title: Optimized method for TAG protein homology modeling: In silico and experimental structural characterization.
abstract: The DNA glycosylases cleave CN glycosyl bond to release a free base and generate abasic sites concurrently. Function and structure of these enzymes in the pathogenic bacterium Acinetobacter baumannii and its closely related species are not well characterized. Inhibition of TAG enzyme is a promising drug design strategy against A. baumannii. Here optimized molecular modeling approaches were used to provide a structural scaffold of TAG. The recombinant TAG protein was expressed and purified to determine oligomeric state using size exclusion chromatography, which showed the existence of TAG protein as monomer (mwt ∼21kDa). Secondary structure and substrate binding were analyzed using CD are in good agreement with the in silico predictions. Near UV-CD spectrum shows the involvement of Tyr residues in substrate recognition. Molecular docking studies were performed to understand the molecular recognition interactions and this knowledge was used to identify the potent inhibitors using virtual screening. Residues crucial for DNA holding and enzyme catalysis are reconfirmed by the in silico mutational studies.
authors.affiliation: Department of Chemistry, Indian Institute of Technology, Roorkee 247667, Uttarakhand, India.
authors.forename: Jyoti Singh
authors.initials: JS
authors.lastname: Tomar
authors.affiliation: Department of Chemistry, Indian Institute of Technology, Roorkee 247667, Uttarakhand, India. Electronic address: rkpedfcy@iitr.ac.in.
authors.forename: Rama Krishna
authors.initials: RK
authors.lastname: Peddinti
mesh_terms: D040981:Acinetobacter baumannii / Q000737:chemistry* / Q000201:enzymology; D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D045647:DNA Glycosylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D062105:Molecular Docking Simulation; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013816:Thermodynamics; D014584:User-Computer Interface
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C025946:3-methyladenine; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D000225:Adenine
keywords: 3-Methyladenine; BER pathway; FoldX algorithm; HhH superfamily