Metabolism of a Non-phenolic β-O-4 Lignin Substructure Model Compound by Coriolus versicolor

A non-phenolic β-O-4 lignin substructure model, 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (I), was metabolized by a ligninolytic culture of Coriolus versicolor. Based on the identification of the metabolic products (II~XI), the following reactions were found to occur in the culture; a) oxidation (III) and reduction (II) at the benzyl (Cα′) position of the substrate (I), b) β-ether cleavage to give arylglycerols (IV, V), and c) Cα-Cβ cleavage of the arylglycerols and/or arylglycerol moiety of the substrate (I). In addition, β-deoxy diol (VI) and γ-formylglycerol (VII) were obtained as degradation products from substrate (I).     

Interaction of Amino Acids with Transition Metal Ions in Solution (I) Solution Structure of l-Lysine with Co(II) and Cu(II) Ions as Studied by Nuclear Magnetic Resonance Spectroscopy

Interaction of l-lysine with Co(II) and Cu(II) ions has been studied using ¹H- and ¹³C-NMR and solution absorption spectrometry. In l-lysine-Co²⁺ solution in D₂O (100: 1 in concentration), coordination interaction of the α-amino and carboxyl groups with Co²⁺ occurs from the neutral to alkaline pD region, whereas no interaction of the ?-amino group was observed throughout the whole pD region. On the other hand, in l-lysine-Cu²⁺ solution, the ?-amino group also takes part in complexation in the higher pD region (pD≧10). Structural changes in complexation of l-lysine with the divalent cations along with pD variations in aqueous solution are discussed. Dissociation constants of the three functional groups were obtained by ¹H-NMR chemical shifts; pKa₁ = 2.2, pKa₂ = 9.5 and pKa₃ = 11.2.     

Piloty’s acid derivative with improved nitroxyl-releasing characteristics

Recent studies have shown that nitroxyl (HNO) (¹HNO/³NO^?), which is the one-electron-reduced form of nitric oxide (NO), has unique biological activities, especially in the cardiovascular system, and HNO-releasing agents may have therapeutic potential. Since few HNO donors are available for use under physiological conditions, we synthesized and evaluated a series of Piloty’s acid (PA) derivatives and evaluated their HNO-releasing activity under physiological conditions. N-Hydroxy-2-nitrobenzenesulfonamide (17) was the most efficient HNO donor among our synthesized PA derivatives, including the lead compound, 2-bromo-N-hydroxybenzenesulfonamide (2). The high HNO-releasing activity is suggested to be due to electronic and steric effects. Compound 17 may be a useful tool for biological experiments.     

Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer

ATP-binding cassette (ABC) transporters couple hydrolysis of ATP with vectorial transport across the cell membrane. We have reconstituted ABC transporter MsbA in nanodiscs of various sizes and lipid compositions to test whether ATPase activity is modulated by the properties of the bilayer. ATP hydrolysis rates, Michaelis-Menten parameters, and dissociation constants of substrate analog ATP-γ-S demonstrated that physicochemical properties of the bilayer modulated binding and ATPase activity. This is remarkable when considering that the catalytic unit is located ~50? from the transmembrane region. Our results validated the use of nanodiscs as an effective tool to reconstitute MsbA in an active catalytic state, and highlighted the close relationship between otherwise distant transmembrane and ATPase modules.     

Structural insights into the glycosyltransferase activity of the Actinobacillus pleuropneumoniae HMW1C-like protein

Glycosylation of proteins is a fundamental process that influences protein function. The Haemophilus influenzae HMW1 adhesin is an N-linked glycoprotein that mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. HMW1 is glycosylated by HMW1C, a novel glycosyltransferase in the GT41 family that creates N-glycosidic linkages with glucose and galactose at asparagine residues and di-glucose linkages at sites of glucose modification. Here we report the crystal structure of Actinobacillus pleuropneumoniae HMW1C (ApHMW1C), a functional homolog of HMW1C. The structure of ApHMW1C contains an N-terminal all α-domain (AAD) fold and a C-terminal GT-B fold with two Rossmann-like domains and lacks the tetratricopeptide repeat fold characteristic of the GT41 family. The GT-B fold harbors the binding site for UDP-hexose, and the interface of the AAD fold and the GT-B fold forms a unique groove with potential to accommodate the acceptor protein. Structure-based functional analyses demonstrated that the HMW1C protein shares the same structure as ApHMW1C and provided insights into the unique bi-functional activity of HMW1C and ApHMW1C, suggesting an explanation for the similarities and differences of the HMW1C-like proteins compared with other GT41 family members.     

