Characterization of Three ��-Galactoside Phosphorylases from Clostridium phytofermentans: DISCOVERY OF d-GALACTOSYL-��1��4-l-RHAMNOSE PHOSPHORYLASE*

We characterized three d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphy1920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; d-Gal-β1→3-d-GalNAc) and lacto-N-biose I (LNB; d-Gal-β1→3-d-GlcNAc), thus indicating that they are d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylases, subclassified as GNB/LNB phosphorylase. In contrast, Cphy1920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with l-rhamnose as the acceptor in the reverse reaction using α-d-galactose 1-phosphate as the donor. The reaction product was d-galactosyl-β1→4-l-rhamnose. The enzyme also showed activity on l-mannose, l-lyxose, d-glucose, 2-deoxy-d-glucose, and d-galactose in this order. When d-glucose derivatives were used as acceptors, reaction products were β-1,3-galactosides. Kinetic parameters of phosphorolytic activity on d-galactosyl-β1→4-l-rhamnose were k_cat = 45 s⁻¹ and K_m = 7.9 mm, thus indicating that these values are common among other phosphorylases. We propose d-galactosyl-β1→4-l-rhamnose phosphorylase as the name for Cphy1920 protein.Phosphorylases are a group of enzymes involved in formation and cleavage of glycoside linkage together with glycoside hydrolases and glycosyl-nucleotide glycosyltransferases (synthases). Phosphorylases, which reversibly phosphorolyze oligosaccharides to produce monosaccharide 1-phosphate, are generally intracellular enzymes showing strict substrate specificity. Physiologically, such strict substrate specificity is considered to be closely related to the environment containing bacteria possessing them. For example, d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylase (GalHexNAcP²; EC 2.4.1.211) from Bifidobacterium longum, an intestinal bacterium, forms part of the pathway metabolizing galacto-N-biose (GNB; d-Gal-β1→3-d-GalNAc) from mucin and lacto-N-biose I (LNB; d-Gal-β1→3-d-GlcNAc) from human milk oligosaccharides, both of which are present in the intestinal environment, with GNB- and LNB-releasing enzymes and GNB/LNB transporter (1–8). Another example is cellobiose phosphorylase from Cellvibrio gilvus, which is a cellulolytic bacterium. Cellobiose phosphorylase forms an important cellulose metabolic pathway with an extracellular cellulase system producing cellobiose (9, 10).The reversible catalytic reaction of phosphorylases is one of the most remarkable features that make them suitable catalysts for practical syntheses of oligosaccharides. An oligosaccharide can be produced from inexpensive material by combining reactions of two phosphorylases, one for phosphorolyzing the material and the other for synthesizing the oligosaccharide, in one pot. Based on this idea, LNB is synthesized on a large (kg) scale using sucrose phosphorylase and GalHexNAcP (11). Practical synthesis methods of trehalose and cellobiose have also been developed (12, 13). However, only 14 kinds of substrate specificities have been reported among phosphorylases (13), thus restricting their use. Therefore, it would be useful to find a phosphorylase with novel activity.GalHexNAcP phosphorolyzes GNB and LNB to produce α-d-galactose 1-phosphate (Gal 1-P) and the corresponding N-acetyl-d-hexosamine. To date, GalHexNAcP is the only phosphorylase known to act on β-galactoside. This enzyme was first found in the cell-free extract of Bifidobacterium bifidum (14) and then in B. longum (1, 15), Clostridium perfringens (16), Propionibacterium acnes (17), and Vibrio vulnificus (18). These studies revealed that GalHexNAcPs were classified into three subgroups based on substrate preference between GNB and LNB. These subgroups are as follows: 1) galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP), showing similar activity on both GNB and LNB (B. longum and B. bifidum); 2) galacto-N-biose phosphorylase (GNBP), preferring GNB to LNB (C. perfringens and P. acnes); and 3) lacto-N-biose I phosphorylase (LNBP), preferring LNB to GNB (V. vulnificus) (18). The ternary structure of GLNBP from B. longum (GLNBP_Bl) has been revealed recently (19). Based on the similarity in ternary structures between GLNBP_Bl and β-galactosidase from Thermus thermophilus, which belongs to glycoside hydrolase family 42 (19, 20), GalHexNAcP homologs are classified as GH112 (glycoside hydrolase family 112), although phosphorylases are glycosyltransferases (21, 22).Clostridium phytofermentans is an anaerobic cellulolytic bacterium. It is found in