Aggarwal Geetika, Prajapati Rameshwar, Tripathy Rajan K, Bajaj Priyanka, Iyengar A R Satvik, Sangamwar Abhay T, Pande Abhay H
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
PLoS One. 2016 Feb 1;11(2):e0147999. doi: 10.1371/journal.pone.0147999. eCollection 2016.
Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1.
人对氧磷酶1（h-PON1）是一种能水解多种底物的血清酶。该酶具有抗炎、抗氧化、抗动脉粥样硬化、抗糖尿病、抗微生物和水解有机磷酸酯的活性。因此，h-PON1是开发针对人类多种疾病治疗干预措施的有力候选物。然而，h-PON1的晶体结构尚未解析，该酶如何水解不同底物的分子细节也尚不清楚。了解h-PON1的催化机制对于开发其治疗用途至关重要。文献表明，h-PON1第192位的R/Q多态性显著调节该酶的底物特异性。为了了解h-PON1第192位氨基酸残基在其各种水解活性中的作用，本研究对第192位进行了位点特异性诱变。使用大肠杆菌表达系统产生突变酶，并将它们的水解活性与一组底物进行比较。对选定的重组h-PON1（rh-PON1）突变体进行分子动力学模拟研究，以了解第192位氨基酸取代对酶活性位点结构特征的影响。我们的结果表明，根据底物类型，第192位特定氨基酸残基的存在会不同程度地改变酶活性位点的微环境，从而导致不同氨基酸残基子集参与底物的结合和加工。该结果推进了我们对h-PON1催化机制的理解。
