The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic
VNG0128C, a hypothetical protein from Halobacterium NRC-1, was chosen for detailed insilico and experimental investigations. Computational exercises revealed that VNG0128C functions as NAD(+) binding protein. The phylogenetic analysis with the homolog sequences of VNG0128C suggested that it could act as UDP-galactose 4-epimerase. Hence, the VNG0128C sequence was modeled using a suitable template and docking studies were performed with NAD and UDP-galactose as ligands. The binding interactions strongly indicate that VNG0128C could plausibly act as UDP-galactose 4-epimerase. In order to validate these insilico results, VNG0128C was cloned in pUC57, subcloned in pET22b(+), expressed in BL21 cells and purified using nickel affinity chromatography. An assay using blue dextran was performed to confirm the presence of NAD binding domain. To corroborate the epimerase like enzymatic role of the hypothetical protein, i.e. the ability of the enzyme to convert UDP-galactose to UDP-glucose, the conversion of NAD to NADH was measured. The experimental assay significantly correlated with the insilico predictions, indicating that VNG0128C has a NAD(+) binding domain with epimerase activity. Consequently, its key role in nucleotide-sugar metabolism was thus established. Additionally, the work highlights the need for a methodical characterization of hypothetical proteins (less studied class of biopolymers) to exploit them for relevant applications in the field of biology.
Hypothetical proteins are functionally uncharacterized proteins with assigned function using sequence annotation tools. Almost half of
the coding regions of several genomes are hypothetical proteins. Therefore, it is of our interest to characterize a hypothetical protein
YVRE from the model system Bacillus subtilis using known data. YVRE is assigned the function as a glucono-lactonase using prediction
and phylogenetic analysis. A molecular dynamics simulated homology model of YVRE (with calcium) using human senescence marker
protein 30 /SMP30 (PDB ID: 3G4E) as template is reported for functional inference. It is observed that the protein possesses bivalent
metal binding domain. Molecular docking studies with the substrate glucono-δ-lactone show YVRE binding with the substrate. This
data was further validated using cloning and sub-cloning in pUC57 and pET22b+ respectively, followed by expression and purification
using nickel affinity chromatography. The activity of YVRE using the substrate glucono-δ-lactone was calculated. The results show the
function of YVRE as a gluconolactonase, with higher preference to zinc than calcium or magnesium. Thus, YVRE is shown to play key
role in three metabolic pathways namely, pentose phosphate pathway, ascorbate and aldarate metabolism, and caprolactam
degradation.
Actinobacteria, conventionally known as actinomycetes are the most unique microorganisms revealing a link between bacteria and fungi. They are highly adaptable to extreme environmental condition and also exhibit a high diversity in metabolic activities. Biochemical, physiological and genetic features are mainly responsible for their higher adoptability to harsh conditions and extra cellular synthesis of wider secondary metabolites in general and enzymes and antibiotics in particular. The limestone quarry and lime powder dwellings are the harsh habitats prevailing in the northern region of Karnataka. These are the typical habitats left behind after the exploration of limestone and lime powder for highly commercial industrial activities such as production of cement and petroleum refining process respectively.
In the present investigation, efforts were made to detect cellulolytic actinobacteria from lime powder dwellings. Actinobacteria confirmed by the basic colony characters, microscopic features, biochemical and physiological properties were screened for the potential cellulolytic activity. In all 54 isolates of actinobacteria were detected and screened to obtain three best cellulolytic actinobacteria, namely DSA22, DSA38 and DSA39. The maximum zone of hydrolysis on carboxymethylcellulose medium was an important criterion to screen the best cellulolytic isolates of actinobacteria. Further, the three best isolates of cellulolytic actinobacteria were screened for maximum production of extra cellular cellulase. The isolate DSA22 with higher enzyme activity (12 IU) was subjected to molecular characterization. Based on 16s rRNA analysis (BioEra Laboratory, Pune, Maharashtra) an isolate DSA 22 was identified as Streptomyces enissocaesiles.
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