User:Adam Mirando/Sandbox 1 - Revision history

Adam Mirando at 13:54, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 13:54:51 GMT

←Older revision		Revision as of 13:54, 29 April 2010
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	[http://www.proteopedia.org/wiki/index.php/1dgj 1DGJ] - Aldehyde Oxidoreductase		[http://www.proteopedia.org/wiki/index.php/1dgj 1DGJ] - Aldehyde Oxidoreductase

		+	[http://www.proteopedia.org/wiki/index.php/2cdu 2CDU] - NADPH oxidase from ''Lactobacillus sanfranciscensis''
	== References ==		== References ==

	<references/>		<references/>

Adam Mirando at 13:49, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 13:49:06 GMT

←Older revision		Revision as of 13:49, 29 April 2010
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	'''Xanthine [http://en.wikipedia.org/wiki/Oxidoreductase oxidoreductase]''' (XOR) is an extensively studied metalloflavoprotein from the molybdenum hydroxylase family that is found in a variety of different organisms, ranging from bacteria to eukaryotes <ref>PMID:11848841</ref>. XORs are dimeric enzymes typically around 280 kDa in size with two interconvertible forms: xanthine dehydrogenase (XDH) [1.17.1.4] and xanthine oxidase (XO) [1.17.3.2]. Conversion between the two forms is mediated through the reversible oxidation of several cysteine residues or irreversible [http://en.wikipedia.org/wiki/Trypsin trypsin] truncation <ref name="structure" />. XOR is involved in purine catabolism, catalyzing the [http://en.wikipedia.org/wiki/Redox oxidation] of [http://en.wikipedia.org/wiki/Hypoxanthine hypoxanthine] and [http://en.wikipedia.org/wiki/Xanthine xanthine] to [http://en.wikipedia.org/wiki/Urate urate] through the extraction of two electrons <ref name="gluarg" />. The transport of these electrons is facilitated by the [http://en.wikipedia.org/wiki/Molybdenum molybdenum] of the <scene name='User:Adam_Mirando/Sandbox_1/Mo_pterin_domain/3'>molybdopterin cofactor</scene>, two		'''Xanthine [http://en.wikipedia.org/wiki/Oxidoreductase oxidoreductase]''' (XOR) is an extensively studied metalloflavoprotein from the molybdenum hydroxylase family that is found in a variety of different organisms, ranging from bacteria to eukaryotes <ref>PMID:11848841</ref>. XORs are dimeric enzymes typically around 280 kDa in size with two interconvertible forms: xanthine dehydrogenase (XDH) [1.17.1.4] and xanthine oxidase (XO) [1.17.3.2]. Conversion between the two forms is mediated through the reversible oxidation of several cysteine residues or irreversible [http://en.wikipedia.org/wiki/Trypsin trypsin] truncation <ref name="structure" />. XOR is involved in purine catabolism, catalyzing the [http://en.wikipedia.org/wiki/Redox oxidation] of [http://en.wikipedia.org/wiki/Hypoxanthine hypoxanthine] and [http://en.wikipedia.org/wiki/Xanthine xanthine] to [http://en.wikipedia.org/wiki/Urate urate] through the extraction of two electrons <ref name="gluarg" />. The transport of these electrons is facilitated by the [http://en.wikipedia.org/wiki/Molybdenum molybdenum] of the <scene name='User:Adam_Mirando/Sandbox_1/Mo_pterin_domain/3'>molybdopterin cofactor</scene>, two
	<scene name='User:Adam_Mirando/Sandbox_1/Fes_clusters/2'>iron sulfur centers</scene>, and a bound		<scene name='User:Adam_Mirando/Sandbox_1/Fes_clusters/2'>iron sulfur centers</scene>, and a bound
-	<scene name='User:Adam_Mirando/Sandbox_1/Fad_domain/4'>FAD</scene> coenzyme. In XDH the electrons are then passed preferentially from the reduced [http://en.wikipedia.org/wiki/FAD flavin] to a final [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NAD<sup>+</sup>] acceptor, creating NADH <ref name="thermo" />. Apart from NADH, XDH may also use O<sub>2</sub> as a final electron acceptor. In contrast, conversion to the XO form precludes NAD<sup>+</sup> from binding, permitting only the use of O<sub>2</sub>. The reduction of O<sub>2</sub> produces substantial amounts of H<sub>2</sub>O<sub>2</sub> and superoxide as byproducts <ref name="gluarg" /><ref name="conver">PMID:15878860</ref>. The ~~prodution~~ of these ~~oxidative species has~~ been implicated in the innate immune response <ref>PMID:12967676</ref> and cardiovascular disease, such as [http://en.wikipedia.org/wiki/Atherosclerosis atherosclerosis] <ref>PMID:12958034</ref>, [http://en.wikipedia.org/wiki/Reperfusion_injury ischemia-reperfusion injury], and chronic heart failure <ref>PMID:14694147</ref> <ref>PMID:12105162</ref>.	+	<scene name='User:Adam_Mirando/Sandbox_1/Fad_domain/4'>FAD</scene> coenzyme. In XDH the electrons are then passed preferentially from the reduced [http://en.wikipedia.org/wiki/FAD flavin] to a final [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NAD<sup>+</sup>] acceptor, creating NADH <ref name="thermo" />. Apart from NADH, XDH may also use O<sub>2</sub> as a final electron acceptor. In contrast, conversion to the XO form precludes NAD<sup>+</sup> from binding, permitting only the use of O<sub>2</sub>. The reduction of O<sub>2</sub> produces substantial amounts of H<sub>2</sub>O<sub>2</sub> and superoxide as byproducts <ref name="gluarg" /><ref name="conver">PMID:15878860</ref>. The products of these enzymes have been implicated in the innate immune response as a balancer of redox potential and antioxidant (urate) provider<ref>PMID:12967676</ref> and cardiovascular disease, such as [http://en.wikipedia.org/wiki/Atherosclerosis atherosclerosis] <ref>PMID:12958034</ref>, [http://en.wikipedia.org/wiki/Reperfusion_injury ischemia-reperfusion injury], and chronic heart failure <ref>PMID:14694147</ref> <ref>PMID:12105162</ref>.

