Structural flexibility and protein adaptation to temperature: Molecular dynamics analysis of malate dehydrogenases of marine molluscs.

Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural "flexibility." However, quantifying flexibility and comparing flexibility across proteins has remained a challenge. To address this issue, we examined temperature effects on cytosolic malate dehydrogenase (cMDH) orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of ∼60 °C. We describe consistent patterns of convergent evolution in adaptation of function [temperature effects on KM of cofactor (NADH)] and structural stability (rate of heat denaturation of activity). To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis, we performed molecular dynamics simulation (MDS) analyses. MDS analyses revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements [root mean square deviation (rmsd) of main-chain atoms]. Root mean square fluctuations (RMSFs) of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs. Regions of sequence involved in ligand binding and catalysis-termed mobile regions 1 and 2 (MR1 and MR2), respectively-showed the largest values for RMSF. Heat-induced changes in RMSF values across the sequence and, importantly, in MR1 and MR2 were greatest in cold-adapted species. MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.

Pathogen proteases and host protease inhibitors in molluscan infectious diseases.

The development of infectious diseases represents an outcome of dynamic interactions between the disease-producing agent's pathogenicity and the host's self-defense mechanism. Proteases secreted by pathogenic microorganisms and protease inhibitors produced by host species play an important role in the process. This review aimed at summarizing major findings in research on pathogen proteases and host protease inhibitors that had been proposed to be related to the development of mollusk diseases. Metalloproteases and serine proteases respectively belonging to Family M4 and Family S8 of the MEROPS system are among the most studied proteases that may function as virulence factors in mollusk pathogens. On the other hand, a mollusk-specific family (Family I84) of novel serine protease inhibitors and homologues of the tissue inhibitor of metalloprotease have been studied for their potential in the molluscan host defense. In addition, research at the genomic and transcriptomic levels showed that more proteases of pathogens and protease inhibitor of hosts are likely involved in mollusk disease processes. Therefore, the pathological significance of interactions between pathogen proteases and host protease inhibitors in the development of molluscan infectious diseases deserves more research efforts.

Glycosidases of marine organisms.

This review discusses the catalytic properties, activity regulation, structure, and functions of O-glycoside hydrolases from marine organisms exemplified by endo-1→3-ß-D-glucanases of marine invertebrates.

Opine dehydrogenases in marine invertebrates.

It is well known today that opine production anaerobic pathways are analogs to the classical glycolytic pathway (lactate production pathway). These pathways, catalyzed by a group of enzymes called opine dehydrogenases (OpDHs), ensure continuous flux of glycolysis and a constant supply of ATP by maintaining the NADH/NAD(+) ratio during exercise and hypoxia, thus regulating the cytosolic redox balance in glycolysis under anoxia. OpDHs are distributed in a wide range of marine invertebrate phyla, including sponges (Porifera). Phylogenetic analyses supported with enzymatic assays strongly indicate that sponge OpDHs constitute an enzyme class unrelated to other OpDHs. Therefore, OpDHs in marine invertebrates are divided into two groups, a mollusk/annelid type and a sponge type, which belongs to the OCD/mu-crystallin family.

Multifunctions of MelB, a fungal tyrosinase from Aspergillus oryzae.

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH(2)OH (reductant), the proenzyme bound dioxygen to give a stable (µ-η(2):η(2) -peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.

Catalytic properties and amino acid sequence of endo-1→3-ß-D-glucanase from the marine mollusk Tapes literata.

A specific 1→3-ß-D-glucanase with molecular mass 37 kDa was isolated in homogeneous state from crystalline style of the commercial marine mollusk Tapes literata. It exhibits maximal activity within the pH range from 4.5 to 7.5 at 45°C. The 1→3-ß-D-glucanase catalyzes hydrolysis of ß-1→3 bonds in glucans as an endoenzyme with retention of bond configuration, and it has transglycosylating activity. The K(m) for hydrolysis of laminaran is 0.25 mg/ml. The enzyme is classified as a glucan endo-(1→3)-ß-D-glucosidase (EC 3.2.1.39). The cDNA encoding this 1→3-ß-D-glucanase from T. literata was sequenced, and the amino acid sequence of the enzyme was determined. The endo-1→3-ß-D-glucanase from T. literata was assigned to the 16th structural family (GHF 16) of O-glycoside hydrolases.