soil and grows optimally at 37 °C (23). Its whole genome sequence has been revealed (GenBank^TM accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000885","term_id":"160426828","term_text":"CP000885"}}CP000885). The bacterium possesses three GalHexNAcP homologous genes (cphy0577, cphy1920, and cphy3030 genes; GenBank^TM accession numbers are {"type":"entrez-protein","attrs":{"text":"ABX40964.1","term_id":"160427401","term_text":"ABX40964.1"}}ABX40964.1, {"type":"entrez-protein","attrs":{"text":"ABX42289.1","term_id":"160428726","term_text":"ABX42289.1"}}ABX42289.1, and {"type":"entrez-protein","attrs":{"text":"ABX43387.1","term_id":"160429824","term_text":"ABX43387.1"}}ABX43387.1, respectively). C. phytofermentans has the ability to utilize a wide range of plant polysaccharides (23), and substrate specificities of these three gene products (Cphy0577, Cphy1920, and Cphy3030 proteins) are considered to be responsible for this ability. Furthermore, the three proteins have not been clearly categorized as GLNBP, GNBP, or LNBP, based on the phylogenetic tree shown in Fig. 1.Open in a separate windowFIGURE 1.Phylogenetic tree of GalHexNAcP homologs in GH112. Multiple alignment was performed using ClustalW2 (available on the World Wide Web). A phylogenetic tree was constructed using Treeview version 1.6.6. The proteins characterized in this study are represented with boldface letters in boxes with a heavy outline. The other proteins are numbered serially in boxes. Characterized GLNBP, GNBP, and LNBP are represented with boldface black letters on a gray background, boldface white letters on a gray background, and boldface white letters on a black background, respectively. Organisms and GenBank^TM accession numbers of numbered proteins are as follows: 1, CPF0553 (C. perfringens ATCC13124, {"type":"entrez-protein","attrs":{"text":"ABG83511.1","term_id":"110674524","term_text":"ABG83511.1"}}ABG83511.1) (16); 2, CPE0573 (C. perfringens str.13, {"type":"entrez-protein","attrs":{"text":"BAB80279.1","term_id":"18144232","term_text":"BAB80279.1"}}BAB80279.1); 3, CPR0537 (C. perfringens SM101, {"type":"entrez-protein","attrs":{"text":"ABG86710.1","term_id":"110683340","term_text":"ABG86710.1"}}ABG86710.1); 4, LnpA2 (B. bifidum JCM1254, {"type":"entrez-protein","attrs":{"text":"BAE95374.1","term_id":"107597820","term_text":"BAE95374.1"}}BAE95374.1) (14, 15); 5, LnpA1 (B. bifidum JCM1254, {"type":"entrez-protein","attrs":{"text":"BAD80752.1","term_id":"56676017","term_text":"BAD80752.1"}}BAD80752.1) (14, 15); 6, GLNBP_Bl (B. longum subsp. longum JCM 1217, {"type":"entrez-protein","attrs":{"text":"BAD80751.1","term_id":"56676015","term_text":"BAD80751.1"}}BAD80751.1) (1, 16); 7, Blon_2174 (B. longum subsp. infantis ATCC 15697, {"type":"entrez-protein","attrs":{"text":"ACJ53235.1","term_id":"213524488","term_text":"ACJ53235.1"}}ACJ53235.1); 8, BL1641 (B. longum NCC2705, {"type":"entrez-protein","attrs":{"text":"AAN25428.1","term_id":"23326930","term_text":"AAN25428.1"}}AAN25428.1); 9, BLD_1765 (B. longum DJO10A, {"type":"entrez-protein","attrs":{"text":"ACD99210.1","term_id":"189429062","term_text":"ACD99210.1"}}ACD99210.1); 10, GnpA (P. acnes JCM6473, {"type":"entrez-nucleotide","attrs":{"text":"AB468065","term_id":"219807264","term_text":"AB468065"}}AB468065) (17); 11, GnpA (P. acnes JCM6425, {"type":"entrez-nucleotide","attrs":{"text":"AB468066","term_id":"219807262","term_text":"AB468066"}}AB468066) (17); 12, PPA0083 (P. acnes KPA171202, {"type":"entrez-protein","attrs":{"text":"AAT81843.1","term_id":"50839176","term_text":"AAT81843.1"}}AAT81843.1); 13, VV2_1091 (V. vulnificus CMCP6, {"type":"entrez-protein","attrs":{"text":"AAO07997.1","term_id":"27359049","term_text":"AAO07997.1"}}AAO07997.1) (18); 14, VVA1614 (V. vulnificus YJ016, {"type":"entrez-protein","attrs":{"text":"BAC97640.1","term_id":"37201820","term_text":"BAC97640.1"}}BAC97640.1); 15, Oter_1377 (Opitutus terrae PB90-1, {"type":"entrez-protein","attrs":{"text":"ACB74662.1","term_id":"177840410","term_text":"ACB74662.1"}}ACB74662.1); 16, BCQ_1989 (B. cereus Q1, {"type":"entrez-protein","attrs":{"text":"ACM12417.1","term_id":"221239707","term_text":"ACM12417.1"}}ACM12417.1); 17, BCAH187_A2105 (Bacillus cereus AH187, {"type":"entrez-protein","attrs":{"text":"ACJ78918.1","term_id":"217064668","term_text":"ACJ78918.1"}}ACJ78918.1).In this study, we characterized the three proteins. We reported that two of them were GalHexNAcPs and that the other was a β-galactoside phosphorylase showing unique substrate specificity.     

