Characterization of a novel 4-guanidinobutyrase from Candida parapsilosis.

Enzymes of the ureohydrolase superfamily are specific in recognizing their substrates. While looking to broaden the substrate specificity of 4-guanidinobutyrase (GBase), we isolated a yeast, typed as Candida parapsilosis (NCIM 3689), that efficiently utilized both 4-guanidinobutyrate (GB) and 3-guanidinopropionate (GP) as a sole source of nitrogen. A putative GBase sequence was identified from its genome upon pBLAST query using the GBase sequence from Aspergillus niger (AnGBase).

Molecular insights into the inhibition of glutamate dehydrogenase by the dicarboxylic acid metabolites.

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD - or NADP -dependent reversible conversion of α-ketoglutarate (AKG) to l-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.

Environmental role of aromatic carboxylesterases.

The carboxylesterases (EC 3.1.1.

Protein expression and secretion by filamentous fungi.

In the search for optimal platforms for protein expression and secretion, filamentous fungi in principle provide some of the best microbial cell factories. They are inherently endowed with the ability to secrete proteins. Fungi belonging to and species are well-studied for industrial production of proteins and enzymes.

High-Throughput Screening of Dye-Ligands for Chromatography.

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc.

Autophagy is important to the acidogenic metabolism of Aspergillus niger.

Significant phenotypic overlaps exist between autophagy and acidogenesis in Aspergillus niger. The possible role of autophagy in the acidogenic growth and metabolism of this fungus was therefore examined and the movement of cytosolic EGFP to vacuoles served to monitor this phenomenon. An autophagy response to typical as well as a metabolic inhibitor-induced nitrogen starvation was observed in A.

2-Oxoglutarate cooperativity and biphasic ammonium saturation of Aspergillus niger NADP-glutamate dehydrogenase are structurally coupled.

NADP-glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoidal 2-oxoglutarate saturation. Despite sharing 88% amino acid identity, the homologous enzyme from Aspergillus terreus (AtGDH) shows hyperbolic 2-oxoglutarate saturation. In order to address the structural origins of this phenomenon, six AnGDH-AtGDH chimeras were constructed and characterized.

Characterization of 4-guanidinobutyrase from Aspergillus niger.

Arginase is the only fungal ureohydrolase that is well documented in the literature. More recently, a novel route for agmatine catabolism in Aspergillus niger involving another ureohydrolase, 4-guanidinobutyrase (GBase), was reported. We present here a detailed characterization of A.

Carbaryl as a Carbon and Nitrogen Source: an Inducible Methylamine Metabolic Pathway at the Biochemical and Molecular Levels in sp. Strain C5pp.

Carbaryl is the most widely used carbamate family pesticide, and its persistent nature causes it to pollute both soil and water ecosystems. Microbes maintain the Earth's biogeochemical cycles by metabolizing various compounds present in the matter, including xenobiotics, as a sole source of carbon, nitrogen, and energy. Soil isolate sp.

Structural basis for the catalytic mechanism and α-ketoglutarate cooperativity of glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is a key enzyme connecting carbon and nitrogen metabolism in all living organisms. Despite extensive studies on GDHs from both prokaryotic and eukaryotic organisms in the last 40 years, the structural basis of the catalytic features of this enzyme remains incomplete. This study reports the structural basis of the GDH catalytic mechanism and allosteric behavior.

Novel Route for Agmatine Catabolism in Aspergillus niger: 4-Guanidinobutyrase Assay.

The enzyme 4-guanidinobutyrase (GBase) catalyzes the hydrolysis of 4-guanidinobutyric acid (GB) to 4-aminobutyric acid (GABA) and urea. Here we describe methods to estimate urea and GABA that were suitably adapted from the published literature. The urea is determined by colorimetric assay using modified Archibald's method.

Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production.

Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus.

Contribution of arginase to manganese metabolism of Aspergillus niger.

Aspects of manganese metabolism during normal and acidogenic growth of Aspergillus niger were explored. Arginase from this fungus was a Mn[II]-enzyme. The contribution of the arginase protein towards A.

Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase.

Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies.

Characterization of succinic semialdehyde dehydrogenase from Aspergillus niger.

The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min(-1) mg(-1), using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+.

Purification, crystallization and preliminary X-ray diffraction analysis of NADP-dependent glutamate dehydrogenase from Aspergillus niger.

Glutamate dehydrogenase (GDH) catalyzes the NAD-dependent or NADP-dependent oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia. This important reversible reaction establishes the link between carbon and nitrogen metabolism. In this study, Aspergillus niger NADP-GDH (AnGDH) has been overexpressed and purified.

Mixed disulfide formation at Cys141 leads to apparent unidirectional attenuation of Aspergillus niger NADP-glutamate dehydrogenase activity.

NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.

High-throughput screening of dye-ligands for chromatography.

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc.

A biochemical correlate of dimorphism in a zygomycete Benjaminiella poitrasii: characterization of purified NAD-dependent glutamate dehydrogenase, a target for antifungal agents.

The fungal organisms, especially pathogens, change their vegetative (Y, unicellular yeast and H, hypha) morphology reversibly for survival and proliferation in the host environment. NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.

A novel selectable marker based on Aspergillus niger arginase expression.

Selectable markers are valuable tools in transforming asexual fungi like Aspergillus niger. An arginase (agaA) expression vector and a suitable arginase-disrupted host would define a novel nutritional marker/selection for transformation. The development of such a marker was successfully achieved in two steps.

Utility of Aspergillus niger citrate synthase promoter for heterologous expression.

Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5' upstream sequence (0.9kb DNA) of citrate synthase gene (citA) from A.

Discriminatory protein binding by a library of 96 new affinity resins: a novel dye-affinity chromatography tool-kit.

Initial acceptance of Cibacron Blue 3G-A based matrices has made dye-ligand affinity chromatography an attractive proposition. This prompted the synthesis and search for new dye structures. A systematic library of 96 affinity resins was generated using novel analogs of Cibacron Blue 3G-A and also by varying spacer lengths for immobilization.

Aspergillus terreus NADP-glutamate dehydrogenase is kinetically distinct from the allosteric enzyme of other Aspergilli.

NADP-Glutamate dehydrogenase (NADP-GDH) located at the interface of carbon and nitrogen metabolism has the potential to dictate fungal carbon flux. NADP-GDH from Aspergillus terreus, itaconate producer and an opportunistic pathogen, was purified to homogeneity using novel reactive dye-affinity resins. The pure enzyme was extensively characterized for its biochemical and kinetic properties and compared with its well studied Aspergillus niger counterpart.

Phosphinothricin resistance in Aspergillus niger and its utility as a selectable transformation marker.

Aspergillus niger is moderately susceptible to inhibition by phosphinothricin (PPT)-a potent inhibitor of glutamine synthetase. This growth inhibition was relieved by L-glutamine. PPT inhibited A.

Delineation of an in vivo inhibitor for Aspergillus glutamate dehydrogenase.

NADP-glutamate dehydrogenase (NADP-GDH) along with glutamine synthetase plays a pivotal role in ammonium assimilation. Specific inhibitors were valuable in defining the importance of glutamine synthetase in nitrogen metabolism. Selective in vivo inhibition of NADP-GDH has so far been an elusive desideratum.