Comparing the in Vivo Function of α-Carboxysomes and β-Carboxysomes in Two Model Cyanobacteria

The carbon dioxide (CO₂)-concentrating mechanism of cyanobacteria is characterized by the occurrence of Rubisco-containing microcompartments called carboxysomes within cells. The encapsulation of Rubisco allows for high-CO₂ concentrations at the site of fixation, providing an advantage in low-CO₂ environments. Cyanobacteria with Form-IA Rubisco contain α-carboxysomes, and cyanobacteria with Form-IB Rubisco contain β-carboxysomes. The two carboxysome types have arisen through convergent evolution, and α-cyanobacteria and β-cyanobacteria occupy different ecological niches. Here, we present, to our knowledge, the first direct comparison of the carboxysome function from α-cyanobacteria (Cyanobium spp. PCC7001) and β-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (C_i; as CO₂ and HCO₃⁻) transporter systems. Despite evolutionary and structural differences between α-carboxysomes and β-carboxysomes, we found that the two strains are remarkably similar in many physiological parameters, particularly the response of photosynthesis to light and external C_i and their modulation of internal ribulose-1,5-bisphosphate, phosphoglycerate, and C_i pools when grown under comparable conditions. In addition, the different Rubisco forms present in each carboxysome had almost identical kinetic parameters. The conclusions indicate that the possession of different carboxysome types does not significantly influence the physiological function of these species and that similar carboxysome function may be possessed by each carboxysome type. Interestingly, both carboxysome types showed a response to cytosolic C_i, which is of higher affinity than predicted by current models, being saturated by 5 to 15 mm C_i. This finding has bearing on the viability of transplanting functional carboxysomes into the C₃ chloroplast.Cyanobacteria inhabit a diverse range of ecological habitats, including both freshwater and marine ecosystems. The flexibility to occupy these different habitats is thought to come in part from the carbon-concentrating mechanism (CCM) present in all species (Badger et al., 2006). The CCM comprises inorganic carbon (C_i; as carbon dioxide [CO₂] and HCO₃⁻) transporters for C_i uptake and protein microbodies called carboxysomes for CO₂ concentration and fixation by Rubisco (Badger and Price, 2003). The CCM is believed to have evolved in response to changes in the absolute and relative levels of CO₂ and oxygen (O₂) in the atmosphere during the evolution of oxygenic photosynthesis in cyanobacteria (Price et al., 2008).There are two main phylogenetic groups within the cyanobacteria based on Rubisco and carboxysome phylogenies; α-cyanobacteria have α-carboxysomes with Form-IA Rubisco, whereas β-cyanobacteria have β-carboxysomes with Form-IB Rubisco (Tabita, 1999; Badger et al., 2002). Rubisco large subunit protein sequences from these two groups are closely related but nevertheless, distinguishable (Supplemental Fig. S1). In general, α-cyanobacteria and β-cyanobacteria occupy a quite different range of ecological habitats. The α-cyanobacteria are mostly marine organisms, with the majority of species living in the open ocean (Badger et al., 2006). Marine α-cyanobacteria live in very stable environments with high pH (pH 8.2) and dissolved carbon levels but low nutrients. They are characterized by small cells, very small genomes (1.6–2.8 Mb), and a few constitutively expressed carbon uptake transporters (Rae et al., 2011; Beck et al., 2012). They have been described as low flux, low energy cyanobacteria with a minimal CCM (Badger et al., 2006). Although these species are slow growing, oceanic cyanobacteria contribute as much as one-half of oceanic primary productivity (Liu et al., 1997, 1999; Field et al., 1998), suggesting that they may contribute up to 25% to net global productivity every year.In comparison, β-cyanobacteria occupy a much more diverse range of habitats, including freshwater, estuarine, and hot springs and never reach the same levels of global abundance (Badger et al., 2006). They are characterized by larger cells, larger genomes (2.2–3.6 Mb), and an array of carbon uptake transporters, including those transporters induced under low C_i (Rae et al., 2011, 2013). In addition to these broadly defined α-groups and β-groups, there are small numbers of α-cyanobacteria that have been termed transitional strains (Price, 2011; Rae et al., 2011). These species (e.g. Cyanobium spp. PCC7001, Synechococcus spp. WH5701, and Cyanobium spp. PCC6307; Supplemental Fig. S1) live in marginal marine and freshwater environments and have a number of characteristics similar to β-cyanobacteria. For example, they have a more diverse range of C_i uptake systems and a significantly larger genome than closely related α-cyanobacteria, and it has been suggested that the additional genes encoding transport systems were acquired by horizontal gene transfer (HGT) from β-cyanobacteria (Rae et al., 2011).Although the carboxysomes from α-cyanobacteria and β-cyanobacteria are very similar in overall structure, in that they share an outer protein shell of common phylogenetic origin (Kerfeld et al., 2005), they are distinguished from each other largely by differences in the proteins, which seem to make up or interact with the interior of the carboxysome compartment (Supplemental Table S1). This finding suggests that their different structures today have