Autophagosomes,phagosomes, autolysosomes,phagolysosomes, autophagolysosomes… Wait,I’m confused

When an autophagosome or an amphisome fuse with a lysosome, the resulting compartment is referred to as an autolysosome. Some people writing papers on the topic of autophagy use the terms “autolysosome” and “autophagolysosome” interchangeably. We contend that these words should be used to denote 2 different compartments, and that it is worthwhile maintaining this distinction—the autophagolysosome has a particular origin in the process of xenophagy that makes it distinct from an autolysosome.     

Autophagosomes,phagosomes, autolysosomes,phagolysosomes, autophagolysosomes… Wait,I’m confused

When an autophagosome or an amphisome fuse with a lysosome, the resulting compartment is referred to as an autolysosome. Some people writing papers on the topic of autophagy use the terms “autolysosome” and “autophagolysosome” interchangeably. We contend that these words should be used to denote 2 different compartments, and that it is worthwhile maintaining this distinction—the autophagolysosome has a particular origin in the process of xenophagy that makes it distinct from an autolysosome.     

Der Einfluß endogener Faktoren auf den Asche-, Kalzium-, Magnesium-, Kalium-, Natrium-, Phosphor-, Zink-, Eisen-, Kupfer- und Mangangehalt der Schweineborste

The objective of the present study was to investigate physiological effects of a marginal copper and iron supply on pigs. Therefore an experiment was conducted with 4 × 12 growing pigs of the crossbreed Pietrain × Deutsche Landrasse. The animals were fed for a period of 119 days with a diet poor of copper (1.5 mg Cu/kg diet) and/or poor of iron (35 mg Fe/kg diet). Control animals were supplied adequately with copper (4.8 mg Cu/kg diet) and iron (85 mg Fe/kg diet). The diet was given according to weight. After reaching an average weight of 102.6 ± 3.5 kg the animals were slaughtered. Due to the activity of the coerulplasmin and katalase enzyme and the haematological parameters, the supply of copper and iron could be classified as marginal. There was no interaction between copper deficiency and iron metabolism. The protein metabolism was unchanged. Low copper intake reduced the copper concentrations in serum, liver, muscle and backfat, and low iron intake reduced the iron concentration in serum, liver and muscle. Marginal copper and iron supply had no relevant effect on either food intake and growth performance or carcass characteristics and meat quality.     

Der Mengen- und Spurenelementgehalt des Rinderhaares als Indikator der Calcium-, Magnesium-, Phosphor-, Kalium-, Natrium-, Eisen-, Zink-, Mangan-, Kupfer-, Molybdän- und Kobaltversorgung

Beyond the energy requirement of maintenance, the assimilated energy, occurring in bioproducts, is linearly proportional to the intake of metabolizable energy in non‐underfed conditions. In contrast, resting metabolic rate is differing between individuals within a population of an animal species. As adaptability to changed environmental conditions may play a role, young bulls were exposed to thermoneutral (18°C) and low (4°C) ambient temperatures and were fed at two feeding levels (1.0 and 1.6 times energy requirement in maintenance) to produce metabolic rate differences, using the same animals, metabolic rate was altered by reducing the sympathetic outflow in each case. Expression of sulfonylurea receptors in circulating mononuclear leukocytes and cells from skeletal muscle (m. semitendinosus) was studied by flow cytom‐etry. Changes of metabolic rate at rest corresponded to the portion of cells with sulfonylurea receptors expression. The data from reducing the sympathetic outflow and those from sulfonylurea receptors expression are useful to explain metabolic rate differences among individuals of an animal population.     

