The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical S-adenosyl-L-methionine (SAM) superfamily was previously predicted based on bioinformatic analysis. A number of these are Cbl-dependent methyltransferases but the details surrounding their reaction mechanisms have remained unclear. In this report we demonstrate the in vitro activity of GenK, a Cbl-dependent radical SAM enzyme that methylates an unactivated sp3 carbon during the biosynthesis of gentamicin, an aminoglycoside antibiotic. Experiments to investigate the stoichiometry of the GenK reaction revealed that one equivalent each of 5′-deoxyadenosine and S-adenosyl-homocysteine are produced for each methylation reaction catalyzed by GenK. Furthermore, isotope-labeling experiments demonstrate that the S-methyl group from SAM is transferred to Cbl and the aminoglycoside product during the course of the reaction. Based on these results, one mechanistic possibility for the GenK reaction can be ruled out and further questions regarding the mechanisms of Cbl-dependent radical SAM methyltransferases, in general, are discussed.

Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been studied. BE-7585A produced by Amycolatopsis orientalis subsp. vinearia BA-07585 has a 2-thiosugar and is a member of the angucycline class of compounds. We report herein the results of our initial efforts to study the biosynthesis of BE-7585A. Spectroscopic analyses verified the structure of BE-7585A, which is closely related to rhodonocardin A. Feeding experiments using 13 C-labeled acetate were carried out to confirm that the angucycline core is indeed polyketide-derived. The results indicated an unusual manner of angular tetracyclic ring construction, perhaps via a Baeyer-Villiger type rearrangement. Subsequent cloning and sequencing led to the identification of the bex gene cluster spanning ~30 kbp. A total of 28 open reading frames, which are likely involved in BE-7585A formation, were identified in the cluster. In view of the presence of a homologue of a thiazole synthase gene (thiG), bexX, in the bex cluster, the mechanism of sulfur incorporation into the 2-thiosugar moiety could resemble that found in thiamin biosynthesis. A glycosyltransferase homologue, BexG2, was heterologously expressed in E. coli. The purified enzyme successfully catalyzed the coupling of 2-thioglucose 6-phoshate and UDP-glucose to produce 2-thiotrehalose 6-phosphate, which is the precursor of the disaccharide unit in BE-7585A. On the basis of these genetic and biochemical experiments, a biosynthetic pathway for BE-7585A can now be proposed. The combined results set the stage for future biochemical studies of 2-thiosugar biosynthesis and BE-7585A assembly.The angucycline class of natural products, which have a characteristic fused four-ring frame assembled in an angular manner, are rich in antibacterial or anticancer activities. 1 BE-7585A (1), produced by Amycolatopsis orientalis subsp. vinearia BA-07585, is an angucycline-type natural product that inhibits thymidylate synthase. 2 In addition to the benz [a]anthraquinone core, BE-7585A also contains the deoxysugar, rhodinose (2), and a disaccharide appendage (4) containing a highly unusual 2-thioglucose (3). While sulfur is an essential element found ubiquitously in living systems, including amino acids, nucleic acids, metal clusters, enzyme cofactors, and many secondary metabolites, it is seldom found in carbohydrates. The rareness of the thiosugar entity prompted us to explore the biosynthesis of BE-7585A, especially the mode of sulfur incorporation into the 2-thiosugar moiety (3). Mechanistic details of several biological sulfur insertion reactions have recently been unraveled. 3 Highly regulated reactive intermediates such as protein persulfide, protein thiocarboxylate or S-adenosylmethioninedependent radical are often generated in the enzymes carrying out such reactions. Whether the same or different mechanisms are used to form the C-S bond in 2-thiosugar biosynthesis is the focus of this study.* T...

Amylose was prepared by enzymatic polymerization of alpha-D-glucose 1-phosphate dipotassium catalyzed by a phosphorylase using two kinds of the primers derived from maltopentaose, and then it was chemically bonded to silica gel to be used as a chiral stationary phase (CSP) in high-performance liquid chromatography. In method I, maltopentaose was first lactonized and allowed to react with (3-aminopropyl)triethoxysilane to form an amide bond. Amylose chains with a desired chain length and a narrow molecular weight distribution were then constructed by the enzymatic polymerization. The resulting amylose bearing a trialkoxysilyl group at the terminal was allowed to react with silica gel for immobilization. In method II, maltopentaose was first oxidized to form a potassium gluconate at the reducing terminal. After the enzymatic polymerization was performed with the potassium gluconate, the amylose end was lactonized to be immobilized to 3-aminopropyl-silanized silica gel through amide bond formation. Two amylose-conjugated silica gels thus obtained were treated with a large excess of 3,5-dimethylphenyl isocyanate to convert hydroxy groups of amylose to corresponding carbamate residues. The CSP derived through method II was superior in chiral recognition to the CSP derived from method I and showed better resolving power and higher durability against solvents such as tetrahydrofuran compared with a coated-type CSP. Influences of degree of polymerization of amylose, the spacer length between amylose and silica gel, and mobile phase compositions on chiral recognition were investigated.