arisen through periods of common and convergent evolution. Certain carboxysome shell proteins from α-carboxysomes and β-carboxysomes show regions of significant sequence homology. These proteins are denoted as CsoS1 to CsoS4 (in α-cyanobacteria) and CcmKLO (in β-cyanobacteria), and the homologous regions have been termed bacterial microcompartment domains (Kerfeld et al., 2010; Rae et al., 2013). Proteins with these domains are also found in bacterial microcompartments in proteobacteria. However, other identified carboxysome proteins do not show any sequence homology between α-carboxysomes and β-carboxysomes but may perform similar functional roles. For example, carbonic anhydrase activity is essential for carboxysome function, but its activity seems to be provided by a range of different proteins (β-CcaA, β-CcmM, and α-CsoSCA; Kupriyanova et al., 2013). Similarly, β-CcmM and α-CsoS2 could play similar roles in organizing the interface between the shell and Rubisco within the carboxysomes (Gonzales et al., 2005; Long et al., 2007).The functioning of a carboxysome relies on a number of biochemical properties associated with the protein microbody structure. These properties include the biochemical/kinetic properties of Rubisco contained within carboxysomes, the conductance of the carboxysome shell to the influx of substrate ribulose-1,5-bisphosphate (RuBP) and the efflux of the carboxylation product phosphoglycerate (PGA), the conductance of the shell to the influx of bicarbonate and the efflux of CO₂, and lastly, the manner in which bicarbonate is converted to CO₂ within the carboxysomes. α-Carboxysomes and β-carboxysomes have the potential to differ in each of these properties. The flux of phosphorylated sugars across the shell has been postulated to be mediated by the pores in the hexameric shell proteins (Yeates et al., 2010; Kinney et al., 2011), which although similar, do differ between the two carboxysomes types. Bicarbonate and CO₂ uptake processes are less well-defined but probably involve aspects of the way in which unique shell interface proteins interact with Rubisco, which also differs in that CsoS2 and CsoSCA are probably the interacting proteins involved in α-carboxysomes (Espie and Kimber, 2011), whereas CcmM and β-carboxysomal CA are variably involved in β-carboxysomes (Long et al., 2010). Finally, the Form-IA and Form-IB Rubisco proteins at the heart of carboxylation, although similar, have the potential to show different kinetic properties. Although Form-IB Rubiscos from β-cyanobacteria are well-characterized, the Form-IA counterparts have received very little attention. In addition, the CCM of very few strains of cyanobacteria have been studied at the level of biochemistry and physiology, and they have been almost exclusively β-cyanobacteria. As a result, there are significant gaps in our knowledge about the similarities and differences in functional traits between α-cyanobacterial and β-cyanobacterial strains. One important question that remains to be answered is whether α-carboxysomes and β-carboxysomes have intrinsic differences in their biochemical properties that influence the nature of the CCM, which is established within each broad cell type.Because of the difficulties in isolating and assaying intact carboxysomes in vitro, the characterization of biochemical properties of carboxysomes is not easily addressed. One way forward is to study the properties of the CCM in detail in a model representative strain from each group and compare their characteristics to contrast the intracellular function of α-cell types and β-cell types. In the past, it has been restricted because of the difficulties in growing many of the open ocean α-cyanobacteria and their very different natures in relation to inorganic transporter composition. However, the availability of α-cyanobacteria transition strains, which grow well in the laboratory, has provided an opportunity to address this question. The α-cyanobacteria Cyanobium spp. PCC7001 (hereafter Cyanobium spp.), in particular, grows in standard freshwater media (BG11) and has growth and photosynthetic performance properties that closely match the model β-cyanobacteria, Synechococcus spp. PCC7942 (hereafter Synechococcus spp.); for this reason, Cyanobium spp. is ideal for a balanced comparison of the in vivo physiological properties of α-carboxysomes and β-carboxysomes in two species with relatively similar C_i-uptake properties.Genome analysis of both strains indicates that Cyanobium spp. have many of the same carbon uptake systems present in Synechococcus spp. (Rae et al., 2011). In using two strains with such similar transport capacities, we aimed to shed light on aspects of the functional properties of carboxysomes in each strain and how these properties affect the operation of the CCM in α-cyanobacteria and β-cyanobacteria. Using both membrane inlet mass spectrometry (MIMS) and silicon oil centrifugation, we investigated C_i pool sizes and CO₂ uptake rates in both species for cells grown at high and low CO₂. Comparative Rubisco properties and photosynthetic rates of each species were determined, and intracellular pools of RuBP and PGA were measured. In addition, we characterized a number of cellular properties to determine differences in the biochemical environments in which each carboxysome type exists. Together, the results provide a unique functional comparison of two distinct carboxysome types from phylogenetically disparate cyanobacteria.     

