Nonaqueous fractionation and overexpression of fluorescent‐tagged enzymes reveals the subcellular sites of L‐theanine biosynthesis in tea

l ‐Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l ‐theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l ‐theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l ‐theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l ‐theanine synthetase and also to determine the subcellular localization of fluorescent‐tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l ‐theanine biosynthesis, and cytosol‐located CsTSI was the key l ‐theanine synthase. In tea shoot tissue, l ‐theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l ‐theanine synthases. In addition, l ‐theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.     

The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis.

Neisseria gonorrhoeae synthesizes a rough lipopolysaccharide that does not contain any of the repetitive units characteristic of the smooth lipopolysaccharide of members of the family Enterobacteriaceae. Three gonococcal homologs of Salmonella serovar typhimurium genes involved in the synthesis of the rhamnose component of the repetitive subunits have been isolated. Gonococcal homologs for rfbB, rfbA, and rfbD were found downstream of the galE gene in a region of the chromosome which shows overall homology with the meningococcal capsule gene complex region D. Sequence alignment demonstrated that the gonococcal gene products have 69, 65, and 54% amino acid identity with the Salmonella proteins RfbB, RfbA, and RfbD. The gonococcal RfbB and RfbA amino acid sequences share even more identical residues (73 and 65%, respectively) with the amino acid sequences derived from Escherichia coli genes o355 and o292, respectively. These genes are clustered with the genes involved in the biosynthesis of enterobacterial common antigen, and o355 is listed in the GenBank and Swiss Protein data banks as rffE (encoding UDP-GlcNAc-2-epimerase). However, complementation studies demonstrated that o355 does not encode the enzyme UDP-GlcNAc-2-epimerase. Gonococcal strains constructed with null mutations in the rfbBAD genes were unchanged in lipopolysaccharide phenotype and in the synthesis of gonococcal carbohydrate-containing C antigen. We were unable to detect any changes in gonococcal phenotype with respect to lipopolysaccharide sialylation, monoclonal-antibody binding, serum sensitivity, or interaction with eukaryotic cells in vitro. We conclude that the absence of a homolog for rfbC precludes the existence of a functional dTDP-rhamnose biosynthesis pathway in the gonococcal strains examined and that these genes are only maintained in N. gonorrhoeae either because of the presence of the galE gene or because of another as yet unrecognized function.     

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis.

To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger syndrome. We found that all enzymes measured, including putative peroxisomal enzymes, are at least as active in the peroxisome-deficient Zellweger mice as in control mice, indicating that mislocalization of enzymes to the cytosol does not lead to decreased activity or degradation. Prompted by these results, we re-examined this aspect in human subjects by specific enzyme activity measurements and immunoblotting with highly specific antisera. Our results show that the previously reported deficiencies of mevalonate kinase and phosphomevalonate kinase activity in livers from human Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol.Our data provide an explanation for the conflicting findings in the literature and show that great care should be taken in the interpretation of data obtained in postmortem material.     

Toward understanding the key enzymes involved in β‐poly (L‐malic acid) biosynthesis by Aureobasidium pullulans ipe‐1

β‐poly (L‐malic acid) (PMLA) is a biopolyester which has attracted industrial interest for its potential application in medicine and other industries. A high dissolved oxygen concentration (DO) was beneficial for PMLA production, while the mechanisms of DO in PMLA biosynthesis by Aureobasidium pullulans are still poorly understood. In this work, the amount of PMLA was first compared when A. pullulans ipe‐1 were cultured under a high DO level (70% saturation) and a low DO level (10% saturation). Meanwhile, the key enzymes involved in different pathways of the precursor L‐malic acid biosynthesis were studied. The results revealed that the activities of glucose‐6‐phosphate dehydrogenase (G6PDH) and phosphoenolpyruvate carboxylase (PEPC) were positively correlated with cell growth and PMLA production, while the activities of phosphofructokinases (PFK), pyruvic carboxylase (PC) and citrate synthetase (CS) did no show such correlations. It indicated that the Pentose Phosphate Pathway (PPP) may play a vital role in cell growth and PMLA biosynthesis. Moreover, the precursor L‐malic acid for PMLA biosynthesis was mainly biosynthesized through phosphoenolpyruvic acid (PEP) via oxaloacetate catalyzed by PEPC. It was also found that low concentration of sodium fluoride (NaF) might impel carbon flux flow to the oxaloacetate through PEP, but inhibit the flux to the oxaloacetate via pyruvic acid.     

