Identification of iron-chelating phenolics contributing to seed coat coloration in soybeans (Glycine max (L.) Merr.) expressing aryloxyalkanoate dioxygenase-12.

Soybeans (Glycine max (L.) Merr.) genetically modified to express aryloxyalkanoate dioxygenase-12 (AAD-12), an enzyme that confers resistance to the herbicide 2,4-D, can sometimes exhibit a darker seed coat coloration than equivalent unmodified soybeans.

Natural Background and Anthropogenic Arsenic Enrichment in Florida Soils, Surface Water, and Groundwater: A Review with a Discussion on Public Health Risk.

Florida geologic units and soils contain a wide range in concentrations of naturally-occurring arsenic. The average range of bulk rock concentrations is 1 to 13.1 mg/kg with concentrations in accessary minerals being over 1000 mg/kg.

Cellulose degradation by polysaccharide monooxygenases.

Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage.

A family of starch-active polysaccharide monooxygenases.

The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand.

Determinants of regioselective hydroxylation in the fungal polysaccharide monooxygenases.

The ubiquitous fungal polysaccharide monooxygenases (PMOs) (also known as GH61 proteins, LPMOs, and AA9 proteins) are structurally related but have significant variation in sequence. A heterologous expression method in Neurospora crassa was developed as a step toward connecting regioselectivity of the chemistry to PMO phylogeny. Activity assays, as well as sequence and phylogenetic analyses, showed that the majority of fungal PMOs fall into three major groups with distinctive active site surface features.

Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases.

The use of cellulases remains a major cost in the production of renewable fuels and chemicals from lignocellulosic biomass. Fungi secrete copper-dependent polysaccharide monooxygenases (PMOs) that oxidatively cleave crystalline cellulose and improve the effectiveness of cellulases. However, the means by which PMOs recognize and cleave their substrates in the plant cell wall remain unclear.

Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases.

Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O(2)-dependent oxidative cleavage of recalcitrant polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N.

Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa.

The high cost of enzymes for saccharification of lignocellulosic biomass is a major barrier to the production of second generation biofuels. Using a combination of genetic and biochemical techniques, we report that filamentous fungi use oxidative enzymes to cleave glycosidic bonds in cellulose. Deletion of cdh-1, the gene encoding the major cellobiose dehydrogenase of Neurospora crassa, reduced cellulase activity substantially, and addition of purified cellobiose dehydrogenases from M.

Extracellular aldonolactonase from Myceliophthora thermophila.

Fungi secrete many different enzymes to deconstruct lignocellulosic biomass, including several families of hydrolases, oxidative enzymes, and many uncharacterized proteins. Here we describe the isolation, characterization, and primary sequence analysis of an extracellular aldonolactonase from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile). The lactonase is a 48-kDa glycoprotein with a broad pH optimum.

Cellodextrin transport in yeast for improved biofuel production.

Fungal degradation of plant biomass may provide insights for improving cellulosic biofuel production. We show that the model cellulolytic fungus Neurospora crassa relies on a high-affinity cellodextrin transport system for rapid growth on cellulose. Reconstitution of the N.

Expression and characterization of the Neurospora crassa endoglucanase GH5-1.

Filamentous fungi secrete a wide range of enzymes, including cellulases and hemicellulases, with potential applications in the production of lignocellulosic biofuels. Of the cellulolytic fungi, Hypocrea jecorina (anamorph Trichoderma reesei) is the best characterized in terms of cellulose degradation, but other cellulolytic fungi, such as the model filamentous fungus Neurospora crassa, can serve a crucial role in building our knowledge about the fungal response to biomass due to the many molecular and genetic tools available for this organism. Here we cloned and expressed GH5-1 (NCU00762), a secreted endoglucanase in N.

Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa.

The filamentous fungus Neurospora crassa is a model laboratory organism, but in nature is commonly found growing on dead plant material, particularly grasses. Using functional genomics resources available for N. crassa, which include a near-full genome deletion strain set and whole genome microarrays, we undertook a system-wide analysis of plant cell wall and cellulose degradation.

N(G)-aminoguanidines from primary amines and the preparation of nitric oxide synthase inhibitors.

A concise, general, and high-yielding method for the preparation of N(G)-aminoguanidines from primary amines is reported. Using available and readily prepared materials, primary amines are converted to protected N(G)-aminoguanidines in a one-pot procedure. The method has been successfully applied to a number of examples including the syntheses of four nitric oxide synthase (NOS) inhibitors.

Design and synthesis of C5 methylated L-arginine analogues as active site probes for nitric oxide synthase.

The role of nitric oxide (NO) as a biological signaling molecule is well established. NO is produced by the nitric oxide synthases (NOSs, EC 1.14.

A convenient gHMQC-based NMR assay for investigating ammonia channeling in glutamine-dependent amidotransferases: studies of Escherichia coli asparagine synthetase B.

X-ray crystal structures of glutamine-dependent amidotransferases in their "active" conformation have revealed the existence of multiple active sites linked by solvent inaccessible intramolecular channels, giving rise to the widely accepted view that ammonia released in a glutaminase site is channeled efficiently into a separate synthetase site where it undergoes further reaction. We now report a very convenient isotope-edited 1H NMR-based assay that can be used to probe the transfer of ammonia between the active sites of amidotransferases and demonstrate its use in studies of Escherichia coli asparagine synthetase B (AS-B). Our NMR results suggest that (i) high glutamine concentrations do not suppress ammonia-dependent asparagine formation in this bacterial asparagine synthetase and (ii) ammonia in bulk solution can react with the thioester intermediate formed during the glutaminase half-reaction by accessing the N-terminal active site of AS-B during catalytic turnover.