[Glutathione-S-transferase as a molecular biomarker of the state of marine organisms influenced by anthropogenic pressure].

The activity ofglutathione-S-transferase (GST), a key phase II enzyme catalyzing the biotransformation of organic electrophilic compounds, was studied in the bivalve Crenomytilus grayanus, the mysid shrimp Neomysis mirabilis, and the flounder Liopsetta pinnifasciata from Peter the Great Bay, Sea of Japan. GST activity was increased in fish and mollusks from polluted areas of the bay, compared to background areas. In mysid shrimps, pollution caused inhibition of the activity of this enzyme. The role of GST in adaptation processes of different species to the current level of pollution is discussed.

Myosin kinase of molluscan smooth muscle. Regulation by binding of calcium to the substrate and inhibition of myorod and twitchin phosphorylation by myosin.

Major contractile proteins were purified from relaxed actomyosin extracted from molluscan catch muscle myofibrils using ammonium sulfate fractionation and divalent cation precipitation. A fraction of this actomyosin was precipitated and purified as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). Another TW-MR complex was obtained via the removal of myosin. These supramolecular complexes and filaments assembled from purified myosin contained an endogenous protein kinase that phosphorylated myosin and myorod. Significantly, the activity of this novel myosin-associated (MA) kinase was inhibited at calcium concentrations of >0.1 microM. After partial purification of the kinase, we established that the inhibition resulted from binding of calcium to the substrate (myosin) and not from the binding to the enzyme (kinase). No inhibition was observed when myorod was used as a substrate, although the latter is identical to the rod portion of myosin lacking the head domains. Phosphorylation sites of myorod were identified, three at the C-terminal tip and three at the N-terminal domain. In the presence of calcium, addition of myosin to the TW-MR complex resulted in inhibition of this phosphorylation, while in the absence of myosin, this inhibition was negligible. Added myosin also inhibited phosphorylation of twitchin by PKA-like kinase, the latter also present in the complex. The opposite was true with the TW-MY-MR complex; that is, phosphorylation of myosin was inhibited by twitchin and/or myorod. Thus, in parallel to the well-established direct activation by calcium, molluscan catch muscle myosin also regulated its own phosphorylation. Therefore, in addition to the established phosphorylation of twitchin by PKA-like kinase, phosphorylation of myosin and myorod by myosin-associated kinase appears to play an important role in the development of the catch state.

Agarase: review of major sources, categories, purification method, enzyme characteristics and applications.

Agarases are the enzymes which catalyze the hydrolysis of agar. They are classified into alpha-agarase (E.C. 3.2.1.158) and beta-agarase (E.C. 3.2.1.81) according to the cleavage pattern. Several agarases have been isolated from different genera of bacteria found in seawater and marine sediments, as well as engineered microorganisms. Agarases have wide applications in food industry, cosmetics, and medical fields because they produce oligosaccharides with remarkable activities. They are also used as a tool enzyme for biological, physiological, and cytological studies. The paper reviews the category, source, purification method, major characteristics, and application fields of these native and gene cloned agarases in the past, present, and future.

Analysis of AchE and LDH in mollusc, Lamellidens marginalis after exposure to chlorpyrifos.

The enzymes Acetylcholinesterase (AchE) and Lactatedehydrogenase (LDH) are used as biological markers in the present study. Enzymes are highly sensitive and used to evaluate the biological effects of organophosphate pesticide chlorpyrifos in freshwater mussel Lamellidens marginalis. The test organisms were exposed to sub-lethal concentration (5 ppm) of chlorpyrifos for 30 days and allowed to recover for seven days. A distinct reduction of the enzyme AchE (34 +/- 3.3 U l(-1)) was found in the treated hepatopancreas. A significant increase in LDH activity in gill, hepatopancreas and muscle was observed. There was a significant recovery in AchE and LDH in the different tissues, after seven days recovery period.. Hence, the changes in the enzymes are found as the best biomarkering tool to evaluate the effect of organophosphate pesticide chlorpyrifos on the aquatic biota.

Adenosine triphosphatase activity in the membranes of the squid nerve fiber.