Adam Mirando at 13:33, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 13:33:56 GMT

←Older revision		Revision as of 13:33, 29 April 2010
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	[[Image:Xanthine Mechanism.png\|thumb\|center\|1000px\|'''Xanthine oxidation mechanism.''' Adapted from Nishino ''et al.'' ''FEBS Journal.'' (2008) 275, 3278-3289]]		[[Image:Xanthine Mechanism.png\|thumb\|center\|1000px\|'''Xanthine oxidation mechanism.''' Adapted from Nishino ''et al.'' ''FEBS Journal.'' (2008) 275, 3278-3289]]

-	Several mechanisms have been suggested for the oxidation of xanthine to urate by xanthine oxidoreductase. However, a substantial amount of data appears to favor a mechanism in which a deprotonated molybdenum hydroxyl attacks the C8 atom of xanthine. This mechanism begins with the extraction of a proton from the hydroxyl of the molybdenum center by Glu1261 <ref>PMID:15265866</ref>, an event computed to occur readily in the presence of the substrate <ref name="theoretical">PMID:17564439</ref>. The electrons from the deprotonated oxygen are then free to attack the electrophilic C8 atom of the bound <scene name='User:Adam_Mirando/Sandbox_1/Xanthine_in_active_site/1'>xanthine</scene>. The formation of glutamic acid stabilizes this structure through hydrogen bond interactions with the N1 atom <ref>PMID:15148401</ref>. Crystalographic data has also suggested possible stabilizing interactions between Arg880 of the active site and enolate tautomerization at C6 <ref name="SubOri">PMID:19109252</ref>. Bond formation between the substrate and the molybdenum center orients a Mo = S moiety equatorially to the substrate, positioning it favorably for a concomitant hydride transfer from xanthine N7 <ref name="gluarg">PMID:18513323</ref>. Extraction of this hydride produces Mo-SH and reduces the Mo center from Mo VI to Mo IV. This intermediate breaks down through electron transfer from the molybdenum center through the iron-sulfur clusters, known as Fe-S I and Fe-S II to the bound FAD, forming FADH<sub>2</sub>. In this mechanism the Fe-S clusters function as electron sinks, maintaining an oxidized Mo-cofactor and a reduced FADH<sub>2</sub>. The Mo atom serves as a transducer between the two electrons passed from the substrate to the single electron of system of the Fe-S clusters. The transfer of electrons can be monitored through the formation of the paramagnetic transient Mo V <ref>PMID:15134930</ref>. Subsequent reduction of NAD<sup>+</sup> to NADH in the case of xanthine dehydrogenases and O<sub>2</sub> to H<sub>2</sub>O<sub>2</sub> regenerates the oxidized FAD. Other mechanisms involving protonated molybdenum hydroxyls have been proposed with similar calculated activation energies (40 kcal/mol). However, the products in these cases have been computationally determined to be less stable that the reactant complex <ref name="theoretical" />.	+	Several mechanisms have been suggested for the oxidation of xanthine to urate by xanthine oxidoreductase. However, a substantial amount of data appears to favor a mechanism in which a deprotonated molybdenum hydroxyl attacks the C8 atom of xanthine. This mechanism begins with the extraction of a proton from the hydroxyl of the molybdenum center by Glu1261 <ref>PMID:15265866</ref>, an event computed to occur readily in the presence of the substrate <ref name="theoretical">PMID:17564439</ref>. The electrons from the deprotonated oxygen are then free to attack the electrophilic C8 atom of the bound <scene name='User:Adam_Mirando/Sandbox_1/Xanthine_in_active_site/1'>xanthine</scene>. The formation of glutamic acid stabilizes this structure through hydrogen bond interactions with the N1 atom <ref>PMID:15148401</ref>. Crystalographic data has also suggested possible stabilizing interactions between Arg880 of the active site and enolate tautomerization at C6 <ref name="SubOri">PMID:19109252</ref>. Bond formation between the substrate and the molybdenum center orients a Mo = S moiety equatorially to the substrate, positioning it favorably for a concomitant hydride transfer from xanthine N7 <ref name="gluarg">PMID:18513323</ref>. Extraction of this hydride produces Mo-SH and reduces the Mo center from Mo VI to Mo IV. This intermediate breaks down through electron transfer from the molybdenum center through the iron-sulfur clusters, known as Fe-S I and Fe-S II to the bound FAD, forming FADH<sub>2</sub>. In this mechanism the Fe-S clusters function as electron sinks, maintaining an oxidized Mo-cofactor and a reduced FADH<sub>2</sub>. The Mo atom serves as a transducer between the two electrons passed from the substrate to the single electron of system of the Fe-S clusters. The transfer of electrons can be monitored through the formation of the paramagnetic transient Mo V <ref>PMID:15134930</ref>. Subsequent reduction of NAD<sup>+</sup> to NADH in the case of xanthine dehydrogenases and O<sub>2</sub> to H<sub>2</sub>O<sub>2</sub> and superoxide for the oxidase regenerates the oxidized FAD. Other mechanisms involving protonated molybdenum hydroxyls have been proposed with similar calculated activation energies (40 kcal/mol). However, the products in these cases have been computationally determined to be less stable that the reactant complex <ref name="theoretical" />.

