The plant cell wall is a complex and dynamic network made mostly of cellulose, hemicelluloses, and pectins. Xyloglucan, the major hemicellulosic component in Arabidopsis thaliana, is biosynthesized in the Golgi apparatus by a series of glycan synthases and glycosyltransferases before export to the wall. A better understanding of the xyloglucan biosynthetic machinery will give clues toward engineering plants with improved wall properties or designing novel xyloglucan-based biomaterials. The xyloglucan-specific α2-fucosyltransferase FUT1 catalyzes the transfer of fucose from GDP-fucose to terminal galactosyl residues on xyloglucan side chains. Here, we present crystal structures of Arabidopsis FUT1 in its apoform and in a ternary complex with GDP and a xylo-oligosaccharide acceptor (named XLLG). Although FUT1 is clearly a member of the large GT-B fold family, like other fucosyltransferases of known structures, it contains a variant of the GT-B fold. In particular, it includes an extra C-terminal region that is part of the acceptor binding site. Our crystal structures support previous findings that FUT1 behaves as a functional dimer. Mutational studies and structure comparison with other fucosyltransferases suggest that FUT1 uses a S2-like reaction mechanism similar to that of protein-O-fucosyltransferase 2. Thus, our results provide new insights into the mechanism of xyloglucan fucosylation in the Golgi.

). The structures of cdMGD1 and cdMGD1:UDP have been deposited in the Protein Data Bank under accession numbers 4WYI and 4X1T, respectively. SUMMARYMonogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP-galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.

A unique feature of chloroplasts is their high content of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which constitute up to 80% of their lipids. These galactolipids are synthesized in the chloroplast envelope membrane through the concerted action of galactosyltransferases, the so-called ‘MGDG synthases (MGDs)’ and ‘DGDG synthases (DGDs),’ which use uridine diphosphate (UDP)-galactose as donor. In Arabidopsis leaves, under standard conditions, the enzymes MGD1 and DGD1 provide the bulk of galactolipids, necessary for the massive expansion of thylakoid membranes. Under phosphate limited conditions, plants activate another pathway involving MGD2/MGD3 and DGD2 to provide additional DGDG that is exported to extraplastidial membranes where they partly replace phospholipids, a phosphate-saving mechanism in plants. A third enzyme system, which relies on the UDP-Gal-independent GGGT (also called SFR2 for SENSITIVE TO FREEZING 2), can be activated in response to a freezing stress. The biosynthesis of galactolipids by these multiple enzyme sets must be tightly regulated to meet the cellular demand in response to changing environmental conditions. The cooperation between MGD and DGD enzymes with a possible substrate channeling from diacylglycerol to MGDG and DGDG is supported by biochemical and biophysical studies and mutant analyses reviewed herein. The fine-tuning of MGDG to DGDG ratio, which allows the reversible transition from the hexagonal II to lamellar α phase of the lipid bilayer, could be a key factor in thylakoid biogenesis.

Mono- and digalactosyldiacylglycerol (MGDG and DGDG) are the most abundant lipids of photosynthetic membranes (thylakoids). In Arabidopsis green tissues, MGD1 is the main enzyme synthesizing MGDG. This monotopic enzyme is embedded in the inner envelope membrane of chloroplasts. DGDG synthesis occurs in the outer envelope membrane. Although the suborganellar localization of MGD1 has been determined, it is still not known how the lipid/glycolipid composition influences its binding to the membrane. The existence of a topological relationship between MGD1 and "embryonic" thylakoids is also unknown. To investigate MGD1 membrane binding, we used a Langmuir membrane model allowing the tuning of both lipid composition and packing. Surprisingly, MGD1 presents a high affinity to MGDG, its product, which maintains the enzyme bound to the membrane. This positive feedback is consistent with the low level of diacylglycerol, the substrate of MGD1, in chloroplast membranes. By contrast, MGD1 is excluded from membranes highly enriched in, or made of, pure DGDG. DGDG therefore exerts a retrocontrol, which is effective on the overall synthesis of galactolipids. Previously identified activators, phosphatidic acid and phosphatidylglycerol, also play a role on MGD1 membrane binding via electrostatic interactions, compensating the exclusion triggered by DGDG. The opposite effects of MGDG and DGDG suggest a role of these lipids on the localization of MGD1 in specific domains. Consistently, MGDG induces the self-organization of MGD1 into elongated and reticulated nanostructures scaffolding the chloroplast membrane.-Sarkis, J., Rocha, J., Maniti, O., Jouhet, J., Vié, V., Block, M. A., Breton, C., Maréchal, E., Girard-Egrot, A. The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts.

