A ruthenium-catalyzed, redox neutral C-O bond cleavage of 2-aryloxy-1-arylethanols was developed that yields cleavage products in 62-98% isolated yield. This reaction is applicable to breaking the key ethereal bond found in lignin-related polymers. The bond transformation proceeds by a tandem dehydrogenation/ reductive ether cleavage. Initial mechanistic investigations indicate that the ether cleavage is most likely an organometallic C-O activation. A catalytic depolymerization of a lignin-related polymer quantitatively yields the corresponding monomer with no added reagent.

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose-and pectin-containing biomass to acetate, CO 2 , and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocelluloseutilizing, biofuel-producing bacterium.Microbial hydrogen production from biomass has been recognized as an important source of renewable energy (15, 47). High-temperature microorganisms are well suited for production of biohydrogen from plant polysaccharides, as anaerobic fermentation is thermodynamically favored at elevated temperatures (17, 43). The extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903, a fermentative anaerobe initially isolated from wood in the flow of a thermal spring in New Zealand, first received attention because of its capacity to utilize cellulose at its optimal growth temperature, 70°C (37). Further work showed that C. saccharolyticus (i) can utilize a wide range of plant materials, including cellulose, hemicellulose, starch, and pectin, (ii) has a very high hydrogen yield (almost 4 mol of H 2 per mol of glucose) (14,20,48), and (iii) can ferment C 5 and C 6 sugars simultaneously. These features have led to the development of bioprocessing schemes based on C. saccharolyticus. For example, H 2 production is now being investigated using a two-step process in which H 2 and acetate are generated from biomass hydrolysates in one bioreactor and the acetate is fed to a second bioreactor and used by phototrophic organisms (Rhodobacter spp.) to produce additional H 2 in the presence of...

In eukaryotes, MogA and MoeA are fused into a single polypeptide chain. The corresponding mammalian protein gephyrin has also been implicated in the anchoring of glycinergic receptors to the cytoskeleton at inhibitory synapses. Based on the structures of MoeA and MogA, gephyrin is surmised to be a highly organized molecule containing at least five domains. This multidomain arrangement could provide a structural basis for its functional diversity. The oligomeric states of MoeA and MogA suggest how gephyrin could assemble into a hexagonal scaffold at inhibitory synapses.

[reaction: see text] Dirhodium caprolactamate [Rh2(cap)4] is an effective catalyst for benzylic oxidation with tert-butyl hydroperoxide (TBHP) under mild conditions. Sodium bicarbonate is the optimal base additive for substrate conversion. Benzylic carbonyl compounds are readily obtained, and a formal synthesis of palmarumycin CP2 using this methodology is described.

Dirhodium caprolactamate [Rh2(cap)4] is a highly effective catalyst for the oxidative Mannich reaction. The reaction proceeds via C-H oxidation of a tertiary amine followed by nucleophilic capture. This green transformation is conducted in protic solvent using inexpensive T-HYDRO (70% t-BuOOH in water). Synthetically valuable gamma-aminoalkyl butenolides are obtained.