	===Hypoxanthine Oxidation Mechanism===		===Hypoxanthine Oxidation Mechanism===

Adam Mirando at 04:26, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 04:26:22 GMT

←Older revision		Revision as of 04:26, 29 April 2010
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	===Xanthine Dehydrogenase/Xanthine Oxidase Conversion===		===Xanthine Dehydrogenase/Xanthine Oxidase Conversion===

-	Xanthine oxidoreductase has two functional forms: xanthine dehydrogenase and xanthine oxidase. This conversion is controlled by the oxidation state of Cys535, Cys992, Cys1316, and Cys1324. When these residues are reduced, the enzyme functions as a dehydrogenase, using NAD<sup>+</sup> as its final receptor. Following chemical modification (ie fluorodinitrobenzene) or oxidation (ie 4,4'-dithiopyridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie [http://en.wikipedia.org/wiki/Dithiothreitol dithiothreitol]) will restore the enzyme to the the dehydrogenase form <ref name="conver" />. Studies involving the C535A/C992R/C1316S triple mutant, however, were unable to convert to the oxidase form. Consequently, crystal structures of this mutant revealed a monomeric structure, in contrast to the normally homodimeric wild type enzyme <ref name="conver" />. The mechanism of this conversion is thought to be the formation of [http://en.wikipedia.org/wiki/Disulfide_bond disulfide bridges] between Cys535 and Cys992 and Cys1317 and Cys1325<ref name="gluarg" /><ref name="conver" />. Crystallographic data have show that residues 535 and 992 are capable of forming disulfide bonds<ref name="gluarg" />. Due to a distance of 15.7 Ǻ between the α-carbons of Cys535 and 992, the formation of a bond would require a substantial conformational change <ref name="structure" />. Further structural analyses reveal a peptide cluster composed of Arg426, Arg334, Trp335, and Phe549 that are tightly packed in the XDH but dispersed following disulfide bond formation. This modification is then transmitted to a loop consisting of residues 422-432 in the FAD domain restricting NAD<sup>+</sup> from binding while opening a channel accessible for O<sub>2</sub> in the now dispersed peptide cluster<ref name="gluarg" />. As a consequence of the loop shifting, Asp429 interactions with the flavin are eliminated while new interactions between the flavin and Arg426 are introduced. These new electrostatic interactions modify the flavin potential in favor of oxidase functiondomain<ref name="structure" /> (<scene name='User:Adam_Mirando/Sandbox_1/Cluster_cys535_xdh/4'>XDH structure</scene> and <scene name='User:Adam_Mirando/Sandbox_1/Xo_cys_cluster/3'>XO structure</scene>: shown are Cys992 (red), Lys537 (cyan, XDH only, Cys535 could not be solved in either structure), peptide cluster (teal), Arg426 (blue), Asp529 (yellow), 422-432 loop (fuchsia), and FAD (silver). Similar effects are also noticed in the case of irreversible conversion to XO by trypsin digestion after Lys551. The cleavage disrupts an interaction between Phe549 and Arg427 resulting in a shift of the 422-432 loop and an exchange of Asp429 for Arg426 <ref name="structure" />. The oxidation ~~Cys1316~~ and ~~1324~~ also eliminates the NAD<sup>+</sup> binding properties of XOR. The two residues are 20.5 Ǻ on the C-terminal tail of the enzyme. The insertion of this tail into the FAD domain appears to be essential for the binding of NAD<sup>+</sup>. Oxidation of the cysteines changes the structure of this loop, preventing its insertion into the FAD domain<ref name="conver" /> (<scene name='User:Adam_Mirando/Sandbox_1/C-terminal_tail_xdh/1'>XDH structure</scene>: shown are Cys1317 (fuchsia), Cys1325 (green), and the C-terminal tail (yellow); C-terminal tail not solved in XO structure).	