-The objective of this work was to determine the apparent digestibility coefficient (ADC) of crude protein, crude energy, fat, and dry matter of fish protein hydrolysate (FPH), made of by-products of Nile tilapia (Oreochromis niloticus) and whole sardines (Cetengraulis edentulus), and to evaluate the productive performance and muscle fiber growth of Nile tilapia post-larvae. Two trials were conducted, the first one to determine the digestibility in 120 fingerlings (70.0±2.0 g), and the second one to evaluate the productive performance of 375 post-larvae, with three days of age, which were distributed in 25 aquaria with 30 L of useful volume. Five diets were prepared based on vegetable ingredients, to which fish were included at 0, 2, 4, 6, and 8% FPH. For the evaluation of muscle growth, eight fish of each experimental unit were used. The ADC values found were: 98.29% for dry matter; 99.28% for crude protein; and 99.13% for gross energy. The best zootechnical response for the productive performance resulted from the treatment with the inclusion of fish hydrolysate at 4.75%. The diets affected the frequency of the muscle fiber diameters, mainly the growth by hyperplasia. FPH can be efficiently used, and its inclusion at 4.75% is indicated in the diets for Nile tilapia in the post-larvae stage.Index terms: Cetengraulis edentulus, Oreochromis niloticus, digestibility, fish meal, nutrition, peptides, pisciculture. Hidrolisado proteico de peixe em dietas para pós-larvas de tilápia-do-niloResumo -O objetivo deste trabalho foi determinar o coeficiente de digestibilidade aparente (CDA) da proteína bruta, da energia bruta, da gordura e da matéria seca de um hidrolisado proteico de peixe (HPP), feito de resíduos de tilápia-do-nilo (Oreochromis niloticus) e sardinha inteira (Cetengraulis edentulus), e avaliar o desempenho produtivo e o crescimento das fibras musculares de pós-larvas de tilápia-do-nilo. Realizaram-se dois ensaios, o primeiro para a determinação da digestibilidade em 120 alevinos (70,0±2,0 g) e, o segundo, para avaliação do desempenho produtivo de 375 pós-larvas, com três dias de idade, distribuídas em 25 aquários (unidade experimental) com volume útil de 30 L. Cinco rações à base de ingredientes vegetais foram elaboradas, às quais se incluíram os peixes a 0, 2, 4, 6 e 8% de HPP. Para a avaliação do crescimento muscular, oito peixes de cada unidade experimental foram utilizados. Os valores de CDA encontrados foram: 98,29% para matéria seca; 99,28% para proteína bruta; e 99,13% para energia bruta. As melhores respostas zootécnicas quanto ao desempenho produtivo resultaram do tratamento com a inclusão do hidrolisado proteico a 4,75%. As dietas influenciaram a frequência do diâmetro das fibras musculares, principalmente o crescimento por hiperplasia. O HPP pode ser eficientemente utilizado, e sua inclusão a 4,75% é indicada em dietas de tilápia-do-nilo na fase de pós-larva.Termos para indexação: Cetengraulis edentulus, Oreochromis niloticus, digestibilidade, farinha de peixe, nutrição, peptídeos, pisci...

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Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate.

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