Der Mengen- und Spurenelementgehalt des Rinderhaares als Indikator der Calcium-, Magnesium-, Phosphor-, Kalium-, Natrium-, Eisen-, Zink-, Mangan-, Kupfer-, Molybdän- und Kobaltversorgung

Many health effects can be attributed to the Mediterranean herb oregano (Origanum vulgare L.) and several studies demonstrated the improving effect on performance, changes in blood count, antibacterial, antifungal and immunmodulating abilities. The majority of these investigations were carried out with processed essential oil, while whole plant material was only used in a few studies. Thus, the aim of the present experiment was to test the effect of increasing proportions of dried oregano in piglet feed on health and performance, with a special focus on immune modulation. A total of 80 male castrated weaned piglets (body weight [BW] 7.9 kg ±1.0 kg) were used in a feeding experiment lasting 5 weeks. They were assigned to 4 experimental groups: a control diet, and three diets with an oregano supplementation at 2 g, 4 g and 8 g per kg feed, respectively, corresponding to 23.5 mg, 46.9 mg and 93.9 mg carvacrol/kg DM. After 3 weeks, half of each group was challenged with 5 µg lipopolysaccharides (LPS) per kg BW. Blood samples were collected 2 h after LPS stimulation and analysed for T-cell phenotypes, granulocyte activity, clinical-chemistry as well as white and red blood count. The results indicate no effects of oregano on performance. In contrast, oregano altered the lymphocyte proportion and the ratio of CD4⁺ and CD8⁺ T-cells as well as the triglyceride concentration in the serum of non-stimulated and in LPS-stimulated piglets. In conclusion, whole plant supplementation of oregano to piglet feed altered immune-related parameters, but did not modulate the acute inflammatory response induced by LPS stimulation.     

The Seven Carbohydrate Bioengineering Meeting,Braunschweig, Germany,April, 22–25, 2007

Stereocontrol in bakers' yeast reduction can be achieved by introduction of a sulfur functional group into substrates. α-Methylthio-β-keto esters are reduced to give exclusively (3S)-3-hydroxy esters. α-Substituted β-keto thiol esters and dithioesters afford (2R,3S)-3-hydroxy esters with high diastereo-and enantioselectivity. Ketones possessing 1,3-dithiane, phenylsulfenyl, or phenylsulfonyl groups at the α-position are transformed also into the corresponding (S)-secondary alcohols. Optically pure (S)-(phenylsulfinyl)acetones can be obtained by kinetic resolution of racemic derivatives with the yeast. Diastereo- and enantioselective reduction of 1,2-diketones leading into (1S,2S)-1,2-diol derivatives can be also achieved by introduction of 1,3-dithiane, phenylsulfenyl or phenylsulfonyl groups into the α-position. Reductions of carbon-carbon double bond of sulfur-functionalized prenyl derivatives provide both chiral (R)- and (S)-C₅-building blocks for terpenoid synthesis. The utility of the reduction products as chiral building blocks is demonstrated in the synthesis of biologically active natural products such as pheromones, sugars, antibiotics etc. by functional group transformation and carbon-carbon bond formation reactions with the aid of sulfur functional groups.     

人口,食物,资源,环境与生物学

人口、食物、资源、环境与生物学钱燕文（中国科学院动物研究所北京１０００８０）人家都说下一个世纪是生物学的世纪,人口、食物、资源、环境也是近２０年来的热门话题,因为这四个方面关系到我们人类的未来。为什么下个世纪是生物学的世纪？也得从人口、食物、资源、环...     

Barley—Botany,production, harvesting,processing, utilization and economics

Barley, probably the oldest cultivated cereal, is widely grown in cooler areas of the world. The annual world production of nearly two and a half billion bushels exceeds that of rye but is less than that of rice, wheat, corn and oats, respectively. Most of the annual 300 million dollar crop of the U.S. is fed to livestock, but about one- third is manufactured into malt.     

胸怀祖国, 忘我工作, 为国争光

刘以训教授是我国著名的生物学家,中国科学院院士。他致力于生殖生物学研究,在排卵、黄体萎缩、精子发生和胚胎植入等系列研究上,成绩卓著,享有国际声誉。读罢此文,钦敬之情油然而生。     

An investigation into the structural,electronic, and non-linear optical properties in CN (N = 20, 24, 26, 28, 30, 32, 34, 36, and 38) fullerene cages

Journal of Molecular Modeling -     

相思子根化学成分研究——相思子醌A,B,D,E,F,G

从民间药用抗肝炎药相思子（ＡｂｒｕｓｐｒｅｃａｔｏｒｉｕｓＬ．）根中分得８个异黄烷醌类化合物,即相思子醌Ａ、Ｂ、Ｄ、Ｅ、Ｆ、Ｇ以及已知化合物３,７二羟基６甲氧基双氢黄酮和２,８二羟基３,４,９,１０四甲氧基紫檀素。用化学转化和光谱学方法包括１Ｈ１ＨＣＯＳＹ、１Ｈ１３ＣＣＯＳＹ、ＣＤ等方法鉴定它们的结构。