Ultrastructural localization of immunoreactive corticotropin, β-lipotropin, α- and β-endorphin in Cells of the human fetal anterior pituitary

Summary Cells immunoreactive with anti--(17–39) ACTH, -(1–24) corticotropin, -LPH, - and -EP were identified in the human fetal anterior pituitary at the ultrastructural level using the peroxidase-antiperoxidase complex method on ultrathin sections.Only one definite cell type was revealed by all these antisera. All granules of each individual immunostained cell reacted regardless of the antiserum used. The immunostained cells occurred in groups and were sometimes located in the wall of the follicle-like structures commonly observed in the fetal anterior pituitary. The cells revealed two main aspects: 1) The largest elements were rich in organelles, and their numerous secretory granules showed significant variations in size (250–500 nm in diameter), electron density of their content and stain-deposit intensity. The ergastoplasm, consisting of irregular tubules, was poorly developed. In the vicinity of the conspicuous Golgi apparatus, organelles related to the GERL complex were commonly observed. Multivesicular bodies were frequent. Some of these cells showed bundles of microfilaments (60 nm in thickness). 2) The smaller cells had an electron-lucent hyaloplasm with sparse organelles; they contained fewer granules and never showed microfilaments.The immunocytological results are consistent with the synthesis of a molecule similar to pro-opiocortin by this type of endocrine cell in human fetuses. Morphological evidence for the maturation process of this precursor and for the secretory activity of these cells and its possible regulation is presented and discussed.Abbreviations used ACTH corticotropin (39 amino acid polypeptide) - -MSH -melanotropin (ACTH [1–13]) - CLIP corticotropin-like intermediate lobe peptide (ACTH [18–39]) - -LPH -lipotropin (91 amino acid polypeptide) - -MSH -melanotropin (-LPH [41–58]) - -EP -endorphin (-LPH [61–91]) - -EP -endorphin (-LPH [61–76]) - PTA phosphotungstic acid Acknowledgements: The authors would like to thank Professors P. Magnin and J. Liaras, Hôpital Edouard Herriot; M. Dumont, Hôpital de la Croix-Rousse; A. Notter and R. Garmier, Hôtel Dieu; M. Bethenod, Hôpital Debrousse, Lyon, and the entire staff whose cooperation enabled samples to be taken under optimal conditions. The authors also thank Professor L. Graf, Research Institute for Pharmaceutical Chemistry (Budapest), and Professor R. Guillemin (Salk Institute, La Jolla) for their generous gift of antigensThis work was supported by a grant from I.N.S.E.R.M., ATP 46.77.78 (P.M. Dubois)     