This investigation deals with the localization of sites of ATPase activity, especially of transport ATPase, in nerve fibers of the squid Doryteuthis plei, at the subcellular level. Splitting of ATP liberates inorganic phosphate which reacts with lead to form a precipitate in the tissue. The reaction was made on nerve fibers fixed with glutaraldehyde. Frozen slices were incubated in Wachstein-Meisel medium containing ATP and Pb(NO(3))(2). Deposits of reaction product were found in the axolemma (towards its axoplasmic side), Schwann cell membranes (mainly at the channels crossing the layer), and mitochondria. Control experiments revealed that no deposits were observed in nerve fibers fixed in osmium tetroxide prior to incubation in the medium containing ATP, or in nerve fibers incubated without substrate or with adenosine monophosphate, adenosine diphosphate, glycerophosphate, or guanosine triphosphate as substrate. For evaluation of transport ATPase activity, these findings were compared with results obtained with nerve fibers treated with G-strophanthin or K-strophanthoside before or after glutaraldehyde fixation. The cardiac glycosides produced a disappearance or diminution of the deposits. The largest inhibitory effect was observed in the axolemma. The findings indicate that the highest ATPase activity is localized in the axolemma and may be due primarily to transport ATPase.

Octopine as an end product of anaerobic glycolysis in the chambered nautilus.

The terminal step in the anaerobic glycolysis of muscle in the chambered nautilus, Nautilus pompilius, is not pyruvate reduction to lactate as in vertebrate muscle. Instead of lactate dehydrogenase, these organisms utilize octopine dehydrogenase (E.C. 1.5.1.11), catalyzing the reductive condensation of pyruvate and arginine, which is dependent on the reduced form of nicotinamide adenine dinucleotide, to form octopine and the oxidized form of the coenzyme. The kinetic properties of octopine dehydrogenase favor the production of octopine, which accumulates under a variety of conditions.

Invertebrate facultative anaerobiosis.

The unique pattern of anaerobic carbohydrate metabolism in invertebrate facultative anaerobes serves to couple other substrate-level phosphorylations to the glycolytic reactions, thus increasing the potential yield of high-energy phosphate compounds. Currently, two important coupling sites can be identified:

Library assembly of mono-, di- and tri-O-sulfated beta-D-xylopyranosides; effect of O-sulfation on pyranose ring conformation.

With molluscan sulfatase-catalyzed de-O-sulfation reactions, a series of mono-, di- and tri-O-sulfated p-nitrophenyl beta-D-xylopyranosides were assembled and applied to a 1H NMR study to examine the effect of O-sulfate groups on the equilibration between pyranose 4C1 and 1C4 conformations.

Thermal plasticity of mitochondria: a latitudinal comparison between Southern Ocean molluscs.

Mitochondrial volume density (Vv((mt,f))), cristae surface density (Sv((im,mt))), cristae surface area (Sv((im,f))) and citrate synthase (CS) activity were analysed as indicators of thermal acclimation in foot muscle of the limpet, Nacella concinna, and the clam, Laternula elliptica, collected from 4 locations within the Southern Ocean, South Georgia (54 degrees S, N. concinna only), Signy (60 degrees S), Jubany (L. elliptica only -62 degrees S) and Rothera (67 degrees S). Animals were acclimated to 0.0 degrees C whilst a sub-set of N. concinna (South Georgia, Signy and Rothera) and L. elliptica (Rothera) were acclimated to 3.0 degrees C. At 0.0 degrees C N. concinna had higher Vv((mt,f)), Sv((im,mt)), Sv((im,f)) and muscle fibre specific CS activity than L. elliptica which correlated with the more active life style of N. concinna. However, mitochondrial density was very low, 1-2% in both species, suggesting that low temperature compensation of mitochondrial density is not a universal evolutionary response of Antarctic marine ectotherms. Both Sv((im,mt)) and Sv((im,f)) were reduced by warm acclimation of N. concinna. South Georgia N. concinna maintained muscle fibre specific CS activity after acclimation, in contrast to N. concinna from Rothera and Signy and L. elliptica from Rothera, indicating that they have the physiological plasticity to respond to their warmer, more variable thermal environment.

o-Diphenol oxidase activity of molluscan hemocyanins.

