Optimized method for TAG protein homology modeling: In silico and experimental structural characterization
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- 作者：Jyoti Singh Tomar ; Rama Krishna Peddinti ; rkpedfcy@iitr.ac.in" class="auth_mail" title="E-mail the corresponding author ; ramakpeddinti@gmail.com" class="auth_mail" title="E-mail the corresponding author
- 关键词：3mA ; 3-methyladenine ; 7mG ; 7-methylguanine ; AlkA ; 3-methyladenine DNA glycosylase II ; BER pathway ; base excision repair pathway ; TAG ; 3-methyladenine DNA glycosylase I
- 刊名：International Journal of Biological Macromolecules
- 出版年：2016
- 出版时间：July 2016
- 年：2016
- 卷：88
- 期：Complete
- 页码：102-112
- 全文大小：2041 K
文摘
The DNA glycosylases cleave C
N 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 ∼21 kDa). 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.
N 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 ∼21 kDa). 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.