DNA-dependent RNA-polymerases from Artemia embryos. characterization of polymerases I and II from nauplius larvae.

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.     

Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices

Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca²⁺-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.     

Structural Analysis and Mutant Growth Properties Reveal Distinctive Enzymatic and Cellular Roles for the Three Major L-Alanine Transaminases of Escherichia coli

In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5′-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.     

Multiple forms of DNA-dependent RNA and polyadenylic acid polymerases from heterotrophically grown Rhodospirillum rubrum.

Three, two major and one minor, distinct RNA polymerases have been isolated and partially purified from heterotrophically grown Rhodospirillum rubrum, a facultative photosynethetic bacterium. Associated with each of these three enzymes is a distinct polyadenylic acid polyemrase. All of these enzyme activities are dependent on DNA templates and are resistant to rifampicin and streptovaricin. The structural subunit composition, the response to various chemical compounds and DNA templates, and the properties of the products of these enzymes are studied in detail and compared with those of similar enzyme activities from other bacterial systems. Several unique features have been observed in the R. rubrum enzyme systems, such as an uneven incorporation of purine and pyrimidine nucleotides by the RNA polymerases, and the presence of a lag period in the polyadenylic acid polymerase activities.     

Methemoglobin Reductase of Bacteria and Bacteroids <Emphasis Type="Italic">Bradyrhizobium lupini</Emphasis>: Purification and Properties

Reductase capable of reducing hemoglobin-like proteins was isolated from nodule bacteria Bradyrhizobium lupini and bacteroids of lupine root nodules. It is similar in some properties to many known methemoglobin reductases reducing animal and plant hemoglobins. It is a NADH-dependent FAD-containing flavoprotein with molecular weight of 87 kDa without metals. The presence of such enzymes in prokaryotes could be an explanation for the physiological activity of both bacterial and eukaryotic hemoglobins expressed in bacterial cells.     

The role of polyunsaturated fatty acids in fish

The physical properties of polyunsaturated and saturated fatty acids are compared in relation to melting points and fluidity. The role of polyunsaturated fatty acids on membrane fluidity and membrane bound enzyme activity is discussed. The influence of the environment, particularly temperature, on poikilothermic animals is considered in relation to membrane fatty acid composition and metabolism. The metabolic role of polyunsaturated fatty acids of the (n-3) series and their interaction with arachidonate metabolism is discussed.     

De novo Biosynthesis of Purines in the Retina: Evolutionary Aspects

The author's own experimental findings and some literature data are summarized concerning the presence in the retina of the key enzymes of de novo biosynthesis of purines, (adenine and guanine), ones of the first organic compounds that appeared on the Earth and are the main components of DNA and RNA, nucleic acids providing protein synthesis. For the first time, highly purified preparations of these enzymes from the nerve tissue have been obtained, and their properties, activity control, and distribution in the photoreceptor cells have been studied. The data obtained are compared with the literature data on these enzymes in liver and on the systems regulating the enzyme activity in E. coli. The light-dependent, genetically determined changes in the key enzyme activities were found in the retina within the 24-h period: these activities significantly differed in the daytime and night periods (biorhythms), which indicates a direct relation of the photoreceptor to the retinal adaptation system. The data on diversity of retinal photoreceptors and their presence in cells of other tissues, as well as possible perspectives of this direction of research are briefly discussed. In each section, the evolutionary aspects of the obtained facts are considered.     