+	Xanthine oxidoreductase has two functional forms: xanthine dehydrogenase and xanthine oxidase. This conversion is controlled by the oxidation state of Cys535, Cys992, Cys1316, and Cys1324. When these residues are reduced, the enzyme functions as a dehydrogenase, using NAD<sup>+</sup> as its final receptor. Following chemical modification (ie fluorodinitrobenzene) or oxidation (ie 4,4'-dithiopyridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie [http://en.wikipedia.org/wiki/Dithiothreitol dithiothreitol]) will restore the enzyme to the the dehydrogenase form <ref name="conver" />. Studies involving the C535A/C992R/C1316S triple mutant, however, were unable to convert to the oxidase form. Consequently, crystal structures of this mutant revealed a monomeric structure, in contrast to the normally homodimeric wild type enzyme <ref name="conver" />. The mechanism of this conversion is thought to be the formation of [http://en.wikipedia.org/wiki/Disulfide_bond disulfide bridges] between Cys535 and Cys992 and Cys1317 and Cys1325<ref name="gluarg" /><ref name="conver" />. Crystallographic data have show that residues 535 and 992 are capable of forming disulfide bonds<ref name="gluarg" />. Due to a distance of 15.7 Ǻ between the α-carbons of Cys535 and 992, the formation of a bond would require a substantial conformational change <ref name="structure" />. Further structural analyses reveal a peptide cluster composed of Arg426, Arg334, Trp335, and Phe549 that are tightly packed in the XDH but dispersed following disulfide bond formation. This modification is then transmitted to a loop consisting of residues 422-432 in the FAD domain restricting NAD<sup>+</sup> from binding while opening a channel accessible for O<sub>2</sub> in the now dispersed peptide cluster<ref name="gluarg" />. As a consequence of the loop shifting, Asp429 interactions with the flavin are eliminated while new interactions between the flavin and Arg426 are introduced. These new electrostatic interactions modify the flavin potential in favor of oxidase functiondomain<ref name="structure" /> (<scene name='User:Adam_Mirando/Sandbox_1/Cluster_cys535_xdh/4'>XDH structure</scene> and <scene name='User:Adam_Mirando/Sandbox_1/Xo_cys_cluster/3'>XO structure</scene>: shown are Cys992 (red), Lys537 (cyan, XDH only, Cys535 could not be solved in either structure), peptide cluster (teal), Arg426 (blue), Asp529 (yellow), 422-432 loop (fuchsia), and FAD (silver). Similar effects are also noticed in the case of irreversible conversion to XO by trypsin digestion after Lys551. The cleavage disrupts an interaction between Phe549 and Arg427 resulting in a shift of the 422-432 loop and an exchange of Asp429 for Arg426 <ref name="structure" />. The oxidation Cys1317 and 1325 also eliminates the NAD<sup>+</sup> binding properties of XOR. The two residues are 20.5 Ǻ on the C-terminal tail of the enzyme. The insertion of this tail into the FAD domain appears to be essential for the binding of NAD<sup>+</sup>. Oxidation of the cysteines changes the structure of this loop, preventing its insertion into the FAD domain<ref name="conver" /> (<scene name='User:Adam_Mirando/Sandbox_1/C-terminal_tail_xdh/1'>XDH structure</scene>: shown are Cys1317 (fuchsia), Cys1325 (green), and the C-terminal tail (yellow); C-terminal tail not solved in XO structure).

