Towards Understanding the Catalytic Mechanism of Human Paraoxonase 1: Experimental and In Silico Mutagenesis Studies.
Human paraoxonase 1 (h-PON1) is a ~45-kDa serum enzyme that can hydrolyze a variety of substrates, including organophosphate (OP) compounds. It is a potential candidate for the development of antidote against OP poisoning in humans. However, insufficient OP-hydrolyzing activity of native enzyme affirms the urgent need to develop improved variant(s) having enhanced OP-hydrolyzing activity. The crystal structure of h-PON1 remains unsolved, and the molecular details of how the enzyme catalyses hydrolysis of different types of substrates are also not clear. Understanding the molecular details of the catalytic mechanism of h-PON1 is essential to engineer better variant(s) of enzyme. In this study, we have used a random mutagenesis approach to increase the OP-hydrolyzing activity of recombinant h-PON1. The mutants not only showed a 10-340-fold increased OP-hydrolyzing activity against different OP substrates but also exhibited differential lactonase and arylesterase activities. In order to investigate the mechanistic details of the effect of observed mutations on the hydrolytic activities of enzyme, molecular docking studies were performed with selected mutants. The results suggested that the observed mutations permit differential binding of substrate/inhibitor into the enzyme's active site. This may explain differential hydrolytic activities of the enzyme towards different substrates.
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab, 160062, India.
Rajan K
RK
Tripathy
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab, 160062, India.
Geetika
G
Aggarwal
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab, 160062, India.
Priyanka
P
Bajaj
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab, 160062, India.
Deepika
D
Kathuria
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab, 160062, India.
Prasad V
PV
Bharatam
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab, 160062, India. apande@niper.ac.in.
Abhay H
AH
Pande
D000595:Amino Acid Sequence; D043303:Aryldialkylphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007783:Lactones / Q000378:metabolism; D062105:Molecular Docking Simulation; D016296:Mutagenesis; D009154:Mutation; D010755:Organophosphates / Q000378:metabolism
D004791:Enzyme Inhibitors; D007783:Lactones; D010755:Organophosphates; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human
Acyl homoserine lactone; Molecular docking; Organophosphate; Random mutagenesis; Recombinant human PON1