Supported ruthenium hydroxide catalysts (Ru(OH)(x)/support) were prepared with three different TiO(2) supports (anatase TiO(2) (TiO(2)(A), BET surface area: 316 m(2) g(-1)), anatase TiO(2) (TiO(2)(B), 73 m(2) g(-1)), and rutile TiO(2) (TiO(2)(C), 3.2 m(2) g(-1))), as well as an Al(2)O(3) support (160 m(2) g(-1)). Characterizations with X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), electron spin resonance (ESR), and X-ray absorption fine structure (XAFS) showed the presence of monomeric ruthenium(III) hydroxide and polymeric ruthenium(III) hydroxide species. Judging from the coordination numbers of the nearest-neighbor Ru atoms and the intensities of the ESR signals, the amount of monomeric hydroxide species increased in the order of Ru(OH)(x)

We propose a new sealed battery operating on a redox reaction between an oxide (O2−) and a peroxide (O22−) with its theoretical specific energy of 2570 Wh kg−1 (897 mAh g−1, 2.87 V) and demonstrate that a Co-doped Li2O cathode exhibits a reversible capacity over 190 mAh g−1, a high rate capability, and a good cyclability with a superconcentrated lithium bis(fluorosulfonyl)amide electrolyte in acetonitrile. The reversible capacity is largely dominated by the O2−/O22− redox reaction between oxide and peroxide with some contribution of the Co2+/Co3+ redox reaction.

In the presence of Mo6+-doped α-MnO2 (Mo–MnO2), various sulfides could efficiently be oxidized to the corresponding sulfoxides as the major products. In addition, Mo–MnO2 could repeatedly be reused.

Spinosyn A is a polyketide-derived macrolide produced by Saccharopolyspora spinosa and is an active ingredient in several commercial insecticides. It is glycosylated by a tri-O-methylated rhamnose at C-9 and a forosamine at C-17. Previous studies indicated that the rhamnose methyltransferases are encoded by the spnH, spnI and spnK genes. To verify the functions of these methyltransferases and to study how they are coordinated to achieve the desired level of methylation of rhamnose, we studied the catalytic properties of the spnH, spnI and spnK gene products and validated their roles in the permethylation process of spinosyn A. Our data reported herein firmly established that SpnH, SpnI, and SpnK are the respective rhamnose 4′-, 2′-, and 3′-Omethyltransferase. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK and SpnH in sequence is productive for the permethylation of the rhamnose moiety. Moreover, the completion of rhamnose permethylation is likely achieved by the proper control of the expression levels of the methyltransferase genes involved. These results set the stage for future exploitation of spinosyn biosynthetic pathway to produce targeted spinosyn derivatives and, perhaps, new analogues.Spinosyn A (SPA, 1) is a polyketide-derived macrolide produced by Saccharopolyspora spinosa, that is an active ingredient in several commercial insecticides. 1 The structures of SPA and its many analogues have a characteristic perhydro-as-indacene core, which is glycosylated by a rhamnose (see 2) at C-9 and a forosamine at C-17. Both the aglycone (AGL, 3) and the sugar appendages contribute to the observed activity of the spinosyns, among which SPA is most potent. 2 Alteration of the tetracyclic nucleus or removal of either deoxy sugars significantly diminishes the pesticidal activity. Even subtle structural variations, such as the methylation pattern of the rhamnose moiety in spinosyns, change the LD 50 by as much as >200- 3 the study of how the corresponding methyltransferases are coordinated to achieve the desired level of methylation of rhamnose has been a focus of this research. We report herein the function and substrate specificity of the three methyltransferases involved in the methylation reactions, the preferred reaction sequence of their catalyzed reactions, and the likely regulation of permethylation of rhamnose in 1.The spinosyn biosynthetic gene cluster had been cloned from S. spinosa. 4 Sequence analysis and gene disruption experiments revealed that the spnH, spnI and spnK genes, 4 all of which show high sequence identity to those encoding S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTs), are involved in the O-methylation of rhamnose in 1. 4,5 As illustrated in Scheme 1, methylations may take place before (2 → 4 → 6, route A) or after (3 → 5 → 6, route B) the attachment of rhamnose to the aglycone (AGL, 3). It is also possible that methylations are the final tailoring steps after both sugars have been coupled to the aglycone (...

Highly negatively charged heteropolyacids (HPAs), in particular H5BW12O40, efficiently promoted saccharification of crystalline cellulose into water‐soluble saccharides in concentrated aqueous solutions (e.g., 82 % total yield and 77 % glucose yield, based on cellulose with a 0.7 M H5BW12O40 solution); the performance was much better than those of previously reported systems with commonly utilized mineral acids (e.g., H2SO4 and HCl) and HPAs (e.g., H3PW12O40 and H4SiW12O40). Besides crystalline cellulose, the present system was applicable to the selective transformation of cellobiose, starch, and xylan to the corresponding monosaccharides such as glucose and xylose. In addition, one‐pot synthesis of levulinic acid and sorbitol directly from cellulose was realized by using concentrated HPA solutions. The present system, concentrated aqueous solutions of highly negatively charged HPAs, was further applicable to saccharification of natural (non‐purified) lignocellulose biomass, such as “rice plant straw”, “oil palm empty fruit bunch (palm EFB) fiber”, and “Japanese cedar sawdust”, giving a mixture of the corresponding water‐soluble saccharides, such as glucose (main product), galactose, mannose, xylose, arabinose, and cellobiose, in high yields (≥77 % total yields of saccharides based on holocellulose). Separation of the saccharides and H5BW12O40 was easy, and the retrieved H5BW12O40 could repeatedly be used without appreciable loss of the high performance.