	== Mechanism ==		== Mechanism ==

Adam Mirando at 04:18, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 04:18:32 GMT

←Older revision		Revision as of 04:18, 29 April 2010
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	===Xanthine Dehydrogenase/Xanthine Oxidase Conversion===		===Xanthine Dehydrogenase/Xanthine Oxidase Conversion===

-	Xanthine oxidoreductase has two functional forms: xanthine dehydrogenase and xanthine oxidase. This conversion is controlled by the oxidation state of Cys535, Cys992, Cys1316, and Cys1324. When these residues are reduced, the enzyme functions as a dehydrogenase, using NAD<sup>+</sup> as its final receptor. Following chemical modification (ie fluorodinitrobenzene) or oxidation (ie 4,4'-dithiopyridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie [http://en.wikipedia.org/wiki/Dithiothreitol dithiothreitol]) will restore the enzyme to the the dehydrogenase form <ref name="conver" />. Studies involving the C535A/C992R/C1316S triple mutant, however, were unable to convert to the oxidase form. Consequently, crystal structures of this mutant revealed a monomeric structure, in contrast to the normally homodimeric wild type enzyme <ref name="conver" />. The mechanism of this conversion is thought to be the formation of [http://en.wikipedia.org/wiki/Disulfide_bond disulfide bridges] between Cys535 and Cys992 and ~~Cys1316~~ and ~~Cys1324~~<ref name="gluarg" /><ref name="conver" />. Crystallographic data have show that residues 535 and 992 are capable of forming disulfide bonds<ref name="gluarg" />. Due to a distance of 15.7 Ǻ between the α-carbons of Cys535 and 992, the formation of a bond would require a substantial conformational change <ref name="structure" />. Further structural analyses reveal a peptide cluster composed of Arg426, Arg334, Trp335, and Phe549 that are tightly packed in the XDH but dispersed following disulfide bond formation. This modification is then transmitted to a loop consisting of residues 422-432 in the FAD domain restricting NAD<sup>+</sup> from binding while opening a channel accessible for O<sub>2</sub> in the now dispersed peptide cluster<ref name="gluarg" />. As a consequence of the loop shifting, Asp429 interactions with the flavin are eliminated while new interactions between the flavin and Arg426 are introduced. These new electrostatic interactions modify the flavin potential in favor of oxidase functiondomain<ref name="structure" /> (<scene name='User:Adam_Mirando/Sandbox_1/Cluster_cys535_xdh/4'>XDH structure</scene> and <scene name='User:Adam_Mirando/Sandbox_1/Xo_cys_cluster/3'>XO structure</scene>: shown are Cys992 (red), Lys537 (cyan, XDH only, Cys535 could not be solved in either structure), peptide cluster (teal), Arg426 (blue), Asp529 (yellow), 422-432 loop (fuchsia), and FAD (silver). Similar effects are also noticed in the case of irreversible conversion to XO by trypsin digestion after Lys551. The cleavage disrupts an interaction between Phe549 and Arg427 resulting in a shift of the 422-432 loop and an exchange of Asp429 for Arg426 <ref name="structure" />. The oxidation Cys1316 and 1324 also eliminates the NAD<sup>+</sup> binding properties of XOR. The two residues are 20.5 Ǻ on the C-terminal tail of the enzyme. The insertion of this tail into the FAD domain appears to be essential for the binding of NAD<sup>+</sup>. Oxidation of the cysteines changes the structure of this loop, preventing its insertion into the FAD domain<ref name="conver" /> (<scene name='User:Adam_Mirando/Sandbox_1/C-terminal_tail_xdh/1'>XDH structure</scene>: shown are Cys1317 (fuchsia), Cys1325 (green), and the C-terminal tail (yellow); C-terminal tail not solved in XO structure).	+	Xanthine oxidoreductase has two functional forms: xanthine dehydrogenase and xanthine oxidase. This conversion is controlled by the oxidation state of Cys535, Cys992, Cys1316, and Cys1324. When these residues are reduced, the enzyme functions as a dehydrogenase, using NAD<sup>+</sup> as its final receptor. Following chemical modification (ie fluorodinitrobenzene) or oxidation (ie 4,4'-dithiopyridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie [http://en.wikipedia.org/wiki/Dithiothreitol dithiothreitol]) will restore the enzyme to the the dehydrogenase form <ref name="conver" />. Studies involving the C535A/C992R/C1316S triple mutant, however, were unable to convert to the oxidase form. Consequently, crystal structures of this mutant revealed a monomeric structure, in contrast to the normally homodimeric wild type enzyme <ref name="conver" />. The mechanism of this conversion is thought to be the formation of [http://en.wikipedia.org/wiki/Disulfide_bond disulfide bridges] between Cys535 and Cys992 and Cys1317 and Cys1325<ref name="gluarg" /><ref name="conver" />. Crystallographic data have show that residues 535 and 992 are capable of forming disulfide bonds<ref name="gluarg" />. Due to a distance of 15.7 Ǻ between the α-carbons of Cys535 and 992, the formation of a bond would require a substantial conformational change <ref name="structure" />. Further structural analyses reveal a peptide cluster composed of Arg426, Arg334, Trp335, and Phe549 that are tightly packed in the XDH but dispersed following disulfide bond formation. This modification is then transmitted to a loop consisting of residues 422-432 in the FAD domain restricting NAD<sup>+</sup> from binding while opening a channel accessible for O<sub>2</sub> in the now dispersed peptide cluster<ref name="gluarg" />. As a consequence of the loop shifting, Asp429 interactions with the flavin are eliminated while new interactions between the flavin and Arg426 are introduced. These new electrostatic interactions modify the flavin potential in favor of oxidase functiondomain<ref name="structure" /> (<scene name='User:Adam_Mirando/Sandbox_1/Cluster_cys535_xdh/4'>XDH structure</scene> and <scene name='User:Adam_Mirando/Sandbox_1/Xo_cys_cluster/3'>XO structure</scene>: shown are Cys992 (red), Lys537 (cyan, XDH only, Cys535 could not be solved in either structure), peptide cluster (teal), Arg426 (blue), Asp529 (yellow), 422-432 loop (fuchsia), and FAD (silver). Similar effects are also noticed in the case of irreversible conversion to XO by trypsin digestion after Lys551. The cleavage disrupts an interaction between Phe549 and Arg427 resulting in a shift of the 422-432 loop and an exchange of Asp429 for Arg426 <ref name="structure" />. The oxidation Cys1316 and 1324 also eliminates the NAD<sup>+</sup> binding properties of XOR. The two residues are 20.5 Ǻ on the C-terminal tail of the enzyme. The insertion of this tail into the FAD domain appears to be essential for the binding of NAD<sup>+</sup>. Oxidation of the cysteines changes the structure of this loop, preventing its insertion into the FAD domain<ref name="conver" /> (<scene name='User:Adam_Mirando/Sandbox_1/C-terminal_tail_xdh/1'>XDH structure</scene>: shown are Cys1317 (fuchsia), Cys1325 (green), and the C-terminal tail (yellow); C-terminal tail not solved in XO structure).