Synthesis of inositol 1,2,3,4,5,6-hexakisphosphate (IP 6 ), also known as phytate, is integral to cellular function in all eukaryotes. Production of IP 6 predominately occurs through phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (IP 5 ) by a 2-kinase. Recent cloning of the gene encoding this kinase from Saccharomyces cerevisiae, designated scIpk1, has identified a cellular role for IP 6 production in the regulation of mRNA export from the nucleus. In this report, we characterize the biochemical and functional parameters of recombinant scIpk1. Purified recombinant scIpk1 kinase activity is highly selective for IP 5 substrate and exhibits apparent K m values of 644 nM and 62.8 M for IP 5 and ATP, respectively. The observed apparent catalytic efficiency (k cat / K m ) of scIpk1 is 31,610 s ؊1 M ؊1 . A sequence similarity search was used to identify an IP 5 2-kinase from the fission yeast Schizosaccharomyces pombe. Recombinant spIpk1 has similar substrate selectivity and catalytic efficiency to its budding yeast counterpart, despite sharing only 24% sequence identity. Cells lacking sc-IPK1 are deficient in IP 6 production and exhibit lethality in combination with a gle1 mutant allele. Both of these phenotypes are complemented by expression of the spIPK1 gene in the sc-ipk1 cells. Analysis of several inactive mutants and multiple sequence alignment of scIpk1, spIpk1, and a putative Candida albicans Ipk1 have identified residues involved in catalysis. This includes two conserved motifs: E(i/l/m)KPKWL(t/y) and LXMTLRDV(t/g)(l/c)(f/y)I. Our data suggest that the mechanism for IP 6 production is conserved across species.Inositol polyphosphates (IPs) 1 in eukaryotic cells are key regulatory molecules whose levels transiently fluctuate in response to diverse cellular stimuli (1, 2). A major route for synthesis of IPs is through activation of phosphatidylinositolspecific phospholipase C. Phospholipase C cleaves lipids such as phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate, a regulator of calcium efflux from the endoplasmic reticulum. The release of a soluble inositol head group from its anchoring lipid also represents the first step in the pathway for generation of more highly phosphorylated inositols (3). The most abundant of these is inositol 1,2,3,4,5,6-hexakisphosphate (IP 6 ), also known as phytate. IP 6 can represent up to 1% of the mass of a plant seed, where it may serve as an antioxidant and a phosphate storage source (4, 5). The role of IP 6 is less clear in mammalian cells, although there is evidence suggesting that it may regulate inflammation, neurotransmission, and cell growth (reviewed in Ref.3). Recently, a metabolic pathway converting inositol 1,4,5-trisphosphate to IP 6 was delineated in budding yeast Saccharomyces cerevisiae cells (6-9). It has been shown that IP 6 also serves as a precursor for diphosphorylated inositols, such as diphosphoryl inositol 1,3,4,5,6-pentakisphosphate (PP-IP 5 ), in both yeast and vertebrate cells (3, 10). Combined in vivo and in vitro...

Microwave irradiation can be used to regulate biocatalysis. Herein, the utilization of hyperthermophilic enzymes in a microwave reactor is reported. While these enzymes are inactive at low temperatures, they can be activated with microwave irradiation. To the best of our knowledge, this is the first illustration of a specific microwave effect in enzymatic catalysis.

We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA ؊ cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.In all molybdenum-containing enzymes, with the exception of nitrogenase, the molybdenum cofactor (Moco) 1 consists of a mononuclear Mo atom coordinated to the cis-dithiolene moiety of molybdopterin (MPT). In Escherichia coli, biosynthesis of Moco begins with the conversion of GTP to a pterin intermediate termed precursor Z, catalyzed by the MoaA and MoaC proteins (1-4). MPT is synthesized from precursor Z by the MoaD and MoaE proteins, which together compose the MPT synthase complex (5, 6). The MoeB protein is involved in reactivating the MoaD subunit of MPT synthase between rounds of MPT synthesis (7,8). Ligation of the Mo atom to MPT requires the MoeA and MogA proteins along with the ModABC molybdate transporter system (9, 10). In E. coli, the cofactor is further modified by the covalent addition of GMP to the MPT phosphate, a reaction catalyzed by the MobA protein (11,12).Previous results from our laboratory verified that E. coli MoeA and MogA are both required for in vivo Mo ligation to the MPT dithiolene. Analysis of crude extracts produced from E. coli moeA Ϫ and mogA Ϫ cells showed the presence of MPT but the absence of MPT-guanine dinucleotide, the biosynthesis of which was found to require prior attachment of Mo to MPT. Recombinantly expressed human sulfite oxidase (SO) purified from both strains contained MPT but was devoid of Mo. In vitro experiments demonstrated the ability of MoeA to activate the recombinant Mo-free, MPT-containing human SO in moeA Ϫ crude extract. MogA was incapable of supporting the same type of activity in mogA Ϫ crude extract. Thus, MoeA appeared to be the protein directly responsible for Mo ligation, ...