Biocatalysts provide a major advantage to bio-based economy over chemical catalysts by catalyzing... more Biocatalysts provide a major advantage to bio-based economy over chemical catalysts by catalyzing various useful transformations in an environment friendly manner along with other major benefits of selectivity, specificity, and low energy consumption. Since last decade, cellulase is the 3rd highest used enzyme in industry in various processes. Xylanase is also one amongst the widely used enzymes, and many industrial applications require synergistic action of both of these enzymes. These applications predominantly include bioethanol production, deinking of waste paper, animal feed processing, food processing, paper and pulp production, removal of fine fibers from textile material (biostoning), and pharmaceuticals. These enzymes are produced by microorganisms (fungi and bacteria), and hence, the microorganisms producing both cellulases and xylanases are in high demand by these industries. This review focuses on the synergistic applications of cellulase and xylanase enzymes across various industrial sectors. It also discusses the potential applications and the need of the microbial systems (fungi and bacteria) secreting both of these enzymes and the future prospects of their development into an integral part of various industrial processes.
Hemoproteins have recently emerged as promising biocatalysts for promoting a variety of carbene t... more Hemoproteins have recently emerged as promising biocatalysts for promoting a variety of carbene transfer reactions including cyclopropanation and Y-H insertion (Y = N, S, Si, B). For these and synthetic carbene transfer catalysts alike, achieving high chemoselectivity toward cyclopropanation in olefin substrates bearing unprotected Y-H groups has proven remarkably challenging due to competition from the more facile carbene Y-H insertion reaction. In this report, we describe the development of a novel artificial metalloenzyme based on an engineered myoglobin incorporating a serine-ligated Co-porphyrin cofactor that is capable of offering high selectivity toward olefin cyclopropanation over N-H and Si-H insertion. Intramolecular competition experiments revealed a distinct and dramatically altered chemoselectivity of the Mb(H64V,V68A,H93S)[Co(ppIX)] variant in carbene transfer reactions compared to myoglobin-based variants containing the native histidine-ligated heme cofactor or other ...
Applied biochemistry and biotechnology, Jan 4, 2017
Human paraoxonase 1 (h-PON1) is a ~45-kDa serum enzyme that can hydrolyze a variety of substrates... more 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 t...
Engineered hemoproteins have recently emerged as promising systems for promoting asymmetric cyclo... more Engineered hemoproteins have recently emerged as promising systems for promoting asymmetric cyclopropanations,b ut variants featuring predictable,c omplementary stereoselectivity in these reactions have remained elusive.I nt his study,arationally driven strategy was implemented and applied to engineer myoglobin variants capable of providing access to 1-carboxy-2-aryl-cyclopropanes with high trans-(1R,2R) selectivity and catalytic activity.T he stereoselectivity of these cyclopropanation biocatalysts complements that of trans-(1S,2S)-selective variants developed here and previously. In combination with whole-cell biotransformations,t hese stereocomplementary biocatalysts enabled the multigram synthesis of the chiral cyclopropane core of four drugs (Tranylcypromine,T asimelteon, Ticagrelor,a nd aT RPV1 inhibitor) in high yield and with excellent diastereo-and enantioselectivity (98-99.9% de;9 6-99.9% ee). These biocatalytic strategies outperform currently available methods to produce these drugs.
The first example of abiocatalytic [2,3]-sigmatropic rearrangement reaction involving allylic sul... more The first example of abiocatalytic [2,3]-sigmatropic rearrangement reaction involving allylic sulfides and diazo reagents (Doyle-Kirmse reaction) is reported. Engineered variants of sperm whale myoglobin catalyze this synthetically valuable C À Cb ond-forming transformation with high efficiency and product conversions across av ariety of sulfide substrates (e.g., aryl-, benzyl-, and alkyl-substituted allylic sulfides) and a-diazo esters.M oreover,t he scope of this myoglobin-mediated transformation could be extended to the conversion of propargylic sulfides to give substituted allenes. Active-site mutations proved effective in enhancing the catalytic efficiency of the hemoprotein in these reactions as well as modulating the enantioselectivity,resulting in the identification of the myoglobin variant Mb(L29S,H64V,V68F), whichi s capable of mediating asymmetric Doyle-Kirmse reactions with an enantiomeric excess up to 71 %. This work extends the toolbox of currently available biocatalytic strategies for the asymmetric formation of carbon-carbon bonds.