	== Mechanism ==		== Mechanism ==

Adam Mirando at 04:15, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 04:15:54 GMT

←Older revision		Revision as of 04:15, 29 April 2010
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	<applet load='1FO4' size='300' frame='true' align='right' caption='Bovine Milk Xanthine Dehydrogenase' />		<applet load='1FO4' size='300' frame='true' align='right' caption='Bovine Milk Xanthine Dehydrogenase' />
-	Bovine xanthine dehydrogenase has the overall dimensions 155 Ǻ x 90 Ǻ x 70 Ǻ in its dimeric form and 100 Ǻ x 90 Ǻ x 70 Ǻ for the individual protomers. The overall structure of the enzyme can be categorized into three key domains. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>N-terminal domain</scene> (green, residues 1- 165) harbors the two [http://en.wikipedia.org/wiki/Iron-sulfur_cluster Fe-S clusters] (shown in yellow). The second, <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>middle domain</scene> (blue, residues 226-531) contains the FAD domain (shown in orange) and the NAD<sup>+</sup>/O<sub>2</sub> binding site. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>C-terminal domain</scene> (purple, residues 590-1332) contains the [http://en.wikipedia.org/wiki/Molybdopterin molybdopterin] cofactor (shown in red) and is positioned close to the interface between the other two domains. This structure allows for interactions between co-factors of the same ~~[http://en.wikipedia.org/wiki/Protomer~~ protomer]. However, closest distance of co-factors between the two subunits is greater than 50 Ǻ, suggesting that the two subunits do not cross communicate <ref name="structure">PMID:11005854</ref>.	+	Bovine xanthine dehydrogenase has the overall dimensions 155 Ǻ x 90 Ǻ x 70 Ǻ in its dimeric form and 100 Ǻ x 90 Ǻ x 70 Ǻ for the individual [http://en.wikipedia.org/wiki/Protomer protomers]. The overall structure of the enzyme can be categorized into three key domains. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>N-terminal domain</scene> (green, residues 1- 165) harbors the two [http://en.wikipedia.org/wiki/Iron-sulfur_cluster Fe-S clusters] (shown in yellow). The second, <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>middle domain</scene> (blue, residues 226-531) contains the FAD domain (shown in orange) and the NAD<sup>+</sup>/O<sub>2</sub> binding site. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>C-terminal domain</scene> (purple, residues 590-1332) contains the [http://en.wikipedia.org/wiki/Molybdopterin molybdopterin] cofactor (shown in red) and is positioned close to the interface between the other two domains. This structure allows for interactions between co-factors of the same protomer. However, closest distance of co-factors between the two subunits is greater than 50 Ǻ, suggesting that the two subunits do not cross communicate <ref name="structure">PMID:11005854</ref>.