Cationic oligopeptides with the repeating sequence L‐lysyl‐L‐alanyl‐L‐alanine: conformational and thermal stability study using optical spectroscopic methods

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L ‐Lys‐L ‐Ala‐L ‐Ala)_n (n = 1, 2, and 3) were measured over a range of temperatures (20–90 °C) in aqueous solution at near‐neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I′ region indicate the presence of extended helical polyproline II (PPII)‐like conformation at room temperature. UV‐ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII‐like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles ? and ψ are within the range ?82 to ?107° and 143–154°, respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII‐like conformation of the Lys‐Ala‐Ala sequence is still preferred, being in equilibrium with an unordered conformer at near‐neutral pH values within the range of temperatures from 20 to 90 °C. This finding was obtained from analysis of the temperature‐dependent spectra using the singular value decomposition method. The study presents KAA‐containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Structure,function, and biosynthesis of nickel‐dependent enzymes

Nickel enzymes, present in archaea, bacteria, plants, and primitive eukaryotes are divided into redox and nonredox enzymes and play key functions in diverse metabolic processes, such as energy metabolism and virulence. They catalyze various reactions by using active sites of diverse complexities, such as mononuclear nickel in Ni‐superoxide dismutase, glyoxylase I and acireductone dioxygenase, dinuclear nickel in urease, heteronuclear metalloclusters in [NiFe]‐carbon monoxide dehydrogenase, acetyl‐CoA decarbonylase/synthase and [NiFe]‐hydrogenase, and even more complex cofactors in methyl‐CoM reductase and lactate racemase. The presence of metalloenzymes in a cell necessitates a tight regulation of metal homeostasis, in order to maintain the appropriate intracellular concentration of nickel while avoiding its toxicity. As well, the biosynthesis and insertion of nickel active sites often require specific and elaborated maturation pathways, allowing the correct metal to be delivered and incorporated into the target enzyme. In this review, the phylogenetic distribution of nickel enzymes will be briefly described. Their tridimensional structures as well as the complexity of their active sites will be discussed. In view of the latest findings on these enzymes, a special focus will be put on the biosynthesis of their active sites and nickel activation of apo‐enzymes.     

The zebrafish erythropoietin: functional identification and biochemical characterization

In the present study, the zebrafish epo cDNA was cloned. The encoded protein displays 90%, 55% and 32% identity to the Epo from carp, fugu and human, respectively. Through RT-PCR, the expression of zepo mRNA was mainly in the heart and liver. In the COS-1 cell transfection experiments, the recombinant zEpo-HA protein was efficiently secreted into the culture medium as a glycoprotein and the carbohydrate moiety can be cleaved by the treatment of peptide-N-glycosidase F (PNGase F). Using the morpholino approach, we showed that zepo morphants displayed severe anemia leading to high mortality during development. Such an effect can be significantly rescued by zepo RNA. Furthermore, in the absence of functional zEpo, the expression of specific markers for adult globin genes, such as alphaA1- and betaA1-globin, but not the embryonic betae1-globin, was affected.     

Preclinical characterization of alginate‐poly‐L‐lysine encapsulated HepaRG for extracorporeal liver supply

We recently demonstrated that HepaRG cells encapsulated into 1.5% alginate beads are capable of self‐assembling into spheroids. They adequately differentiate into hepatocyte‐like cells, with hepatic features observed at Day 14 post‐encapsulation required for external bioartificial liver applications. Preliminary investigations performed within a bioreactor under shear stress conditions and using a culture medium mimicking acute liver failure (ALF) highlighted the need to reinforce beads with a polymer coating. We demonstrated in a first step that a poly‐l ‐lysine coating improved the mechanical stability, without altering the metabolic activities necessary for bioartificial liver applications (such as ammonia and lactate elimination). In a second step, we tested the optimized biomass in a newly designed perfused dynamic bioreactor, in the presence of the medium model for pathological plasma for 6 h. Performances of the biomass were enhanced as compared to the steady configuration, demonstrating its efficacy in decreasing the typical toxins of ALF. This type of bioreactor is easy to scale up as it relies on the number of micro‐encapsulated cells, and could provide an adequate hepatic biomass for liver supply. Its design allows it to be integrated into a hybrid artificial/bioartificial liver setup for further clinical studies regarding its impact on ALF animal models.     

