Characterization of hypothetical protein VNG0128C from Halobacterium NRC-1 reveals GALE like activity and its involvement in Leloir pathway of galactose metabolism.
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.
a Department of Biotechnology , PES Institute of Technology , Bangalore , India.
S V
SV
Reshma
Nitish
N
Sathyanarayanan
H G
HG
Nagendra
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D005690:Galactose / Q000378:metabolism*; D006217:Halobacterium / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D016415:Sequence Alignment; D014534:UDPglucose 4-Epimerase / Q000378:metabolism*
D001426:Bacterial Proteins; D009243:NAD; D014534:UDPglucose 4-Epimerase; D005690:Galactose
Halobacterium NRC-1; NAD+ dependent dehydratase/epimerase; UDP-galactose 4-epimerase; hypothetical protein