Localization of α-Galactosidase in an Alkalophilic Strain of Micrococcus†

Localization of α-galactosidase in an alkalophilic strain of Micrococcus was investigated in relation to the cell membrane as a permeability barrier. The most α-galactosidase appered to be intracellular; only about 4% of α-galactosidase was released by lysozyme or freeze-thaw treatments of the whole cells. The enzyme activity was not inhibited by treatment of the whole cells with diazo-7-amino-1,3-naphthalene disulfonic acid (NDS) which penetrated the cell wall but not the cytoplasmic membrane. The enzyme activity of the whole cells increased about four-fold by toluene-acetone treatment which caused an alteration in the membrane permeability. The enzyme in such cells became to be relatively sensitive to pH. These results showed that cell membrane played a protective role as a permeability barrier against alkaline environment.     

Regulation of calcium in pancreatic α- and β-cells in health and disease

The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca(2+)-influx via voltage-gated Ca(2+)-channels, elevation of [Ca(2+)](i) and initiation of Ca(2+)-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca(2+)-channel present in α- and β-cells and the modulation of hormone secretion by Ca(2+)-dependent and -independent processes. We also consider how subtle changes in Ca(2+)-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes.     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Attempt at Affinity Labeling of α- and β- Amylases by α- and β-d-Glucopyranosides and α- and β-Maltooligosaccharides with 2,3-Epoxypropyl Residue as Aglycone: Specific Inactivation of β-Amylases

Among 2,3-epoxypropyl α-d-glucopyranoside and 2,3-epoxypropyl α-maltooligosaccharides and the β-anomers, 2,3-epoxypropyl α-d-glucopyranoside (α-EPG) strongly inactivated the β-amylases [EC 3.2.1.2] of sweet potato, barley, and Bacillus, cereus, in addition to soybean β amylase [J. Biochem., 99, 1631 (1986)]. However, none of the compounds used inactivated any α-amylases [EC 3.2.1.1] of porcine pancreas, Aspergillus oryzae, or Bacillus amyloliquefaciens. Irreversible incorporation of ¹⁴C-labeled α-EPG into β-amylases was stoichiometric, i.e., one α-EPG per active site of the enzyme was bound, and the inactivations were almost complete. The results suggest that α-EPG is an affinity labeling reagent selective for β-amylase. Slow inactivations by the other compounds were also observed, depending on the difference of source of β amylase.     

Lusitropic effects of α- and β-adrenergic stimulation in amphibian heart

The effects of and -adrenergic stimulation in amphibian superfused hearts and ventricular strips were studied. Superfusion with 3×10^–8 M isoproterenol produced a positive inotropic effect, as detected by a 92±24% increase in the maximal rate of contraction and a positive lusitropic effect characterized by a decrease in both the ratio (23±5%) and the half relaxation time (t_1/2) (19±4%). The mechanical behavior induced by the -agonist was associated with an increase in the intracellular cAMP levels from control values of 173±19 to 329±28 nmol/mg wet tissue. Hearts superfused with³²P in the presence of isoproterenol showed a significant increase in Tn 1 phosphorylation (from 151±13 to 240±44 pmol³²P/mg MF protein) without consistent changes in phosphorylation of C-protein. In sarcoplasmic reticulum membrane vesicles, no phospholamban phosphorylation was detected either by -adrenergic stimulation of superfused hearts or when phosphorylation conditions were optimized by direct treatment of the vesicles with cAMP-dependent protein kinase (PKA) and [y ³²P] ATP.The effect of -adrenergic stimulation on ventricular strips was studied at 30 and 22°C. At 30°C, the effects of 10^–5 to 10^–4M phenylephrine on myocardial contraction and relaxation were diminished to non significant levels by addition of propranolol. At 22°C, blockage with propranolol left a remanent positive inotropic effect (10% of the total effect of phenylephrine) and changed the phenylephrine-induced positive lusitropic effect into a negative lusitropic action. These propranolol-resistant effects were abolished by prazosin. Our results suggest that in amphibian heart, both the inotropic and lusitropic responses to catecholamines are mainly due to a -adrenergic stimulation which predominates over the -adrenergic response. Phospholamban phosphorylation seems not to be involved in mediating the positive lusitropic effect of -adrenergic agents whereas phosphorylation of troponin 1 may play a critical role.     