A global characterization and identification of multifunctional enzymes

Multi-functional enzymes are enzymes that perform multiple physiological functions. Characterization and identification of multi-functional enzymes are critical for communication and cooperation between different functions and pathways within a complex cellular system or between cells. In present study, we collected literature-reported 6,799 multi-functional enzymes and systematically characterized them in structural, functional, and evolutionary aspects. It was found that four physiochemical properties, that is, charge, polarizability, hydrophobicity, and solvent accessibility, are important for characterization of multi-functional enzymes. Accordingly, a combinational model of support vector machine and random forest model was constructed, based on which 6,956 potential novel multi-functional enzymes were successfully identified from the ENZYME database. Moreover, it was observed that multi-functional enzymes are non-evenly distributed in species, and that Bacteria have relatively more multi-functional enzymes than Archaebacteria and Eukaryota. Comparative analysis indicated that the multi-functional enzymes experienced a fluctuation of gene gain and loss during the evolution from S. cerevisiae to H. sapiens. Further pathway analyses indicated that a majority of multi-functional enzymes were well preserved in catalyzing several essential cellular processes, for example, metabolisms of carbohydrates, nucleotides, and amino acids. What's more, a database of known multi-functional enzymes and a server for novel multi-functional enzyme prediction were also constructed for free access at http://bioinf.xmu.edu.cn/databases/MFEs/index.htm.     

Homochiral bifunctional L‐prolinamide‐ and L‐bis‐prolinamide‐catalyzed asymmetric aldol reactions performed in wet solvent‐free conditions

In this study, the novel bifunctional homochiral thiourea‐L‐prolinamides 1–4 , tertiary amino‐L‐prolinamide 5 , and bis‐L‐prolinamides 6 and 7 were prepared from enantiomerically pure (11R,12R)‐11,12‐diamino‐9,10‐dihydro‐9,10‐ethanoanthracene 8 and (11S,12S)‐11,12‐diamino‐9,10‐dihydro‐9,10‐ethanoanthracene ent‐8 . Highly enantioselective and diastereoselective aldolic intermolecular reactions (up to 95% enantiomeric excess, 93:7 anti/syn) between aliphatic ketones (20 equiv) and a range of aromatic aldehydes (1 equiv) were successfully carried out in the presence of water (10 equiv) and monochloroacetic acid (10 mol%), solvent‐free conditions, at room temperature over 24 h using organocatalysts 1–7 (5 mol%). Stereoselective induction using density functional theory–based methods was consistent with the experimental data.     

The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase

We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.     

Thermally induced cyclization of L‐isoleucyl‐L‐alanine in solid state: Effect of dipeptide structure on reaction temperature and self‐assembly

Thermal treatment of short‐chain oligopeptides is able to initiate the process of their self‐assembly with the formation of organic nanostructures with unique properties. On the other hand, heating can lead to a chemical reaction with the formation of new substances with specific properties and ability to form structures with different morphology. Therefore, in order to have a desired process, researcher needs to find its temperature range. In the present work, cyclization of _L‐isoleucyl‐_L‐alanine dipeptide in the solid state upon heating was studied. Kinetic parameters of this reaction were estimated within the approaches of the nonisothermal kinetics. The correlation between side chain structure of dipeptides and temperature of their cyclization in the solid state was found for the first time. This correlation may be used to predict the temperature, at which dipeptide self‐assembly changes to chemical reaction. The differences in self‐assembly of linear and cyclic dipeptides were demonstrated using atomic force microscopy. The effect of dipeptide concentration in a source solution and an organic solvent used on self‐assembly of dipeptides was shown. The new information obtained on the thermal properties and self‐assembly of linear and cyclic forms of _L‐isoleucyl‐_L‐alanine may be useful for the design of new nanomaterials based on oligopeptides, as well as for the synthesis of cyclic oligopeptides.     

L‐Cysteine capped CdTe–CdS core–shell quantum dots: preparation,characterization and immuno‐labeling of HeLa cells

Functionalized CdTe–CdS core–shell quantum dots (QDs) were synthesized in aqueous solution via water‐bathing combined hydrothermal method using L‐cysteine (L‐Cys) as a stabilizer. This method possesses both the advantages of water‐bathing and hydrothermal methods for preparing high‐quality QDs with markedly reduced synthesis time, and better stability than a lone hydrothermal method. The QDs were characterized by transmission electronic microscopy and powder X‐ray diffraction and X‐ray photoelectron spectroscopy. The CdTe–CdS QDs with core–shell structure showed both enhanced fluorescence and better photo stability than nude CdTe QDs. After conjugating with antibody rabbit anti‐CEACAM8 (CD67), the as‐prepared l ‐Cys capped CdTe–CdS QDs were successfully used as fluorescent probes for the direct immuno‐labeling and imaging of HeLa cells. It was indicated that this kind of QD would have application potential in bio‐labeling and cell imaging. Copyright © 2009 John Wiley & Sons, Ltd.     