Applied biochemistry and biotechnology, Jan 30, 2016
Organophosphate (OP) compounds are neurotoxic chemicals, and current treatments available for OP-... more 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;R1...
Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The en... more 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.
SsoPox, a ~35 kDa enzyme from Sulfolobus solfataricus, can hydrolyze and inactivate a variety of ... more SsoPox, a ~35 kDa enzyme from Sulfolobus solfataricus, can hydrolyze and inactivate a variety of organophosphate (OP)-compounds. The enzyme is a potential candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. However, the therapeutic use of recombinant SsoPox suffers from certain limitations associated with the use of recombinant protein pharmaceuticals. Some of these limitations could be overcome by conjugating SsoPox enzyme with polyethylene glycol (PEG). In this study, we report generation and in vitro characterization of N-terminal mono-PEGylated rSsoPox(2p) (a variant of rSsoPox(wt) having enhanced OP-hydrolyzing activity). The enzyme was PEGylated with mPEG-propionaldehyde and the PEGylated protein was isolated using ion-exchange chromatography. Compared with the unmodified enzyme, mono-PEGylation of rSsoPox results in improvement in the thermostability and protease resistance of the enzyme. PEGylated rSsoPox(2p) can be developed as a candidate for the prevention / treatment of OP-poisoning.
Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide ... more Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate.
Organophosphorus hydrolase (OPH) is a ∼38kDa enzyme encoded by opd gene of Flavobacterium sp. The... more Organophosphorus hydrolase (OPH) is a ∼38kDa enzyme encoded by opd gene of Flavobacterium sp. The enzyme can hydrolyze and inactivate variety of organophosphate (OP)-compounds, including chemical warfare nerve agents. Thus, OPH is a strong candidate for the development of therapeutic intervention against OP-poisoning in humans and other animals. It is also a promising bio-decontaminating agent for clean-up of OP-contaminated objects and areas. For successful commercial application, long-term storage stability of purified OPH enzyme is important. In this study we have cloned and expressed recombinant OPH (r-OPH) in Escherichiacoli and the effect of different excipients on the long-term storage stability of purified enzyme was analyzed. The enzyme was stored in either aqueous solution or in lyophilized form at 25°C for 60days in the presence or absence of different excipients and the stability of the enzyme was determined by monitoring the paraoxon-hydrolyzing activity. Our results suggest that, (a) maltose, trehalose, arginine and proline were most effective in stabilizing the enzyme when stored in aqueous buffer at 25°C, and (b) maltose, trehalose, and mannose exerted maximum stabilization effect when the enzyme was stored in lyophilized form at 25°C for 60days. The study shows that common excipients can be used to stabilize purified OPH enzyme in order to store it for long period of time under different storage conditions. The results of this study can be used to develop formulation(s) of OPH enzyme for commercial use.
Human PON1 (h-PON1) is a multifaceted enzyme and can hydrolyze (and inactivate) a wide range of s... more Human PON1 (h-PON1) is a multifaceted enzyme and can hydrolyze (and inactivate) a wide range of substrates. The enzyme shows anti-inflammatory, antioxidative, antiatherogenic, ant-diabetic, antimicrobial, and organophosphate (OP)-detoxifying properties. However, there are certain limitations regarding large-scale production and use of h-PON1 as a therapeutic candidate. These include difficulties in producing recombinant h-PON1 (rh-PON1) using microbial expression system, low hydrolytic activity of wild-type h-PON1 towards certain substrates, and low storage stability of the purified enzyme. This review summarizes the work done in our laboratory to address these limitations. Our results show that (a) optimized polynucleotide sequence encoding rh-PON1 can express the protein in an active form inE. coliand can be used to generate variant of the enzyme having enhanced hydrolytic activity, (b)in vitrorefolding of rh-PON1 enzyme can dramatically increase the yield of an active enzyme, (c)...
Human paraoxonase 1 (h-PON1) is a Ca(2+)-dependent serum enzyme that catalyzes the hydrolysis of ... more Human paraoxonase 1 (h-PON1) is a Ca(2+)-dependent serum enzyme that catalyzes the hydrolysis of different types of substrates. The crystal structure of h-PON1 is not solved yet and the molecular details of how the enzyme catalyzes different types of reactions are not clear. Literature suggests that the amino acid residues at positions 192 and 115 are important for various hydrolytic activities of h-PON1. It is proposed that catalytic residue H115 (and H134) mediates the lactonase and the arylesterase activities of the enzyme while the amino acid residue at position 192 modulates various other hydrolytic activities of the enzyme. However, the relationship between these two residues in the hydrolytic activities of h-PON1 is not studied in detail. In this study, we have expressed and purified the wild-type recombinant h-PON1 (rh-PON1(wt)) and its point mutants differing in the amino acid residues at positions 192 and/or 115 using an Escherichia coli expression system. The hydrolytic a...
Human paraoxonase 1 (h-PON1) hydrolyzes variety of substrates and the hydrolytic activities of en... more Human paraoxonase 1 (h-PON1) hydrolyzes variety of substrates and the hydrolytic activities of enzyme can be broadly grouped into three categories; arylesterase, phosphotriesterase, and lactonase. Current models of the catalytic mechanism of h-PON1 suggest that catalytic residues H115 and H134 mediate the lactonase and arylesterase activities of the enzyme. H-PON1 is a strong candidate for the development of catalytic bioscavenger for organophosphate poisoning in humans. Recently, Gupta et al. (Nat. Chem. Biol. 2011. 7, 120) identified amino acid substitutions that significantly increased the activity of chimeric-PON1 variant (4E9) against some organophosphate nerve agents. In this study we have examined the effect of these (L69G/S111T/H115W/H134R/R192K/F222S/T332S) and other substitutions (H115W/H134R and H115W/H134R/R192K) on the hydrolytic activities of recombinant h-PON1 (rh-PON1) variants. Our results show that the substitutions resulted in a significant increase in the organophosphatase activity of all the three variants of rh-PON1 enzyme while had a variable effect on the lactonase/arylesterase activities. The results suggest that H residues at positions 115 and 134 are not always needed for the lactonase/arylesterase activities of h-PON1 and force a reconsideration of the current model(s) of the catalytic mechanism of h-PON1.
Apolipoprotein-derived peptides are promising candidates for the treatment of various inflammator... more Apolipoprotein-derived peptides are promising candidates for the treatment of various inflammatory conditions and the main mechanism proposed for the protective action of these peptides includes binding to pro-inflammatory lipid mediators with high affinity and facilitating their sequestration/metabolism/clearance in the body. Molecules that act as pro-inflammatory lipid mediators differ considerably in their molecular structures, chemical compositions and physicochemical properties. Importance of the properties of pro-inflammatory lipid mediators on the biological activity of apolipoprotein-derived peptides has not been studied in detail. In this study, we characterized the physicochemical properties of aggregates containing lyso-PAF, acetyl-PAF and butanoyl-PAF, three closely related pro-inflammatory lipid mediators, and studied their interaction with peptides derived from the C-terminal domains of human apolipoprotein E. These PAF-analogs differ only in the chemical composition of the functional groups they carry at the sn-2 positions. Our results show that physicochemical properties of aggregates containing lyso-PAF, acetyl-PAF and butanoyl-PAF differ considerably and affect their apolipoprotein-derived peptides-binding capacity.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2013
Paraoxonase 1 (PON1) is an HDL-associated enzyme and exhibits anti-inflammatory, anti-diabetic, a... more Paraoxonase 1 (PON1) is an HDL-associated enzyme and exhibits anti-inflammatory, anti-diabetic, and anti-atherogenic properties. Association of PON1 to HDL particles increases the stability and activity of PON1 and is important for the normal functioning of the enzyme. HDL particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein constituent of HDL that facilitates various biological activities of HDL. In many disease conditions the oxidized phospholipid (Ox-PL) content of HDL is found to be increased and an inverse correlation between the activity of PON1 and oxidation of the HDL is observed. However, the molecular details of the inhibitory action of the Ox-PL-containing HDL on the function of PON1 are not clear yet. In this study we have assembled reconstituted HDL (rHDL) particles with and without Ox-PL and compared their effect on the structure and function of (13)C-labeled recombinant PON1 ((13)C-rPON1) by employing attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectroscopy and enzymatic assay. Our results show that the presence of the Ox-PL in the rHDL particles alters the structure of rPON1 and decreases its lactonase activity.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2012
High density lipoprotein (HDL) particles are made up of lipid and protein constituents and apolip... more High density lipoprotein (HDL) particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein component that facilitates various biological activities of HDL particles. Increase in Ox-PL content of HDL particles makes them 'dysfunctional' and such modified HDL particles not only lose their athero-protective properties but also acquire pro-atherogenic and pro-inflammatory functions. The details of Ox-PL-induced alteration in the molecular properties of HDL particles are not clear. Paraoxonase 1 (PON1) is an HDL-associated enzyme that possesses anti-inflammatory and anti-atherogenic properties; and many of the athero-protective functions of HDL are attributed to the associated PON1. In this study we have characterized the physicochemical properties of reconstituted HDL (rHDL) particles containing varying amounts of Ox-PL and have compared their PON1 stimulation capacity. Our results show that increased Ox-PL content (a) modifies the physicochemical properties of the lipid domain of the rHDL particles, (b) decreases the stability and alters the conformation as well as orientation of apoA-I molecules on the rHDL particles, and (c) decreases the PON1 stimulation capacity of the rHDL particles. Our data indicate that the presence of Ox-PLs destabilizes the structure of the HDL particles and modifies their function.
Human paraoxonase 1 (h-PON1) is a ~40 kDa multi-tasking enzyme that plays a major role in determi... more Human paraoxonase 1 (h-PON1) is a ~40 kDa multi-tasking enzyme that plays a major role in determining individual susceptibility towards various disease conditions. It is a strong candidate for the development of therapeutic intervention for various diseases and other conditions in humans. However, purified h-PON1 is unstable and there is a need to find condition(s) that can increase the shelf life of the enzyme. In this report, we present the results of our investigation on the effect of excipients on the stability of bacterially produced human PON1 when stored under different storage conditions. Our results show that (a) glycine and serine are most effective in stabilizing the enzyme when stored in aqueous buffer at 25 °C for 30 days, and (b) trehalose, maltose, and BSA exerted maximum stabilization effect when the enzyme was stored in freeze-dried form at 25 °C for 60 days. Results of this study can be used to increase the shelf life of purified h-PON1 enzyme.
Biocatalysts provide a major advantage to bio-based economy over chemical catalysts by catalyzing... more Biocatalysts provide a major advantage to bio-based economy over chemical catalysts by catalyzing various useful transformations in an environment friendly manner along with other major benefits of selectivity, specificity, and low energy consumption. Since last decade, cellulase is the 3rd highest used enzyme in industry in various processes. Xylanase is also one amongst the widely used enzymes, and many industrial applications require synergistic action of both of these enzymes. These applications predominantly include bioethanol production, deinking of waste paper, animal feed processing, food processing, paper and pulp production, removal of fine fibers from textile material (biostoning), and pharmaceuticals. These enzymes are produced by microorganisms (fungi and bacteria), and hence, the microorganisms producing both cellulases and xylanases are in high demand by these industries. This review focuses on the synergistic applications of cellulase and xylanase enzymes across various industrial sectors. It also discusses the potential applications and the need of the microbial systems (fungi and bacteria) secreting both of these enzymes and the future prospects of their development into an integral part of various industrial processes.
Hemoproteins have recently emerged as promising biocatalysts for promoting a variety of carbene t... more Hemoproteins have recently emerged as promising biocatalysts for promoting a variety of carbene transfer reactions including cyclopropanation and Y-H insertion (Y = N, S, Si, B). For these and synthetic carbene transfer catalysts alike, achieving high chemoselectivity toward cyclopropanation in olefin substrates bearing unprotected Y-H groups has proven remarkably challenging due to competition from the more facile carbene Y-H insertion reaction. In this report, we describe the development of a novel artificial metalloenzyme based on an engineered myoglobin incorporating a serine-ligated Co-porphyrin cofactor that is capable of offering high selectivity toward olefin cyclopropanation over N-H and Si-H insertion. Intramolecular competition experiments revealed a distinct and dramatically altered chemoselectivity of the Mb(H64V,V68A,H93S)[Co(ppIX)] variant in carbene transfer reactions compared to myoglobin-based variants containing the native histidine-ligated heme cofactor or other ...
Applied biochemistry and biotechnology, Jan 4, 2017
Human paraoxonase 1 (h-PON1) is a ~45-kDa serum enzyme that can hydrolyze a variety of substrates... more 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 t...
Engineered hemoproteins have recently emerged as promising systems for promoting asymmetric cyclo... more Engineered hemoproteins have recently emerged as promising systems for promoting asymmetric cyclopropanations,b ut variants featuring predictable,c omplementary stereoselectivity in these reactions have remained elusive.I nt his study,arationally driven strategy was implemented and applied to engineer myoglobin variants capable of providing access to 1-carboxy-2-aryl-cyclopropanes with high trans-(1R,2R) selectivity and catalytic activity.T he stereoselectivity of these cyclopropanation biocatalysts complements that of trans-(1S,2S)-selective variants developed here and previously. In combination with whole-cell biotransformations,t hese stereocomplementary biocatalysts enabled the multigram synthesis of the chiral cyclopropane core of four drugs (Tranylcypromine,T asimelteon, Ticagrelor,a nd aT RPV1 inhibitor) in high yield and with excellent diastereo-and enantioselectivity (98-99.9% de;9 6-99.9% ee). These biocatalytic strategies outperform currently available methods to produce these drugs.
The first example of abiocatalytic [2,3]-sigmatropic rearrangement reaction involving allylic sul... more The first example of abiocatalytic [2,3]-sigmatropic rearrangement reaction involving allylic sulfides and diazo reagents (Doyle-Kirmse reaction) is reported. Engineered variants of sperm whale myoglobin catalyze this synthetically valuable C À Cb ond-forming transformation with high efficiency and product conversions across av ariety of sulfide substrates (e.g., aryl-, benzyl-, and alkyl-substituted allylic sulfides) and a-diazo esters.M oreover,t he scope of this myoglobin-mediated transformation could be extended to the conversion of propargylic sulfides to give substituted allenes. Active-site mutations proved effective in enhancing the catalytic efficiency of the hemoprotein in these reactions as well as modulating the enantioselectivity,resulting in the identification of the myoglobin variant Mb(L29S,H64V,V68F), whichi s capable of mediating asymmetric Doyle-Kirmse reactions with an enantiomeric excess up to 71 %. This work extends the toolbox of currently available biocatalytic strategies for the asymmetric formation of carbon-carbon bonds.
Applied biochemistry and biotechnology, Jan 30, 2016
Organophosphate (OP) compounds are neurotoxic chemicals, and current treatments available for OP-... more 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;R1...
Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The en... more 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.
SsoPox, a ~35 kDa enzyme from Sulfolobus solfataricus, can hydrolyze and inactivate a variety of ... more SsoPox, a ~35 kDa enzyme from Sulfolobus solfataricus, can hydrolyze and inactivate a variety of organophosphate (OP)-compounds. The enzyme is a potential candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. However, the therapeutic use of recombinant SsoPox suffers from certain limitations associated with the use of recombinant protein pharmaceuticals. Some of these limitations could be overcome by conjugating SsoPox enzyme with polyethylene glycol (PEG). In this study, we report generation and in vitro characterization of N-terminal mono-PEGylated rSsoPox(2p) (a variant of rSsoPox(wt) having enhanced OP-hydrolyzing activity). The enzyme was PEGylated with mPEG-propionaldehyde and the PEGylated protein was isolated using ion-exchange chromatography. Compared with the unmodified enzyme, mono-PEGylation of rSsoPox results in improvement in the thermostability and protease resistance of the enzyme. PEGylated rSsoPox(2p) can be developed as a candidate for the prevention / treatment of OP-poisoning.
Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide ... more Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate.
Organophosphorus hydrolase (OPH) is a ∼38kDa enzyme encoded by opd gene of Flavobacterium sp. The... more Organophosphorus hydrolase (OPH) is a ∼38kDa enzyme encoded by opd gene of Flavobacterium sp. The enzyme can hydrolyze and inactivate variety of organophosphate (OP)-compounds, including chemical warfare nerve agents. Thus, OPH is a strong candidate for the development of therapeutic intervention against OP-poisoning in humans and other animals. It is also a promising bio-decontaminating agent for clean-up of OP-contaminated objects and areas. For successful commercial application, long-term storage stability of purified OPH enzyme is important. In this study we have cloned and expressed recombinant OPH (r-OPH) in Escherichiacoli and the effect of different excipients on the long-term storage stability of purified enzyme was analyzed. The enzyme was stored in either aqueous solution or in lyophilized form at 25°C for 60days in the presence or absence of different excipients and the stability of the enzyme was determined by monitoring the paraoxon-hydrolyzing activity. Our results suggest that, (a) maltose, trehalose, arginine and proline were most effective in stabilizing the enzyme when stored in aqueous buffer at 25°C, and (b) maltose, trehalose, and mannose exerted maximum stabilization effect when the enzyme was stored in lyophilized form at 25°C for 60days. The study shows that common excipients can be used to stabilize purified OPH enzyme in order to store it for long period of time under different storage conditions. The results of this study can be used to develop formulation(s) of OPH enzyme for commercial use.
Human PON1 (h-PON1) is a multifaceted enzyme and can hydrolyze (and inactivate) a wide range of s... more Human PON1 (h-PON1) is a multifaceted enzyme and can hydrolyze (and inactivate) a wide range of substrates. The enzyme shows anti-inflammatory, antioxidative, antiatherogenic, ant-diabetic, antimicrobial, and organophosphate (OP)-detoxifying properties. However, there are certain limitations regarding large-scale production and use of h-PON1 as a therapeutic candidate. These include difficulties in producing recombinant h-PON1 (rh-PON1) using microbial expression system, low hydrolytic activity of wild-type h-PON1 towards certain substrates, and low storage stability of the purified enzyme. This review summarizes the work done in our laboratory to address these limitations. Our results show that (a) optimized polynucleotide sequence encoding rh-PON1 can express the protein in an active form inE. coliand can be used to generate variant of the enzyme having enhanced hydrolytic activity, (b)in vitrorefolding of rh-PON1 enzyme can dramatically increase the yield of an active enzyme, (c)...
Human paraoxonase 1 (h-PON1) is a Ca(2+)-dependent serum enzyme that catalyzes the hydrolysis of ... more Human paraoxonase 1 (h-PON1) is a Ca(2+)-dependent serum enzyme that catalyzes the hydrolysis of different types of substrates. The crystal structure of h-PON1 is not solved yet and the molecular details of how the enzyme catalyzes different types of reactions are not clear. Literature suggests that the amino acid residues at positions 192 and 115 are important for various hydrolytic activities of h-PON1. It is proposed that catalytic residue H115 (and H134) mediates the lactonase and the arylesterase activities of the enzyme while the amino acid residue at position 192 modulates various other hydrolytic activities of the enzyme. However, the relationship between these two residues in the hydrolytic activities of h-PON1 is not studied in detail. In this study, we have expressed and purified the wild-type recombinant h-PON1 (rh-PON1(wt)) and its point mutants differing in the amino acid residues at positions 192 and/or 115 using an Escherichia coli expression system. The hydrolytic a...
Human paraoxonase 1 (h-PON1) hydrolyzes variety of substrates and the hydrolytic activities of en... more Human paraoxonase 1 (h-PON1) hydrolyzes variety of substrates and the hydrolytic activities of enzyme can be broadly grouped into three categories; arylesterase, phosphotriesterase, and lactonase. Current models of the catalytic mechanism of h-PON1 suggest that catalytic residues H115 and H134 mediate the lactonase and arylesterase activities of the enzyme. H-PON1 is a strong candidate for the development of catalytic bioscavenger for organophosphate poisoning in humans. Recently, Gupta et al. (Nat. Chem. Biol. 2011. 7, 120) identified amino acid substitutions that significantly increased the activity of chimeric-PON1 variant (4E9) against some organophosphate nerve agents. In this study we have examined the effect of these (L69G/S111T/H115W/H134R/R192K/F222S/T332S) and other substitutions (H115W/H134R and H115W/H134R/R192K) on the hydrolytic activities of recombinant h-PON1 (rh-PON1) variants. Our results show that the substitutions resulted in a significant increase in the organophosphatase activity of all the three variants of rh-PON1 enzyme while had a variable effect on the lactonase/arylesterase activities. The results suggest that H residues at positions 115 and 134 are not always needed for the lactonase/arylesterase activities of h-PON1 and force a reconsideration of the current model(s) of the catalytic mechanism of h-PON1.
Apolipoprotein-derived peptides are promising candidates for the treatment of various inflammator... more Apolipoprotein-derived peptides are promising candidates for the treatment of various inflammatory conditions and the main mechanism proposed for the protective action of these peptides includes binding to pro-inflammatory lipid mediators with high affinity and facilitating their sequestration/metabolism/clearance in the body. Molecules that act as pro-inflammatory lipid mediators differ considerably in their molecular structures, chemical compositions and physicochemical properties. Importance of the properties of pro-inflammatory lipid mediators on the biological activity of apolipoprotein-derived peptides has not been studied in detail. In this study, we characterized the physicochemical properties of aggregates containing lyso-PAF, acetyl-PAF and butanoyl-PAF, three closely related pro-inflammatory lipid mediators, and studied their interaction with peptides derived from the C-terminal domains of human apolipoprotein E. These PAF-analogs differ only in the chemical composition of the functional groups they carry at the sn-2 positions. Our results show that physicochemical properties of aggregates containing lyso-PAF, acetyl-PAF and butanoyl-PAF differ considerably and affect their apolipoprotein-derived peptides-binding capacity.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2013
Paraoxonase 1 (PON1) is an HDL-associated enzyme and exhibits anti-inflammatory, anti-diabetic, a... more Paraoxonase 1 (PON1) is an HDL-associated enzyme and exhibits anti-inflammatory, anti-diabetic, and anti-atherogenic properties. Association of PON1 to HDL particles increases the stability and activity of PON1 and is important for the normal functioning of the enzyme. HDL particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein constituent of HDL that facilitates various biological activities of HDL. In many disease conditions the oxidized phospholipid (Ox-PL) content of HDL is found to be increased and an inverse correlation between the activity of PON1 and oxidation of the HDL is observed. However, the molecular details of the inhibitory action of the Ox-PL-containing HDL on the function of PON1 are not clear yet. In this study we have assembled reconstituted HDL (rHDL) particles with and without Ox-PL and compared their effect on the structure and function of (13)C-labeled recombinant PON1 ((13)C-rPON1) by employing attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectroscopy and enzymatic assay. Our results show that the presence of the Ox-PL in the rHDL particles alters the structure of rPON1 and decreases its lactonase activity.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2012
High density lipoprotein (HDL) particles are made up of lipid and protein constituents and apolip... more High density lipoprotein (HDL) particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein component that facilitates various biological activities of HDL particles. Increase in Ox-PL content of HDL particles makes them 'dysfunctional' and such modified HDL particles not only lose their athero-protective properties but also acquire pro-atherogenic and pro-inflammatory functions. The details of Ox-PL-induced alteration in the molecular properties of HDL particles are not clear. Paraoxonase 1 (PON1) is an HDL-associated enzyme that possesses anti-inflammatory and anti-atherogenic properties; and many of the athero-protective functions of HDL are attributed to the associated PON1. In this study we have characterized the physicochemical properties of reconstituted HDL (rHDL) particles containing varying amounts of Ox-PL and have compared their PON1 stimulation capacity. Our results show that increased Ox-PL content (a) modifies the physicochemical properties of the lipid domain of the rHDL particles, (b) decreases the stability and alters the conformation as well as orientation of apoA-I molecules on the rHDL particles, and (c) decreases the PON1 stimulation capacity of the rHDL particles. Our data indicate that the presence of Ox-PLs destabilizes the structure of the HDL particles and modifies their function.
Human paraoxonase 1 (h-PON1) is a ~40 kDa multi-tasking enzyme that plays a major role in determi... more Human paraoxonase 1 (h-PON1) is a ~40 kDa multi-tasking enzyme that plays a major role in determining individual susceptibility towards various disease conditions. It is a strong candidate for the development of therapeutic intervention for various diseases and other conditions in humans. However, purified h-PON1 is unstable and there is a need to find condition(s) that can increase the shelf life of the enzyme. In this report, we present the results of our investigation on the effect of excipients on the stability of bacterially produced human PON1 when stored under different storage conditions. Our results show that (a) glycine and serine are most effective in stabilizing the enzyme when stored in aqueous buffer at 25 °C for 30 days, and (b) trehalose, maltose, and BSA exerted maximum stabilization effect when the enzyme was stored in freeze-dried form at 25 °C for 60 days. Results of this study can be used to increase the shelf life of purified h-PON1 enzyme.
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Papers by Priyanka Bajaj