SummaryMembers of the BAHD family of plant acyl transferases are very versatile catalytically, and are thought to be able to evolve new substrate specificities rapidly. Acylation of anthocyanins occurs in many plant species and affects anthocyanin stability and light absorption in solution. The versatility of BAHD acyl transferases makes it difficult to identify genes encoding enzymes with defined substrate specificities on the basis of structural homology to genes of known catalytic function alone. Consequently, we have used a modification to standard functional genomics strategies, incorporating co-expression profiling with anthocyanin accumulation, to identify genes encoding three anthocyanin acyl transferases from Arabidopsis thaliana. We show that the activities of these enzymes influence the stability of anthocyanins at neutral pH, and some acylations also affect the anthocyanin absorption maxima. These properties make the BAHD acyl transferases suitable tools for engineering anthocyanins for an improved range of biotechnological applications.

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.

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Polyamines are small flexible organic polycations found in almost all cells. They likely existed in the last universal common ancestor of all extant life, and yet relatively little is understood about their biological function, especially in bacteria and archaea. Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may contain instead an alternative polyamine, sym-homospermidine. We demonstrate that homospermidine synthase (HSS) has evolved vertically, primarily in the ␣-Proteobacteria, but enzymatically active, diverse HSS orthologues have spread by horizontal gene transfer to other bacteria, bacteriophage, archaea, eukaryotes, and viruses. By expressing diverse HSS orthologues in Escherichia coli, we demonstrate in vivo the production of co-products diaminopropane and N 1 -aminobutylcadaverine, in addition to sym-homospermidine. We show that sym-homospermidine is required for normal growth of the ␣-proteobacterium Rhizobium leguminosarum. However, sym-homospermidine can be replaced, for growth restoration, by the structural analogues spermidine and sym-norspermidine, suggesting that the symmetrical or unsymmetrical form and carbon backbone length are not critical for polyamine function in growth. We found that the HSS enzyme evolved from the alternative spermidine biosynthetic pathway enzyme carboxyspermidine dehydrogenase. The structure of HSS is related to lysine metabolic enzymes, and HSS and carboxyspermidine dehydrogenase evolved from the aspartate family of pathways. Finally, we show that other bacterial phyla such as Cyanobacteria and some ␣-Proteobacteria synthesize sym-homospermidine by an HSS-independent pathway, very probably based on deoxyhypusine synthase orthologues, similar to the alternative homospermidine synthase found in some plants. Thus, bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine.Polyamines are primordial, small flexible organic polycations found in almost all cells of bacteria, archaea, and eukaryotes (1). In bacteria and archaea, the key polyamines (see Fig. 1A) are the triamines spermidine, sym-norspermidine, and sym-homospermidine (referred to herein as norspermidine and homospermidine), and occasionally more than one triamine can be found in the same cell. In eukaryotes, which contain spermidine (and in some plants, yeasts, and animals, the tetraamine spermine), polyamines are required for growth, cell proliferation, and normal cellular physiology. Polyamine biosynthesis is essential in the fungi Saccharomyces cerevisiae (2), Schizosaccharomyces pombe (3), Aspergillus nidulans (4), and Ustilago maydis (5), the kinetoplastid parasites Trypanosoma brucei (6) and Leishmania donovani (7), and the diplomonad parasite Giardia lamblia (8). In mouse, polyamines are essential for early embryo development (9, 10), and they are also essential for seed development in the flowering plant Arabidopsis thaliana (11).The universal distribution of polyamines suggests that...

dRuminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an ␣-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14 -21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for ␣-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the ␣-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with ␣-(1,6) regioselectivity, leading to the formation of product structures up to [Hex] 12 and [Hex] 8 , respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.T he human gut is colonized by a complex, diverse, and dynamic community of microbes that continuously interact with the host (30). The majority belongs to only four bacterial divisions, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, whereas other minor taxonomic divisions are quite diverse (19,43,62). Several ecological studies have shown that microbial symbionts have adapted to maximize metabolic access to a wide variety of dietary and host-derived carbohydrates (glycans), and competition for these nutrients is considered a major factor shaping the structure-function of the microbiota (33). Recently, a metagenomic analysis of gut microbial communities in humans proposed three predominant variants, or "enterotypes," dominated by Bacteroides, Prevotella, and Ruminococcus (3). A controlledfeeding study showed that enterotype partitioning associates with long-term diets (61). Furthermore, the ability to selectively use prebiotics carbohydrates, ranging from oligosaccharides to polysaccharides, provides a competitive advantage over other bacteria in this ecosystem (28). These studies highlight the importance of understanding precisely how nutrient metabolism serves to maintain a symbiotic relationship between gut bacteria and the host. The genomes of gut bacteria encode a wide array of carbohydrateactive enzymes (CAZymes) that d...

Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.—Gunning, A. P., Kirby, A. R., Fuell, C., Pin, C., Tailford L. E., Juge, N. Mining the “glycocode”—exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy.

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Intestinal γδ T-cell receptor-bearing intraepithelial lymphocytes (γδ IELs) play a multifaceted role in maintaining mucosal homeostasis. In order to investigate the relationship between O-glycosylation and inflammation, we carried out an in-depth mass spectrometric comparison of the intestinal O-glycosylation profile of mice lacking γδ IELs (TCRδ(-/-)) and of their wild-type (WT) littermates. A total of 69 nonsulfated and 59 sulfated compositional types of O-glycans were identified in the small intestine and colon of TCRδ(-/-) and WT mice. Our results demonstrated structural differences in intestinal glycosylation in TCRδ(-/-) mice compared with WT littermates. TCRδ(-/-) colons contained a lower proportion of core-2 structures and an increased proportion of core-1 structures whereas TCRδ(-/-) small intestines had a decreased percentage of core-3 structures. The glycan antennae in TCRδ(-/-) colon and small intestine showed altered structural diversity compared with WT mice. There were significant differences in the sialylated species between the TCRδ(-/-) and WT mice with the sialylated Tn antigen found exclusively in the TCRδ(-/-)small intestine, whereas the sulfation pattern remained mostly unchanged. These findings provide novel molecular insights underpinning the role of γδ IELs in maintaining gut homeostasis.