Activity, location, and role of asparate aminotransferase and alanine aminotransferase isoenzymes in leaves with C4 pathway photosynthesis

Further evidence has been provided that C₄-pathway species characterized by having low malic enzyme activity contain exceptionally high activities of aspartate and alanine aminotransferases. The total activity of both enzymes is distributed about equally between mesophyll and bundle sheath cells. However, the activity in the two cell types is due to different isoenzymes. In addition to the one quantitatively major isoenzyme associated with each cell type there were at least two additional isozymes of each aminotransferase detectable in the different species examined. Increases in activity of both aminotransferases of ten-fold or more were observed during greening of leaves of dark-grown plants. This increased activity was due specifically to the two quantitatively major isoenzymes associated, respectively, with the mesophyll and bundle sheath cells of green leaves, providing further evidence for their specific role in photosynthesis. Apparently, neither the aspartate nor alanine aminotransferases of mesophyll cells was associated with chloroplasts or other subcellular organelles. However, the major aspartate aminotransferase isoenzyme of bundle sheath cells was associated with mitochondria. These findings are discussed in relation to the probable role of aspartate and alanine aminotransferases in C₄-pathway photosynthesis.     

Biostructural theory of the living systems

Eugen Macovschi is among the few scientists who tried, and partly succeeded, to explain the differences between "dead" and "living" in biological sciences. He discovered and characterized the so-called biostructure of the living bodies and worked out a biostructural theory, which is the first supramolecular conception in biology. Nevertheless, complex biological systems are currently considered only from the molecular point of view, although they may be regarded as specific phenomena on highly structured bodies within the four-dimensional Universe. According to Macovschi, the biostructure provides organisms with life properties and controls their life processes and chemical changes. Nevertheless, plant cells or bacterial ones differ much from the animal or human cells. In fact, there are various biostructures which are related with cell properties. Hence, this theory creates confusions and cannot be easily used to explain all the properties of the biosystems. Consequently, it is our goal to highlight the principles, advantages, limitations, and applications of the biostructural theory, which might support new ideas and theories in modern life sciences.     

Plant β-Galactosidases: Physiological Significance and Recent Advances in Technological Applications

The β-galactosidase (BGAL) is one of the oldest ubiquitous enzymes, known for more than 100 years. The enzyme is known to perform various functions in different organisms, however with similar mode of action. There is an immense literature available related to bacterial, fungal as well as animal BGAL compared to plant BGAL. Initially, it was believed that lactose is the only substrate for the enzyme. Later, it was observed that enzyme specificity is due to hydrolysable bond rather than the substrate. The present review is based on the role of BGAL in plants, the in vivo substrates and their physiological significances. Similarity as well as dissimilarity with BGAL from bacterial as well as fungal sources is also discussed. Plant BGAL would be best suited for industrial applications because of its easy availability, cost effectiveness and easy adaptability.     

Taking aim on bacterial pathogens: from phage therapy to enzybiotics

The bactericidal activity of bacteriophages has been used to treat human infections for years as an alternative or a complement to antibiotic therapy. Nowadays, endolysins (phage-encoded enzymes that break down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle) have been used successfully to control antibiotic-resistant pathogenic bacteria in animal models. Their cell wall binding domains target the enzymes to their substrate, and their corresponding catalytic domains are able to cleave bonds in the peptidoglycan network. Recent research has not only revealed the surprising rich structural catalytic diversity of these murein hydrolases but has also yielded insights into their modular organization, their three-dimensional structures, and their mechanism of recognition of bacterial cell wall. These results allow endolysins to be considered as effective antimicrobials with potentially important applications in medicine and biotechnology.     

Protease inhibitors: use to increase plant tolerance to insects and pathogens

The review deals with analysis of the possibility of the use of genes of inhibitors of proteolytic enzymes of plants to increase plant tolerance to insect pests and phytopathogens. The idea of using protease inhibitors for plant defense is strongly supported, first, by their wide distribution in plant tissues and high activity towards various proteolytic enzymes of insects, bacteria and fungi. The results obtained for the last years indicate that the genetic engineering approach is perspective for solving of this kind of problems. The main losses and advantages of the discussed approach are also considered. The described approach for increase of plant tolerance to insects and pathogens has few advantages as compared to traditional ones and belongs to ecologically pure technologies.     