The heparin-binding domain of IGFBP-2 has insulin-like growth factor binding-independent biologic activity in the growing skeleton

Insulin-like growth factor-binding protein 2 (IGFBP-2) is a member of a family of six highly conserved IGFBPs that are carriers for the insulin-like growth factors (IGFs). IGFBP-2 levels rise during rapid neonatal growth and at the time of peak bone acquisition. In contrast, Igfbp2(-/-) mice have low bone mass accompanied by reduced osteoblast numbers, low bone formation rates, and increased PTEN expression. In the current study, we postulated that IGFBP-2 increased bone mass partly through the activity of its heparin-binding domain (HBD). We synthesized a HBD peptide specific for IGFBP-2 and demonstrated in vitro that it rescued the mineralization phenotype of Igfbp2(-/-) bone marrow stromal cells and calvarial osteoblasts. Consistent with its cellular actions, the HBD peptide ex vivo stimulated metacarpal periosteal expansion. Furthermore, administration of HBD peptide to Igfbp2(-/-) mice increased osteoblast number, suppressed marrow adipogenesis, restored trabecular bone mass, and reduced bone resorption. Skeletal rescue in the Igfbp2(-/-) mice was characterized by reduced PTEN expression followed by enhanced Akt phosphorylation in response to IGF-I and increased β-catenin signaling through two mechanisms: 1) stimulation of its cytosolic accumulation and 2) increased phosphorylation of serine 552. We conclude that the HBD peptide of IGFBP-2 has anabolic activity by activating IGF-I/Akt and β-catenin signaling pathways. These data support a growing body of evidence that IGFBP-2 is not just a transport protein but rather that it functions coordinately with IGF-I to stimulate growth and skeletal acquisition.     

Isolation of rice dwarf mutants with ectopic deposition of phenolic components including lignin in parenchyma cell walls of internodes

Rice internodes must have the proper shape to support high-yielding panicles. The shape of internodes is controlled by various factors involved in their formation, such as developmental patterns, cell division, cell elongation, and cell wall biosynthesis. To understand the regulation of internode development, we screened dwarf mutants to identify those with a phenotype of ectopic deposits of phenolic components in parenchyma cell walls of internodes. We named these mutants ectopic deposition of phenolic components1 (edp1). Two alleles were identified, edp1-1 and edp1-2. Furthermore, these mutants showed disordered cell files in internode parenchyma. These abnormal phenotypes were very similar to that of a previously reported dwarf50 (d50) mutant. Genetic analyses of edp1 mutants revealed that the edp1 loci are distinct from d50. Our results indicate that analyses of edp1 mutants as well as the d50 mutant will be useful for understanding the molecular mechanisms behind ectopic deposition of cell wall phenolic components in internode parenchyma cells and the regulation of internode development.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

The present investigation is concerned with l-glutamic acid production in the presence of pyrrolidone carboxylic acid and glucose in Bacillus megaterium st. 6126. This strain does not grow on dl-pyrrolidone carboxylic acid (dl-PCA)¹⁾ as the sole source of carbon and nitrogen. The optimal concentration of yeast extract required for the maximal production of l-glutamic acid was 0.005% under the conditions used. As the yeast extract concentration was increased, growth increased proportionally; but the l-glutamic acid production did not exceed the control’s to which glucose and ammonium chloride had been added. l-Glutamic acid produced by both growing cultures and resting cells was derived from glucose and ammonium salt of dl-PCA. Isotope experiments suggested that the l-glutamic acid produced was partially derived from ammonium salt of dl-PCA in the growing culture which had been supplemented with d-glucose-U-¹⁴C or dl-PCA-1-¹⁴C and that ammonium salt of dl-PCA was consumed as the source of nitrogen and carbon for l-glutamic acid.