The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

In Vivo Cross-linking Reveals Principally Oligomeric Forms of α-Synuclein and β-Synuclein in Neurons and Non-neural Cells

Aggregation of α-synuclein (αSyn) in neurons produces the hallmark cytopathology of Parkinson disease and related synucleinopathies. Since its discovery, αSyn has been thought to exist normally in cells as an unfolded monomer. We recently reported that αSyn can instead exist in cells as a helically folded tetramer that resists aggregation and binds lipid vesicles more avidly than unfolded recombinant monomers (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107–110). However, a subsequent study again concluded that cellular αSyn is an unfolded monomer (Fauvet, B., Mbefo, M. K., Fares, M. B., Desobry, C., Michael, S., Ardah, M. T., Tsika, E., Coune, P., Prudent, M., Lion, N., Eliezer, D., Moore, D. J., Schneider, B., Aebischer, P., El-Agnaf, O. M., Masliah, E., and Lashuel, H. A. (2012) J. Biol. Chem. 287, 15345–15364). Here we describe a simple in vivo cross-linking method that reveals a major ∼60-kDa form of endogenous αSyn (monomer, 14.5 kDa) in intact cells and smaller amounts of ∼80- and ∼100-kDa forms with the same isoelectric point as the 60-kDa species. Controls indicate that the apparent 60-kDa tetramer exists normally and does not arise from pathological aggregation. The pattern of a major 60-kDa and minor 80- and 100-kDa species plus variable amounts of free monomers occurs endogenously in primary neurons and erythroid cells as well as neuroblastoma cells overexpressing αSyn. A similar pattern occurs for the homologue, β-synuclein, which does not undergo pathogenic aggregation. Cell lysis destabilizes the apparent 60-kDa tetramer, leaving mostly free monomers and some 80-kDa oligomer. However, lysis at high protein concentrations allows partial recovery of the 60-kDa tetramer. Together with our prior findings, these data suggest that endogenous αSyn exists principally as a 60-kDa tetramer in living cells but is lysis-sensitive, making the study of natural αSyn challenging outside of intact cells.     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.     

Differences between α- and β-Chain Mutants of Human Haemoglobin and between α- and β-Thalassaemia. Possible Duplication of the α-Chain Gene

Human adult haemoglobin consists of two unlike pairs of polypeptide chains, and can be described as α₂β₂. Amino-acid substitutions in either of the two types of chain result in α- and β-chain variants. In thalassaemia, which causes a lowered production of haemoglobin, the α or the β chain can be affected, the result being α- or β-thalassaemia. There is a quantitative difference in the proportion of α- and β-chain variants to normal haemoglobin in the respective heterozygotes, and there is also a difference in the pattern of inheritance of α- and β-thalassaemia: these could possibly be explained by assuming that man has one gene for the β- and two for the α-chain.     

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Abstract

Rat C6 glioma cells have both β₁- and β₂-adrenergic receptors in ～ 7:3 ratio. When the cells were exposed to the β-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [³H] CGP-12177 to intact cells. Using the β₂-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both β₁ and β₂ receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of ～ 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [¹²⁵I] iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous β₁-and β₂-adrenergic receptors in the same cell line.     

