Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K _m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K _m value was 6.80 mM. In the presence of 3 M KHCO₃ and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K _m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.     

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.     

Secreted Nucleobindin-2 Inhibits 3T3-L1 Adipocyte Differentiation

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.     

Structural Characterization of Arabidopsis Leaf Arabinogalactan Polysaccharides

Proteins decorated with arabinogalactan (AG) have important roles in cell wall structure and plant development, yet the structure and biosynthesis of this polysaccharide are poorly understood. To facilitate the analysis of biosynthetic mutants, water-extractable arabinogalactan proteins (AGPs) were isolated from the leaves of Arabidopsis (Arabidopsis thaliana) plants and the structure of the AG carbohydrate component was studied. Enzymes able to hydrolyze specifically AG were utilized to release AG oligosaccharides. The released oligosaccharides were characterized by high-energy matrix-assisted laser desorption ionization-collision-induced dissociation mass spectrometry and polysaccharide analysis by carbohydrate gel electrophoresis. The Arabidopsis AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-d-galactan side chains. The β-(1→6)-galactan side chains vary in length from one to over 20 galactosyl residues, and they are partly substituted with single α-(1→3)-l-arabinofuranosyl residues. Additionally, a substantial proportion of the β-(1→6)-galactan side chain oligosaccharides are substituted at the nonreducing termini with single 4-O-methyl-glucuronosyl residues via β-(1→6)-linkages. The β-(1→6)-galactan side chains are occasionally substituted with α-l-fucosyl. In the fucose-deficient murus1 mutant, AGPs lack these fucose modifications. This work demonstrates that Arabidopsis mutants in AGP structure can be identified and characterized. The detailed structural elucidation of the AG polysaccharides from the leaves of Arabidopsis is essential for insights into the structure-function relationships of these molecules and will assist studies on their biosynthesis.Arabinogalactans (AGs) are structurally complex large-branched polysaccharides attached to Hyp residues of many plant cell wall polypeptides. Most proteins glycosylated with AGs (AGPs) have both AG glycosylated domains (glycomodules) and structural or enzymatic domains. However, typical AGPs commonly contain less than 10% protein, suggesting that the AG is the functional part of the molecule (Clarke et al., 1979; Fincher et al., 1983; Kieliszewski and Lamport, 1994; Borner et al., 2003; Xu et al., 2008). Hyp is the most characteristic amino acid present at the glycosylated domain of the AGP, but other amino acids such as Ser, Ala, and Thr are also very common. Type II AG polysaccharides share common structural features based on a β-(1→3)-galactan backbone with β-(1→6)-linked galactan side chains and can be found both on AGPs and rhamnogalacturonan-I (RG-I) pectin (Renard et al., 1991). The galactopyranosyl (Galp) residues can be further substituted with l-arabinofuranosyl (l-Araf) and occasionally also l-rhamnosyl (l-Rha), l-fucosyl (l-Fuc), and glucuronosyl (GlcA; with or without 4-O-methylation) residues (Tsumuraya et al., 1988; Tan et al., 2004; Tryfona et al., 2010). (Sugars mentioned in this work belong to the D-series unless otherwise stated.)The structure of AGs is poorly characterized, and this is mainly due to the great heterogeneity of glycan structures, not only between different AGPs but also even on the same peptide sequence in the same tissue (Estévez et al., 2006). The glycan structure can also be different depending on the developmental stage and tissue type (Tsumuraya et al., 1988), adding to the great heterogeneity of these molecules and therefore limiting their detailed characterization. Molecular and biochemical evidence has indicated that AGPs have specific functions during root formation, promotion of somatic embryogenesis (van Hengel et al., 2002), and attraction of pollen tubes to the style (Cheung et al., 1995). In addition, enhanced secretion efficiency or stability in the cell wall are properties that the AG may confer on the glycosylated protein (Borner et al., 2003). However, it has been difficult to differentiate one species of AGP from another in plant tissues and to assign specific roles to individual AGPs.l-Fuc is present in AGPs in Arabidopsis (Arabidopsis thaliana; van Hengel et al., 2002), radish (Raphanus sativus; Nakamura et al., 1984; Tsumuraya et al., 1984a, 1984b, 1988), and several other dicot plants such as thyme (Thymus vulgaris; Chun et al., 2001) and celery (Apium graveolens; Lin et al., 2011). Reduction in l-Fuc by 40% in roots of murus1 (mur1) plants resulted in a decrease of 50% in root cell elongation, and eel lectin binding assays suggested that the phenotype was the result of alterations in the composition of root AGPs (van Hengel and Roberts, 2002). An α-(1→2)-fucosyltransferase (FUT) activity for radish primary root AGPs has been described, where an α-l-Araf-(1→3)-β-Galp-(1→6)-Galp trisaccharide was used as exogenous substrate acceptor to mimic an AG polysaccharide in the enzymatic assay (Misawa et al., 1996). Linkage analysis, reactivity with eel lectin, and digestion with α-(1→2)-fucosidase indicated that the l-Fuc residues added are terminal and attached via an α-linkage to the C-2 position of an adjacent l-Araf residue (Nakamura et al., 1984; Tsumuraya et al., 1984a, 1984b, 1988). Recently, Wu et al. (2010) identified AtFUT4 and AtFUT6 genes encoding FUT proteins specific to AGPs, but the structures of the fucosylated AG generated have not been fully characterized.To gain insights into the synthesis and function of plant AGPs, it would be useful to have mutants altered in their carbohydrate moieties. However, no AG-specific biosynthetic mutants have been characterized, and this, among other reasons, is due to the very limited knowledge of the structure of Arabidopsis AGs (Qu et al., 2008). Moreover, characterization of AG in candidate mutants remains challenging. Even though the structures of some AGs have been proposed using NMR and sugar linkage analyses, the complete structural elucidation of a native AG still remains a formidable task, because NMR spectroscopy and methylation analysis have been largely used to provide information regarding the amount and type of linkages between adjacent glycosyl residues, and AG heterogeneity can confound attempts to build complete structural models. Recently, a modular structure was proposed for AGs on heterologously expressed proteins in tobacco (Nicotiana tabacum; Tan et al., 2010). Tan et al. (2010) proposed that approximately 15-residue repeating blocks of decorated β-(1→3)-trigalactosyl subunits connected by β-(1→6)-linkages were the building blocks of type II AG polysaccharides and concluded that these molecules are far less complex than commonly supposed. Most characterized β-(1→6)-galactan side chains in AGs are reported to be short, of one or two residues (Neukom and Markwalder, 1975; Gane et al., 1995; Gaspar et al., 2001). On the contrary, there are reports of long β-(1→6)-galactan side chains in radish root AGPs (Haque et al., 2005). Similarly, we recently found evidence that wheat (Triticum aestivum) flour endosperm AGP extracts contained long β-(1→6)-galactan side chains heavily substituted with l-Araf at C-3 (Tryfona et al., 2010). This partial structure of the carbohydrate component of wheat flour AGP isolated from water extracts of wheat endosperm was elucidated utilizing a combination of analytical approaches, such as the use of enzymes able to release oligosaccharides specifically from AGs, high-energy matrix-assisted laser desorption ionization (MALDI)-collision-induced dissociation (CID) mass spectrometry (MS), and polysaccharide analysis by carbohydrate gel electrophoresis (PACE; Tryfona et al., 2010). In this work, we applied these techniques to study the carbohydrate component of Arabidopsis leaf AGPs. AG-specific enzyme digestion products were analyzed by PACE and MS, allowing a partial structure to be proposed. We show that endogenous Arabidopsis leaf AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-galactan side chains. These side chains are substituted with l-Araf residues via α-(1→3)-linkages and can vary in length from one up to at least 20 Galp residues. We also found that the β-(1→6)-galactan side chains are substituted mainly with 4-O-methyl-glucuronosyl (4-O-Me-GlcA) at their nonreducing termini, while occasional l-Fuc substitutions were also present via α-(1→2)-linkages on l-Araf residues. In addition, AG oligosaccharides from leaves of the mur1 mutant were identified, and their structures were compared with those isolated from wild-type plants.     