Adam Mirando at 04:10, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 04:10:19 GMT

←Older revision		Revision as of 04:10, 29 April 2010
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-	'''Xanthine [http://en.wikipedia.org/wiki/Oxidoreductase oxidoreductase]''' (XOR) is an extensively studied metalloflavoprotein that is found in a variety of different organisms, ranging from bacteria to eukaryotes <ref>PMID:11848841</ref>. XORs are dimeric enzymes typically around 280 kDa in size with two interconvertible forms: xanthine dehydrogenase (XDH) [1.17.1.4] and xanthine oxidase (XO) [1.17.3.2]. Conversion between the two forms is mediated through the reversible oxidation of several cysteine residues or irreversible [http://en.wikipedia.org/wiki/Trypsin trypsin] truncation <ref name="structure" />. XOR is involved in purine catabolism, catalyzing the [http://en.wikipedia.org/wiki/Redox oxidation] of [http://en.wikipedia.org/wiki/Hypoxanthine hypoxanthine] and [http://en.wikipedia.org/wiki/Xanthine xanthine] to [http://en.wikipedia.org/wiki/Urate urate] through the extraction of two electrons <ref name="gluarg" />. The transport of these electrons is facilitated by the [http://en.wikipedia.org/wiki/Molybdenum molybdenum] of the <scene name='User:Adam_Mirando/Sandbox_1/Mo_pterin_domain/3'>molybdopterin cofactor</scene>, two	+	'''Xanthine [http://en.wikipedia.org/wiki/Oxidoreductase oxidoreductase]''' (XOR) is an extensively studied metalloflavoprotein from the molybdenum hydroxylase family that is found in a variety of different organisms, ranging from bacteria to eukaryotes <ref>PMID:11848841</ref>. XORs are dimeric enzymes typically around 280 kDa in size with two interconvertible forms: xanthine dehydrogenase (XDH) [1.17.1.4] and xanthine oxidase (XO) [1.17.3.2]. Conversion between the two forms is mediated through the reversible oxidation of several cysteine residues or irreversible [http://en.wikipedia.org/wiki/Trypsin trypsin] truncation <ref name="structure" />. XOR is involved in purine catabolism, catalyzing the [http://en.wikipedia.org/wiki/Redox oxidation] of [http://en.wikipedia.org/wiki/Hypoxanthine hypoxanthine] and [http://en.wikipedia.org/wiki/Xanthine xanthine] to [http://en.wikipedia.org/wiki/Urate urate] through the extraction of two electrons <ref name="gluarg" />. The transport of these electrons is facilitated by the [http://en.wikipedia.org/wiki/Molybdenum molybdenum] of the <scene name='User:Adam_Mirando/Sandbox_1/Mo_pterin_domain/3'>molybdopterin cofactor</scene>, two
	<scene name='User:Adam_Mirando/Sandbox_1/Fes_clusters/2'>iron sulfur centers</scene>, and a bound		<scene name='User:Adam_Mirando/Sandbox_1/Fes_clusters/2'>iron sulfur centers</scene>, and a bound
	<scene name='User:Adam_Mirando/Sandbox_1/Fad_domain/4'>FAD</scene> coenzyme. In XDH the electrons are then passed preferentially from the reduced [http://en.wikipedia.org/wiki/FAD flavin] to a final [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NAD<sup>+</sup>] acceptor, creating NADH <ref name="thermo" />. Apart from NADH, XDH may also use O<sub>2</sub> as a final electron acceptor. In contrast, conversion to the XO form precludes NAD<sup>+</sup> from binding, permitting only the use of O<sub>2</sub>. The reduction of O<sub>2</sub> produces substantial amounts of H<sub>2</sub>O<sub>2</sub> and superoxide as byproducts <ref name="gluarg" /><ref name="conver">PMID:15878860</ref>. The prodution of these oxidative species has been implicated in the innate immune response <ref>PMID:12967676</ref> and cardiovascular disease, such as [http://en.wikipedia.org/wiki/Atherosclerosis atherosclerosis] <ref>PMID:12958034</ref>, [http://en.wikipedia.org/wiki/Reperfusion_injury ischemia-reperfusion injury], and chronic heart failure <ref>PMID:14694147</ref> <ref>PMID:12105162</ref>.		<scene name='User:Adam_Mirando/Sandbox_1/Fad_domain/4'>FAD</scene> coenzyme. In XDH the electrons are then passed preferentially from the reduced [http://en.wikipedia.org/wiki/FAD flavin] to a final [http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide NAD<sup>+</sup>] acceptor, creating NADH <ref name="thermo" />. Apart from NADH, XDH may also use O<sub>2</sub> as a final electron acceptor. In contrast, conversion to the XO form precludes NAD<sup>+</sup> from binding, permitting only the use of O<sub>2</sub>. The reduction of O<sub>2</sub> produces substantial amounts of H<sub>2</sub>O<sub>2</sub> and superoxide as byproducts <ref name="gluarg" /><ref name="conver">PMID:15878860</ref>. The prodution of these oxidative species has been implicated in the innate immune response <ref>PMID:12967676</ref> and cardiovascular disease, such as [http://en.wikipedia.org/wiki/Atherosclerosis atherosclerosis] <ref>PMID:12958034</ref>, [http://en.wikipedia.org/wiki/Reperfusion_injury ischemia-reperfusion injury], and chronic heart failure <ref>PMID:14694147</ref> <ref>PMID:12105162</ref>.