Syntheses and spectroscopic properties of α- and β-phosphatidylcholines and phosphatidylethanolamines

The widely used partial synthesis of phospholipids via deacylation of naturally occurring phospholipids, followed by reacylation with fatty acid anhydrides, is accompanied by phosphoryl migration. The resulting mixture of α- and β-phospholipids was separated by short-column chromatography. Milder acylation procedures in which no phosphoryl migration occurs, were developed. 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine was prepared in 50% yield by acylation of sn-glycero-3-phosphocholine (GPC) with N-linoleoylimidazole. Detailed NMR and infrared spectra of α- and β-phosphatidylcholines (PCs) and -ethanolamines (PEs) are reported and the differences between isomers discussed.     

Characterization of the α- and β-Mannosidases of Porphyromonas gingivalis

Mannose is an important sugar in the biology of the Gram-negative bacterium Porphyromonas gingivalis. It is a major component of the oligosaccharides attached to the Arg-gingipain cysteine proteases, the repeating units of an acidic lipopolysaccharide (A-LPS), and the core regions of both types of LPS produced by the organism (O-LPS and A-LPS) and a reported extracellular polysaccharide (EPS) isolated from spent culture medium. The organism occurs at inflamed sites in periodontal tissues, where it is exposed to host glycoproteins rich in mannose, which may be substrates for the acquisition of mannose by P. gingivalis. Five potential mannosidases were identified in the P. gingivalis W83 genome that may play a role in mannose acquisition. Four mannosidases were characterized in this study: PG0032 was a β-mannosidase, whereas PG0902 and PG1712 were capable of hydrolyzing p-nitrophenyl α-d-mannopyranoside. PG1711 and PG1712 were α-1→3 and α-1→2 mannosidases, respectively. No enzyme function could be assigned to PG0973. α-1→6 mannobiose was not hydrolyzed by P. gingivalis W50. EPS present in the culture supernatant was shown to be identical to yeast mannan and a component of the medium used for culturing P. gingivalis and was resistant to hydrolysis by mannosidases. Synthesis of O-LPS and A-LPS and glycosylation of the gingipains appeared to be unaffected in all mutants. Thus, α- and β-mannosidases of P. gingivalis are not involved in the harnessing of mannan/mannose from the growth medium for these biosynthetic processes. P. gingivalis grown in chemically defined medium devoid of carbohydrate showed reduced α-mannosidase activity (25%), suggesting these enzymes are environmentally regulated.     

Coexpression of α-l-arabinofuranosidase and β-glucosidase in Saccharomyces cerevisiae

Monoterpenes are important aroma compounds in grape varieties such as Muscat, Gewürztraminer and Riesling, and are present as either odourless, glycosidically bound complexes or free aromatic monoterpenes. Commercial enzymes can be used to release the monoterpenes, but they commonly consist of crude extracts that often have unwanted and unpredictable side-effects on wine aroma. This project aims to address these problems by the expression and secretion of the Aspergillus awamoriα-l-arabinofuranosidase in combination with either the β-glucosidases from Saccharomycopsis fibuligera or from Aspergillus kawachii in the industrial yeast Saccharomyces cerevisiae VIN13. The concentration of five monoterpenes was monitored throughout alcoholic fermentation of Gewürztraminer grapes. The recombinant yeast strains that caused an early boost in the geraniol concentration led to a reduction in the final geraniol levels due to the downregulation of the sterol biosynthetic pathway. Monoterpene concentrations were also analysed 9 and 38 days after racking and the performance of the VB2 and VAB2 recombinant strains was similar, and in many cases, better than that of a commercial enzyme used in the same experiment. The results were backed by sensorial analysis, with the panel preferring the aroma of the wines produced by the VAB2 strain.     