Osteocyte network; a negative regulatory system for bone mass augmented by the induction of Rankl in osteoblasts and Sost in osteocytes at unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.     

Effect of mycelial morphology on ergothioneine production during liquid fermentation of Lentinula edodes

The morphological type of a microorganism generally influences its metabolite production. In the present study, we investigated the effects of the mycelial morphology of shiitake (Lentinula edodes) on the production of 2-mercaptohistidine trimethylbetaine (ergothioneine, ESH) during liquid fermentation. Analyses of the distribution of ESH in mycelial cells of different morphological types revealed that the ESH content of pellets obtained from the liquid fermentation media was much greater than the content in the free mycelia and clumps. The concentration of ESH in pellets on day 15 of liquid fermentation reached 0.79 mg/g dry weight (DW), which is approximately three times the concentration found in mycelia clumps (0.28 mg/g DW) and free mycelia (0.31 mg/g DW). Macroscopic image analysis of the development and morphological changes of the pellets during a liquid fermentation period of up to 25 days indicated that pellet growth showed a highly positive correlation with the increase in ESH concentration (r ² = 0.9851). A reduced agitation rate of 50 rpm for the culture medium was suitable for pellet formation and size enlargement. The results obtained in this work would be helpful in guiding the intentional manipulation of the distribution and enrichment of ESH in L. edodes through changes in liquid fermentation conditions.