Characterization of human paraoxonase 1 variants suggest that His residues at 115 and 134 positions are not always needed for the lactonase/arylesterase activities of the enzyme

Bajaj, Priyanka; Tripathy, Rajan K.; Aggarwal, Geetika; Pande, Abhay H.

doi:10.1002/pro.2380

Cited by 14 publications

(17 citation statements)

References 34 publications

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“…The mutagenized plasmids were amplified in E . coli DH5α cells, purified and the DNA sequence of the mutants was confirmed by bi-directional DNA sequencing (Eurofinn, India) [ 27 – 28 ]. The plasmids were then transformed into E .…”

Section: Methodsmentioning

confidence: 99%

“…The assays were performed in flat bottom 96-well plates at 25°C in a total volume of 200 μl using a Molecular Devices SPECTRAmax PLUS Microplate spectrophotometer [ 27 – 28 ]. In all assays, the appropriate blank ( i .…”

Section: Methodsmentioning

confidence: 99%

“…e . the product formed) was calculated and expressed as μMole/min/mg of the protein [ 27 – 28 ]. The specific activity of rh-PON1 (wt) was taken as 100% and the activities of the mutant proteins were then calculated with respect to the control.…”

Section: Methodsmentioning

confidence: 99%

“…Although, this variant of chi-PON1 (G2E6) shares ~ 85% sequence similarity with h-PON1 (at amino acid level), there is a considerable difference in the catalytic activities of these enzymes [ 25 – 28 ]. On the basis of the information gained from in silico analysis and enzymatic characterization of h-PON1 and Chi-PON1 different mechanisms explaining various hydrolytic activities of h-PON1 have been proposed [ 24 , 27 – 28 , 29 – 32 ]. It has been proposed that the His residue at position 115 (H115) plays a pivotal role in carrying out the lactonase/arylesterase activities of the enzyme, and the replacement of H with W at position 115 causes significant decrease in these activities of the enzyme [ 25 , 28 , 31 , 33 ].…”

Section: Introductionmentioning

confidence: 99%

“…It has been proposed that the His residue at position 115 (H115) plays a pivotal role in carrying out the lactonase/arylesterase activities of the enzyme, and the replacement of H with W at position 115 causes significant decrease in these activities of the enzyme [ 25 , 28 , 31 , 33 ]. However, results from our earlier studies have shown that H115 residue of h-PON1 may not always be required for mediating the lactonase/arylesterase activities of the enzyme [ 27 – 28 ].…”

Section: Introductionmentioning

confidence: 99%

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Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192

et al. 2016

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192

et al. 2016

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Organophosphate-Hydrolyzing Enzymes as First-Line of Defence Against Nerve Agent-Poisoning: Perspectives and the Road Ahead

Iyengar

Pande

2016

Protein J

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Nerve agents (NAs) are extremely neurotoxic synthetic organophosphate (OP) compounds exploited as weapons of mass destruction in terrorist attacks and chemical warfare. Considering the current world scenario, there is a persistent threat of NA-exposure to military personals and civilians. Various prophylactic and post-exposure treatments (such as atropine and oximes) available currently for NA-poisoning are inadequate and unsatisfactory and suffer from severe limitations. Hence, developing safe and effective treatment(s) against NA-poisoning is a critical necessity. With regards to counteracting NA-toxicity, the OP-hydrolyzing enzymes (OPHEs), which can hydrolyze and inactivate a variety of NAs, have emerged as promising candidates for the development of prophylactic therapy against NA-poisoning. However, there are many hurdles to be crossed before these enzymes can be brought to therapeutic use in humans. In this article, we have reviewed the various advancements in the field of development of OPHEs as prophylactic against NA-poisoning. The article majorly focuses on the toxic effects of NAs, various available therapies to counteract NA poisoning, the current status of OPHEs and attempts made to improve the various properties of these enzymes. Further, we have also briefly discussed about the prospective work that is needed to be undertaken for developing these OPHEs into those suitable for use in humans.

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Refolded Recombinant Human Paraoxonase 1 Variant Exhibits Prophylactic Activity Against Organophosphate Poisoning

Bajaj

Tripathy

Aggarwal

et al. 2016

Appl Biochem Biotechnol

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Organophosphate (OP) compounds are neurotoxic chemicals, and current treatments available for OP-poisoning are considered as unsatisfactory and inadequate. There is an urgent need for the development of more effective treatment(s) for OP-poisoning. Human paraoxonase 1 (h-PON1) is known to hydrolyze a variety of OP-compounds and is a leading candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. Non-availability of effective system(s) for the production of recombinant h-PON1 (rh-PON1) makes it hard to produce improved variant(s) of this enzyme and analyze their in vivo efficacy in animal models. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop variant(s) of h-PON1. Recently, we have developed a procedure to produce active rh-PON1 enzymes by using E. coli expression system. In this study, we have characterized the OP-hydrolyzing properties of refolded rh-PON1(wt) and rh-PON1(H115W;R192K) variant. Our results show that refolded rh-PON1(H115W;R192K) variant exhibit enhanced OP-hydrolyzing activity in in vitro and ex vivo assays and exhibited prophylactic activity in mouse model of OP-poisoning, suggesting that refolded rh-PON1 can be developed as a therapeutic candidate.

show abstract

Characterization of human paraoxonase 1 variants suggest that His residues at 115 and 134 positions are not always needed for the lactonase/arylesterase activities of the enzyme

Cited by 14 publications

References 34 publications

Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192

Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192

Organophosphate-Hydrolyzing Enzymes as First-Line of Defence Against Nerve Agent-Poisoning: Perspectives and the Road Ahead

Refolded Recombinant Human Paraoxonase 1 Variant Exhibits Prophylactic Activity Against Organophosphate Poisoning

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