Synthesis and reactions of α- and β-d-glucopyranosyluronic esters of amino acids

The synthesis of the fully benzylated α- and β-d-glucopyranosyluronic esters of 1-benzyl N-benzyloxycarbonyl-l-aspartic and -glutamic acids and N-(tert-butoxycarbonyl)-l-phenylalanine, followed by hydrogenolysis, afforded the respective anomers of the 1-O-acyl-d-glucopyranuronic acids 2, 7, and 12. Esterification of both anomers of the N-acetylated derivatives of 2 and 7 by diazomethane was accompanied by glycosyl-bond cleavage, and, in the case of the α anomers, with concomitant 1→2 acyl migration to give, after O-acetylation, the 2-O-acyl O-acetyl methyl ester derivatives 5 and 10, respectively. Similarly, 12α yielded methyl 1,3,4-tri-O-acetyl-2-O-[N-(tert-butoxycarbonyl)-l-phenylalanyl]-d-glucopyranuronate and an analogue having a furanurono-6,3-lactone structure. Esterification of the C-5 carboxyl group, in 1-O-acyl-α-d-glucopyranuronic acids by methanol in the presence of the BF_3^?-MeOH reagent (1–1.5 equiv.) proceeded without acyl migration. By using this procedure, followed by acetylation, the N-acetylated derivative of 7α afforded methyl 2,3,4-tri-O-acetyl-1-O-(1-methyl N-acetyl-l-glutam-5-oyl)-α-d-glucopyranuronate, and 12α gave methyl 2,3,4-tri-O-acetyl-1-O-(N-acetyl-l-phenylalanyl)-α-d-glucopyranuronate; the formation of the latter involved cleavage of the tert-butoxycarbonyl group by BF₃, followed by N-acetylation in the next step.     

Investigation of Intramolecular Dynamics and Conformations of α-, β- and γ-Synuclein

The synucleins are a family of natively unstructured proteins consisting of α-, β-, and γ-synuclein which are primarily expressed in neurons. They have been linked to a wide variety of pathologies, including neurological disorders, such as Parkinson’s disease (α-synuclein) and dementia with Lewy bodies (α- and β-synuclein), as well as various types of cancers (γ-synuclein). Self-association is a key pathological feature of many of these disorders, with α-synuclein having the highest propensity to form aggregates, while β-synuclein is the least prone. Here, we used a combination of fluorescence correlation spectroscopy and single molecule Förster resonance energy transfer to compare the intrinsic dynamics of different regions of all three synuclein proteins to investigate any correlation with putative functional or dysfunctional interactions. Despite a relatively high degree of sequence homology, we find that individual regions sample a broad range of diffusion coefficients, differing by almost a factor of four. At low pH, a condition that accelerates aggregation of α-synuclein, on average smaller diffusion coefficients are measured, supporting a hypothesis that slower intrachain dynamics may be correlated with self-association. Moreover, there is a surprising inverse correlation between dynamics and bulkiness of the segments. Aside from this observation, we could not discern any clear relationship between the physico-chemical properties of the constructs and their intrinsic dynamics. This work suggests that while protein dynamics may play a role in modulating self-association or interactions with other binding partners, other factors, particularly the local cellular environment, may be more important.     

Enzymatic synthesis of cyclohexyl-α and β-d-glucosides catalyzed by α- and β-glucosidase in a biphase system

Two secondary alcohol glucosides, cyclohexyl-α-d-glucoside and cyclohexyl-β-d-glucoside, were synthesized via the condensation reaction of cyclohexanol with d-glucose in a biphase system catalyzed by α-glucosidase and β-glucosidase, respectively. The effects of pH, water content, glucose concentration and metal ions on the yield of glucosides were studied. The optimum catalytic conditions established for α-glucosidase was 25% (v/v) water content, 2.5 mol/L glucose concentration and pH 2.0, and for β-glucosidase was 30% (v/v) water content, 2.0 mol/L glucose and pH 5.0. The maximum yield of glucoside was 13.3 mg/mL for cyclohexyl-α-d-glucoside and 8.9 mg/mL for cyclohexyl-β-d-glucoside. Synthesis progress was monitored by TLC and quantitatively analyzed by pre-derived capillary gas chromatography (GC). The retention time was 12.34 min for the α isomer and 12.96 min for the β isomer, respectively. With an anomeric purity of more than 99.5%, the two glucosides display excellent site-specific catalysis by α- and β-glucosidase. Herein, we present a general method to produce anomerically pure glucosides via a one-step bio-reaction in a biphase system. This method could potentially be applied in glucosylation of primary and secondary alcohols or other reactions requiring glucosylation.