Diphenoloxidase activities of two molluscan hemocyanins, isolated from the marine snails Rapana venosa and garden snails Helix vulgaris were studied using o-diphenol and L-Dopa as substrates. The dimers of H. vulgaris Hc show both, diphenol (K(m)=2.86 mM and K(cat)=4.48) and L-Dopa activity due to a more open active sites of the enzyme and better access of the substrates. The K(m) value of molluscan H. vulgaris Hc is very close to those of Helix pomatia and Sepia officinalis Hcs, but several times higher compared to those of Rapana and Octopus Hcs. Also HvH has a very high enzyme activity compared with other molluscan Hcs. Kinetic measurements with native RvH and both structural subunits, RvH1 and RvH2, show that RvH and only one structural subunit, RvH2, exhibited only o-diphenol activity, but no L-Dopa oxidizing activity.

Oxidative stress and expression of chaperones in aging mollusks.

The mechanisms of aging are not well understood in animals with continuous growth such as fish, reptiles, amphibians and numerous invertebrates, including mollusks. We studied the effects of age on oxidative stress, cellular defense mechanisms (including two major antioxidant enzymes, superoxide dismutase (SOD) and catalase), and molecular chaperones in two mollusks--eastern oysters Crassostrea virginica and hard clams Mercenaria mercenaria. In order to detect the age-related changes in these parameters, correction for the effects of size was performed where appropriate to account for growth-related dilution. Fluorescent age pigments accumulated with age in both species. Protein carbonyls did not change with age or size indicating that they are not a good marker of aging in mollusks possibly due to the fast turnover and degradation of oxidized proteins in growing tissues. SOD did not show a compensatory increase with aging in either species, while catalase significantly decreased with age. Mitochondrial heat shock protein (HSP60) decreased with age in mollusks suggesting an age-related decline in mitochondrial chaperone protection. In contrast, changes in cytosolic chaperones were species-specific. HSP70 increased and HSP90 declined with age in clams, whereas in oysters HSP70 expression did not change, and HSP90 increased with aging.

Expression and characterization of two secreted His6-tagged endo-beta-1,4-glucanases from the mollusc Ampullaria crossean in Pichia pastoris.

Two endo-beta-1,4-glucanase cDNAs, eg27I and eg27II, from the mollusc Ampullaria crossean were expressed in Pichia pastoris cells. The secreted His6-tagged proteins were purified in a single chromatography step. The purified recombinant EG27I and EG27II showed enzymatic activity on carboxylmethyl cellulose sodium salt at 15.31 U/mg and 12.40 U/mg, respectively. The optimum pH levels of the recombinant EG27I and EG27II were 5.5 and 5.5-6.0, respectively, and the optimum temperatures were 50 degrees C and 50 degrees C-55 degrees C, respectively. The pH stability study revealed that both EG27I and EG27II showed their highest stability at pH 8.0. Analysis of their thermostability indicated that both EG27I and EG27II were relatively stable up to 40 degrees C. Site-directed mutagenesis of Asp43 and Asp153 of both EG27I and EG27II showed that the two Asp residues are critical for the enzymatic activity.

Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis.

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. The gene encoding Locusta migratoria manilensis AK was cloned and expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 358 mg L(-1) of LB medium. Some parameters for the renatured and soluble forms of AK, including Km, Kd, specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest kcat and stronger synergistic substrate binding. The first report of a concise purification method enables the enzyme to be prepared in large quantities. This research should enable further detailed investigations of the enzymatic mechanism by site directed mutagenesis techniques.

Antibacterial, tyrosinase, and DNA photocleavage studies of some triazolylnucleosides.

In order to explore the biological potential, some synthesized triazolylnucleosides were evaluated for their antibacterial, tyrosinase and DNA photocleavage activities. Triazolylnucleosides (5-12) were screened against Staphylococcus aureus (ATCC 6538), gram-positive and Escherichia coli (ATCC 10536), gram-negative bacterial strains. Among the series, compound 9 exhibited a significant level of antibacterial activity against both strains at higher concentration in reference to the standard drug, Levofloxacin. Tyrosinase activity and inhibition of these compounds were also studied, and it has been found that compounds 8 and 11 displayed more than 50% inhibitory activity. In addition, six compounds (7-12) were evaluated for their DNA photocleavage activity. The compounds 8 and 12 exhibited excellent DNA photocleavage activity at a concentration of 10 µg and may be used as template for antitumor drugs in the future.