GH11 xylanases: Structure/function/properties relationships and applications

For technical, environmental and economical reasons, industrial demands for process-fitted enzymes have evolved drastically in the last decade. Therefore, continuous efforts are made in order to get insights into enzyme structure/function relationships to create improved biocatalysts. Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of the heteroxylans constituting the lignocellulosic plant cell wall. Due to their variety, xylanases have been classified in glycoside hydrolase families GH5, GH8, GH10, GH11, GH30 and GH43 in the CAZy database. In this review, we focus on GH11 family, which is one of the best characterized GH families with bacterial and fungal members considered as true xylanases compared to the other families because of their high substrate specificity. Based on an exhaustive analysis of the sequences and 3D structures available so far, in relation with biochemical properties, we assess biochemical aspects of GH11 xylanases: structure, catalytic machinery, focus on their "thumb" loop of major importance in catalytic efficiency and substrate selectivity, inhibition, stability to pH and temperature. GH11 xylanases have for a long time been used as biotechnological tools in various industrial applications and represent in addition promising candidates for future other uses.     

Isolation and Partial Characterization of Transfer RNAs from Astragalus bisulcatus

A procedure has been developed for the isolation of transfer RNA from the selenium accumulator plant Astragalus bisulcatus. This material appears free of interfering phenolic compounds, has a high guanosine to cytidine ratio, shows a major and modified nucleoside composition characteristic of plant transfer RNAs, and exhibits chromatographic and electrophoretic properties similar to transfer RNAs from other well studied bacterial and plant systems. RNAs isolated from A. bisulcatus seedlings incubated in the presence of ⁷⁵Se indicate some incorporation of radioactivity into the transfer RNAs, but at extremely low levels. The transfer RNAs were active in accepting amino acids, although their over-all levels of activity appeared low when compared with those from a homologous Escherichia coli aminoacylation reaction system.     

Changes in the isoform composition of the cytoskeletal protein titn--adaptation process in hibernation

Changes in the isoform composition of the elastic protein titin from skeletal and cardiac muscles of hibernating ground squirrels were revealed for the first time. It was shown that, upon hibernation, the molecular mass of titin decreases and its functional properties change as compared with the active state of the animal. The physiological significance of the changes in titin isoform composition for the inhibition of muscle contractile activity upon hibernation is discussed in connection with similar changes during some cardiomyopathies.     

Functional and structural characterization of a cation-dependent O-methyltransferase from the cyanobacterium Synechocystis sp. strain PCC 6803

The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation- and S-adenosyl-l-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di- and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys(3) close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed.     

Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production

Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed. Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1. This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB. Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B. napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI. The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct. However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with enhanced agronomic, animal feed and human nutritional values.     

锰氧化细菌的生理生态功能与作用机制研究进展

锰的生物地球化学循环过程与全球尺度的营养元素循环紧密联系,是影响全球生态平衡及气候变化的重要因素之一。在自然界中,锰元素主要以氧化锰和含氧酸盐的矿物形式存在,近年来的研究观点普遍认为细菌介导的氧化作用是自然环境中锰氧化物形成的主要原因。锰氧化细菌广泛分布于海洋、锰矿土壤等生态系统,近期在植物微生态系统中也被发现,其生理生态功能未知。细菌的锰氧化过程是一个复杂的过程,多铜氧化酶和过氧化物(氢)酶是参与该过程的主要酶类,但关于其催化机制的认识尚不成熟。本综述系统探讨锰氧化细菌的种类和分布、细菌锰氧化作用的生理生态功能、参与细菌锰氧化作用的功能酶及其分子机制,总结这一研究领域所取得的成果和仍未解决的科学问题,并对今后的发展方向进行展望。     

Brachyurins--serine collagenolytic enzymes from crabs

The properties of brachyurins, proteolytic enzymes belonging to a new subfamily of chymotrypsin-like proteases, are considered. These enzymes, found in various species of crustacean, exhibit mixed substrate specificity and a marked collagenolytic activity. The enzymatic and physicochemical properties of brachyurins I and their primary and spatial structures are discussed in detail. A separate chapter is devoted to the preparations of collagenases from the hepatopancreas of king crab: their action on the damaged skin and use in medicine.