title: Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs.
abstract: The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, Km values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative property against BSA and Cu(2+)/H2O2 model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies.
authors.affiliation: Laboratory of Enzymology and Recombinant DNA Technology, Department of Microbiology, Maharshi Dayanand University, Rohtak 124001, Haryana, India.
authors.forename: Deepti
authors.initials: D
authors.lastname: Singh
authors.affiliation: Laboratory of Enzymology and Recombinant DNA Technology, Department of Microbiology, Maharshi Dayanand University, Rohtak 124001, Haryana, India.
authors.forename: Surender
authors.initials: S
authors.lastname: Rawat
authors.affiliation: School of Computational & Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Mohd
authors.initials: M
authors.lastname: Waseem
authors.affiliation: School of Computational & Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Sunita
authors.initials: S
authors.lastname: Gupta
authors.affiliation: School of Computational & Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Andrew
authors.initials: A
authors.lastname: Lynn
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Mukesh
authors.initials: M
authors.lastname: Nitin
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Nirala
authors.initials: N
authors.lastname: Ramchiary
authors.affiliation: Laboratory of Enzymology and Recombinant DNA Technology, Department of Microbiology, Maharshi Dayanand University, Rohtak 124001, Haryana, India. Electronic address: kekulsharma@gmail.com.
authors.forename: Krishna Kant
authors.initials: KK
authors.lastname: Sharma
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001711:Biotransformation; D003198:Computer Simulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000502:physiology; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D062105:Molecular Docking Simulation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D015008:Yersinia enterocolitica / Q000201:enzymology* / Q000235:genetics
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D011994:Recombinant Proteins; D042845:Laccase
keywords: Biotransformation; Laccase; MD simulation; NSAIDs; Yersinia enterocolitica