths of non-substrate-cycle enzymes are negligible compared to those of substrate cycles. In fasting animals, on the other hand, where unidirectional Pyr----Glc flux is much greater than Glc----Pyr flux, upstream enzymes (near Pyr) have a regulatory advantage over downstream enzymes (near Glc). In this circumstance, control strength of each substrate-cycle enzyme is inversely related to rate limitingness between its substrate and the pathway substrate. Because the Pyr/PEP cycle is significantly rate limiting, the control strength of the Pyr----PEP limb is much greater than that of pyruvate kinase and all downstream enzymes. In the glycolytic pathway of bovine sperm, strong product inhibition of hexokinase detracts greatly from its rate limitingness and control strength, which are very small despite its position at the beginning of the pathway and its large free energy. Because the glucose-transport-hexokinase segment is not rate limiting, phosphofructo 1-kinase has almost as much control strength as it would have as the first enzyme of the pathway, and because the F6P/FDP cycle is only moderately rate limiting, Fru-1,6-P2ase and enzymes further downstream have substantial control strengths. When glycolysis is accelerated by stimulation of phosphofructo 1-kinase, control strength shifts from phosphofructo-1-kinase and all downstream enzymes to the transporthesokinase segment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Directing enzyme devolution for biosynthesis of alkanols and 1,n-alkanediols from natural polyhydroxy compounds

Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as “specialists”. However, some enzymes are “generalists” that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols, including 1,2-butanediol, 1,2,4-butanetriol, erythritol, 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays, and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals.