The Experts below are selected from a list of 60 Experts worldwide ranked by ideXlab platform
Meir Wilchek - One of the best experts on this subject based on the ideXlab platform.
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thiol disulfide organization in Alliin Lyase Alliinase from garlic allium sativum
Protein Science, 2008Co-Authors: Lev Weiner, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Linda J W Shimon, Felix Frolow, Aharon RabinkovAbstract:Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5′-phosphate-dependent enzymes. There are 10 cysteine residues per Alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S—S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored —SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two —SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that Alliinase activity does not require free —SH groups. This allowed the oriented conjugation of Alliinase, via the —SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the Alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.
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two structures of Alliinase from alliium sativum l apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the plp cofactor
Journal of Molecular Biology, 2007Co-Authors: Linda J W Shimon, Aharon Rabinkov, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:Abstract Alliinase (Alliin Lyase EC 4.4.1.4), a PLP-dependent α, β-eliminating Lyase, constitutes one of the major protein components of garlic ( Alliium sativum L. ) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid Alliin ((+S)-allyl- L -cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl- L -cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo -enzyme was crystallized in tetragonal form. These structures of Alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to “short chain complex vacuole type” oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the e-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo -form is nearly identical to that seen in the holo -form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.
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A spectrophotometric assay for allicin, Alliin, and Alliinase (Alliin Lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates.
Analytical biochemistry, 2002Co-Authors: Talia Miron, Irina Shin, David Mirelman, Guy Feigenblat, Lev Weiner, Meir Wilchek, Aharon RabinkovAbstract:Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid Alliin with the enzyme Alliinase (Alliin Lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and Alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of Alliin and allicin, as well as of Alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of Alliin and allicin concentrations in their pure form as well as of Alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, Alliin, and Alliinase in garlic preparations.
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Alliin Lyase Alliinase from garlic allium sativum crystallization and preliminary x ray characterization
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Linda J W Shimon, Aharon Rabinkov, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:The enzyme Alliinase has been isolated from garlic bulbs and crystallized. The crystals belong to space group P21, with unit-cell parameters a = 70.191, b = 127.006, c = 108.085 A, β = 93.384°. They diffract to 2.2 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function suggests that the crystals contain two dimeric molecules per asymmetric unit.
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A spectrophotometric assay for allicin and Alliinase (Alliin Lyase) activity: reaction of 2-nitro-5-thiobenzoate with thiosulfinates.
Analytical biochemistry, 1998Co-Authors: Talia Miron, Aharon Rabinkov, David Mirelman, Lev Weiner, Meir WilchekAbstract:Allicin (diallylthiosulfinate) is the main biologically active component of freshly crushed garlic cloves. It is produced upon the interaction of the nonprotein amino acid Alliin with the enzyme Alliinase (Alliin Lyase, EC 4.4.1.4). A simple and rapid spectrophotometric procedure for determination of allicin and Alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin, is described. NTB reacts with the activated disulfide bond --S(O)-S--; of allicin, forming the mixed-disulfide allylmercapto-NTB, as characterized by NMR. The method can be used for determination of allicin and total thiosulfinates in garlic preparations and garlic-derived products. The method was applied for determination of pure Alliinase activity and for the activity of the enzyme in crude garlic extracts.
David Mirelman - One of the best experts on this subject based on the ideXlab platform.
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thiol disulfide organization in Alliin Lyase Alliinase from garlic allium sativum
Protein Science, 2008Co-Authors: Lev Weiner, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Linda J W Shimon, Felix Frolow, Aharon RabinkovAbstract:Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5′-phosphate-dependent enzymes. There are 10 cysteine residues per Alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S—S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored —SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two —SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that Alliinase activity does not require free —SH groups. This allowed the oriented conjugation of Alliinase, via the —SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the Alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.
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two structures of Alliinase from alliium sativum l apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the plp cofactor
Journal of Molecular Biology, 2007Co-Authors: Linda J W Shimon, Aharon Rabinkov, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:Abstract Alliinase (Alliin Lyase EC 4.4.1.4), a PLP-dependent α, β-eliminating Lyase, constitutes one of the major protein components of garlic ( Alliium sativum L. ) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid Alliin ((+S)-allyl- L -cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl- L -cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo -enzyme was crystallized in tetragonal form. These structures of Alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to “short chain complex vacuole type” oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the e-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo -form is nearly identical to that seen in the holo -form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.
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A spectrophotometric assay for allicin, Alliin, and Alliinase (Alliin Lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates.
Analytical biochemistry, 2002Co-Authors: Talia Miron, Irina Shin, David Mirelman, Guy Feigenblat, Lev Weiner, Meir Wilchek, Aharon RabinkovAbstract:Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid Alliin with the enzyme Alliinase (Alliin Lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and Alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of Alliin and allicin, as well as of Alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of Alliin and allicin concentrations in their pure form as well as of Alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, Alliin, and Alliinase in garlic preparations.
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Alliin Lyase Alliinase from garlic allium sativum crystallization and preliminary x ray characterization
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Linda J W Shimon, Aharon Rabinkov, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:The enzyme Alliinase has been isolated from garlic bulbs and crystallized. The crystals belong to space group P21, with unit-cell parameters a = 70.191, b = 127.006, c = 108.085 A, β = 93.384°. They diffract to 2.2 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function suggests that the crystals contain two dimeric molecules per asymmetric unit.
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A spectrophotometric assay for allicin and Alliinase (Alliin Lyase) activity: reaction of 2-nitro-5-thiobenzoate with thiosulfinates.
Analytical biochemistry, 1998Co-Authors: Talia Miron, Aharon Rabinkov, David Mirelman, Lev Weiner, Meir WilchekAbstract:Allicin (diallylthiosulfinate) is the main biologically active component of freshly crushed garlic cloves. It is produced upon the interaction of the nonprotein amino acid Alliin with the enzyme Alliinase (Alliin Lyase, EC 4.4.1.4). A simple and rapid spectrophotometric procedure for determination of allicin and Alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin, is described. NTB reacts with the activated disulfide bond --S(O)-S--; of allicin, forming the mixed-disulfide allylmercapto-NTB, as characterized by NMR. The method can be used for determination of allicin and total thiosulfinates in garlic preparations and garlic-derived products. The method was applied for determination of pure Alliinase activity and for the activity of the enzyme in crude garlic extracts.
Aharon Rabinkov - One of the best experts on this subject based on the ideXlab platform.
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thiol disulfide organization in Alliin Lyase Alliinase from garlic allium sativum
Protein Science, 2008Co-Authors: Lev Weiner, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Linda J W Shimon, Felix Frolow, Aharon RabinkovAbstract:Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5′-phosphate-dependent enzymes. There are 10 cysteine residues per Alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S—S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored —SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two —SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that Alliinase activity does not require free —SH groups. This allowed the oriented conjugation of Alliinase, via the —SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the Alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.
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two structures of Alliinase from alliium sativum l apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the plp cofactor
Journal of Molecular Biology, 2007Co-Authors: Linda J W Shimon, Aharon Rabinkov, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:Abstract Alliinase (Alliin Lyase EC 4.4.1.4), a PLP-dependent α, β-eliminating Lyase, constitutes one of the major protein components of garlic ( Alliium sativum L. ) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid Alliin ((+S)-allyl- L -cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl- L -cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo -enzyme was crystallized in tetragonal form. These structures of Alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to “short chain complex vacuole type” oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the e-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo -form is nearly identical to that seen in the holo -form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.
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A spectrophotometric assay for allicin, Alliin, and Alliinase (Alliin Lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates.
Analytical biochemistry, 2002Co-Authors: Talia Miron, Irina Shin, David Mirelman, Guy Feigenblat, Lev Weiner, Meir Wilchek, Aharon RabinkovAbstract:Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid Alliin with the enzyme Alliinase (Alliin Lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and Alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of Alliin and allicin, as well as of Alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of Alliin and allicin concentrations in their pure form as well as of Alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, Alliin, and Alliinase in garlic preparations.
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Alliin Lyase Alliinase from garlic allium sativum crystallization and preliminary x ray characterization
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Linda J W Shimon, Aharon Rabinkov, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:The enzyme Alliinase has been isolated from garlic bulbs and crystallized. The crystals belong to space group P21, with unit-cell parameters a = 70.191, b = 127.006, c = 108.085 A, β = 93.384°. They diffract to 2.2 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function suggests that the crystals contain two dimeric molecules per asymmetric unit.
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A spectrophotometric assay for allicin and Alliinase (Alliin Lyase) activity: reaction of 2-nitro-5-thiobenzoate with thiosulfinates.
Analytical biochemistry, 1998Co-Authors: Talia Miron, Aharon Rabinkov, David Mirelman, Lev Weiner, Meir WilchekAbstract:Allicin (diallylthiosulfinate) is the main biologically active component of freshly crushed garlic cloves. It is produced upon the interaction of the nonprotein amino acid Alliin with the enzyme Alliinase (Alliin Lyase, EC 4.4.1.4). A simple and rapid spectrophotometric procedure for determination of allicin and Alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin, is described. NTB reacts with the activated disulfide bond --S(O)-S--; of allicin, forming the mixed-disulfide allylmercapto-NTB, as characterized by NMR. The method can be used for determination of allicin and total thiosulfinates in garlic preparations and garlic-derived products. The method was applied for determination of pure Alliinase activity and for the activity of the enzyme in crude garlic extracts.
Felix Frolow - One of the best experts on this subject based on the ideXlab platform.
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thiol disulfide organization in Alliin Lyase Alliinase from garlic allium sativum
Protein Science, 2008Co-Authors: Lev Weiner, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Linda J W Shimon, Felix Frolow, Aharon RabinkovAbstract:Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5′-phosphate-dependent enzymes. There are 10 cysteine residues per Alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S—S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored —SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two —SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that Alliinase activity does not require free —SH groups. This allowed the oriented conjugation of Alliinase, via the —SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the Alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.
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two structures of Alliinase from alliium sativum l apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the plp cofactor
Journal of Molecular Biology, 2007Co-Authors: Linda J W Shimon, Aharon Rabinkov, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:Abstract Alliinase (Alliin Lyase EC 4.4.1.4), a PLP-dependent α, β-eliminating Lyase, constitutes one of the major protein components of garlic ( Alliium sativum L. ) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid Alliin ((+S)-allyl- L -cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl- L -cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo -enzyme was crystallized in tetragonal form. These structures of Alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to “short chain complex vacuole type” oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the e-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo -form is nearly identical to that seen in the holo -form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.
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Alliin Lyase Alliinase from garlic allium sativum crystallization and preliminary x ray characterization
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Linda J W Shimon, Aharon Rabinkov, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:The enzyme Alliinase has been isolated from garlic bulbs and crystallized. The crystals belong to space group P21, with unit-cell parameters a = 70.191, b = 127.006, c = 108.085 A, β = 93.384°. They diffract to 2.2 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function suggests that the crystals contain two dimeric molecules per asymmetric unit.
Talia Miron - One of the best experts on this subject based on the ideXlab platform.
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thiol disulfide organization in Alliin Lyase Alliinase from garlic allium sativum
Protein Science, 2008Co-Authors: Lev Weiner, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Linda J W Shimon, Felix Frolow, Aharon RabinkovAbstract:Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5′-phosphate-dependent enzymes. There are 10 cysteine residues per Alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S—S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored —SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two —SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that Alliinase activity does not require free —SH groups. This allowed the oriented conjugation of Alliinase, via the —SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the Alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.
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two structures of Alliinase from alliium sativum l apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the plp cofactor
Journal of Molecular Biology, 2007Co-Authors: Linda J W Shimon, Aharon Rabinkov, Irina Shin, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:Abstract Alliinase (Alliin Lyase EC 4.4.1.4), a PLP-dependent α, β-eliminating Lyase, constitutes one of the major protein components of garlic ( Alliium sativum L. ) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid Alliin ((+S)-allyl- L -cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl- L -cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo -enzyme was crystallized in tetragonal form. These structures of Alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to “short chain complex vacuole type” oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the e-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo -form is nearly identical to that seen in the holo -form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.
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A spectrophotometric assay for allicin, Alliin, and Alliinase (Alliin Lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates.
Analytical biochemistry, 2002Co-Authors: Talia Miron, Irina Shin, David Mirelman, Guy Feigenblat, Lev Weiner, Meir Wilchek, Aharon RabinkovAbstract:Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid Alliin with the enzyme Alliinase (Alliin Lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and Alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of Alliin and allicin, as well as of Alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of Alliin and allicin concentrations in their pure form as well as of Alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, Alliin, and Alliinase in garlic preparations.
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Alliin Lyase Alliinase from garlic allium sativum crystallization and preliminary x ray characterization
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Linda J W Shimon, Aharon Rabinkov, David Mirelman, Talia Miron, Meir Wilchek, Felix FrolowAbstract:The enzyme Alliinase has been isolated from garlic bulbs and crystallized. The crystals belong to space group P21, with unit-cell parameters a = 70.191, b = 127.006, c = 108.085 A, β = 93.384°. They diffract to 2.2 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function suggests that the crystals contain two dimeric molecules per asymmetric unit.
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A spectrophotometric assay for allicin and Alliinase (Alliin Lyase) activity: reaction of 2-nitro-5-thiobenzoate with thiosulfinates.
Analytical biochemistry, 1998Co-Authors: Talia Miron, Aharon Rabinkov, David Mirelman, Lev Weiner, Meir WilchekAbstract:Allicin (diallylthiosulfinate) is the main biologically active component of freshly crushed garlic cloves. It is produced upon the interaction of the nonprotein amino acid Alliin with the enzyme Alliinase (Alliin Lyase, EC 4.4.1.4). A simple and rapid spectrophotometric procedure for determination of allicin and Alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin, is described. NTB reacts with the activated disulfide bond --S(O)-S--; of allicin, forming the mixed-disulfide allylmercapto-NTB, as characterized by NMR. The method can be used for determination of allicin and total thiosulfinates in garlic preparations and garlic-derived products. The method was applied for determination of pure Alliinase activity and for the activity of the enzyme in crude garlic extracts.