Methionine biosynthesis in higher plants: biochemical and molecular characterization of the transsulfuration pathway enzymes

In plants, the transfer of the sulfur atom between cysteine and homocysteine, the direct precursor of methionine, is ensured by two chloroplastic enzymes, cystathionine γ-synthase and cystathionine β-lyase. These proteins have been purified to homogeneity from spinach chloroplasts and their biochemical properties determined. Cystathionine γ-synthase and cystathionine β-lyase are tetramers and are typical pyridoxal 5′-phosphate-dependent proteins. These enzymes are targets for the potent inhibitors of methionine synthesis that are lethal for plants. An Arabidopsis thaliana cDNA encoding chloroplastic cystathionine β-lyase was isolated by functional complementation of a bacterial mutant and cloned in a pET expression vector in order to transform Escherichia coli cells. Preliminary observations of the active site of the purified recombinant enzyme have been performed by characterization of the interaction between i) pyridoxal 5′-phosphate and the polypeptide chain, and ii) the active site-directed inhibitor aminoethoxyvinylglycine and the bound cofactor. This study will be developed further by crystallographic analyses.     

Vitamin E biosynthesis: functional characterization of the monocot homogentisate geranylgeranyl transferase

The biosynthesis of the tocotrienol and tocopherol forms of vitamin E is initiated by prenylation of homogentisate. Geranylgeranyl diphosphate (GGDP) is the prenyl donor for tocotrienol synthesis, whereas phytyl diphosphate (PDP) is the prenyl donor for tocopherol synthesis. We have previously shown that tocotrienol synthesis is initiated in monocot seeds by homogentisate geranylgeranyl transferase (HGGT). This enzyme is related to homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol synthesis. Here we show that monocot HGGT is localized in the plastid and expressed primarily in seed endosperm. Despite the close structural relationship of monocot HGGT and HPT, these enzymes were found to have distinct substrate specificities. Barley (Hordeum vulgare cv. Morex) HGGT expressed in insect cells was six times more active with GGDP than with PDP, whereas the Arabidopsis HPT was nine times more active with PDP than with GGDP. However, only small differences were detected in the apparent Km values of barley HGGT for GGDP and PDP. Consistent with its in vitro substrate properties, barley HGGT generated a mixture of tocotrienols and tocopherols when expressed in the vitamin E-null vte2-1 mutant lacking a functional HPT. Relative levels of tocotrienols and tocopherols produced in vte2-1 differed between organs and growth stages, reflective of the composition of plastidic pools of GGDP and PDP. In addition, HGGT was able to functionally substitute for HPT to rescue vte2-1-associated phenotypes, including reduced seed viability and increased fatty acid oxidation of seed lipids. Overall, we show that monocot HGGT is biochemically distinct from HPT, but can replace HPT in important vitamin E-related physiological processes.     

Physiological and biochemical characterization of NERICA‐L‐44: a novel source of heat tolerance at the vegetative and reproductive stages in rice

The predicted increase in the frequency and magnitude of extreme heat spikes under future climate can reduce rice yields significantly. Rice sensitivity to high temperatures during the reproductive stage is well documented while the same during the vegetative stage is more speculative. Hence, to identify and characterize novel heat‐tolerant donors for both the vegetative and reproductive stages, 71 rice accessions, including approximately 75% New Rice for Africa (NERICAs), were phenotyped across field experiments during summer seasons in Delhi, India, and in a controlled environment study at International Rice Research Institute , Philippines. NERICA‐L‐44 (NL‐44) recorded high seedling survival (52%) and superior growth and greater reproductive success exposed to 42.2°C (sd ± 2.3) under field conditions. NL‐44 and the heat‐tolerant check N22 consistently displayed lower membrane damage and higher antioxidant enzymes activity across leaves and spikelets. NL‐44 recorded 50–60% spikelet fertility, while N22 recorded 67–79% under controlled environment temperature of 38°C (sd ±1.17), although both had about 87% fertility under extremely hot field conditions. N22 and NL‐44, exposed to heat stress (38°C), had similar pollen germination percent and number of pollen tubes reaching the ovary. NL‐44 maintained low hydrogen peroxide production and non‐photochemical quenching (NPQ) with high photosynthesis while N22 avoided photosystem II damage through high NPQ under high‐temperature stress. NL‐44 with its reproductive stage resilience to extreme heat stress, better antioxidant scavenging ability in both vegetative tissue and spikelets and superior yield and grain quality is identified as a novel donor for increasing heat tolerance at both the vegetative and reproductive stages in rice.