Adam Mirando at 03:33, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 03:33:06 GMT

←Older revision		Revision as of 03:33, 29 April 2010
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-	<applet load='3BDJ' size='300' frame='true' align='right' caption='Bovine Milk Xanthine Dehydrogenase ~~with Bound Alloxanthine~~' />	+	<applet load='3BDJ' size='300' frame='true' align='right' caption='Bovine Milk Xanthine Dehydrogenase' />
	== Overview ==		== Overview ==

Adam Mirando at 03:30, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 03:30:26 GMT

←Older revision		Revision as of 03:30, 29 April 2010
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	== Structure ==		== Structure ==

-	<applet load='1FO4' size='300' frame='true' align='right' caption='~~Insert caption here~~' />	+	<applet load='1FO4' size='300' frame='true' align='right' caption='Bovine Milk Xanthine Dehydrogenase' />
	Bovine xanthine dehydrogenase has the overall dimensions 155 Ǻ x 90 Ǻ x 70 Ǻ in its dimeric form and 100 Ǻ x 90 Ǻ x 70 Ǻ for the individual protomers. The overall structure of the enzyme can be categorized into three key domains. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>N-terminal domain</scene> (green, residues 1- 165) harbors the two [http://en.wikipedia.org/wiki/Iron-sulfur_cluster Fe-S clusters] (shown in yellow). The second, <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>middle domain</scene> (blue, residues 226-531) contains the FAD domain (shown in orange) and the NAD<sup>+</sup>/O<sub>2</sub> binding site. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>C-terminal domain</scene> (purple, residues 590-1332) contains the [http://en.wikipedia.org/wiki/Molybdopterin molybdopterin] cofactor (shown in red) and is positioned close to the interface between the other two domains. This structure allows for interactions between co-factors of the same [http://en.wikipedia.org/wiki/Protomer protomer]. However, closest distance of co-factors between the two subunits is greater than 50 Ǻ, suggesting that the two subunits do not cross communicate <ref name="structure">PMID:11005854</ref>.		Bovine xanthine dehydrogenase has the overall dimensions 155 Ǻ x 90 Ǻ x 70 Ǻ in its dimeric form and 100 Ǻ x 90 Ǻ x 70 Ǻ for the individual protomers. The overall structure of the enzyme can be categorized into three key domains. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>N-terminal domain</scene> (green, residues 1- 165) harbors the two [http://en.wikipedia.org/wiki/Iron-sulfur_cluster Fe-S clusters] (shown in yellow). The second, <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>middle domain</scene> (blue, residues 226-531) contains the FAD domain (shown in orange) and the NAD<sup>+</sup>/O<sub>2</sub> binding site. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>C-terminal domain</scene> (purple, residues 590-1332) contains the [http://en.wikipedia.org/wiki/Molybdopterin molybdopterin] cofactor (shown in red) and is positioned close to the interface between the other two domains. This structure allows for interactions between co-factors of the same [http://en.wikipedia.org/wiki/Protomer protomer]. However, closest distance of co-factors between the two subunits is greater than 50 Ǻ, suggesting that the two subunits do not cross communicate <ref name="structure">PMID:11005854</ref>.

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	===Substrate Binding and Intermediate Stabilization===		===Substrate Binding and Intermediate Stabilization===

-	<applet load='3BDJ' size='300' frame='true' align='right' caption='~~Insert caption here~~' />	+	<applet load='3BDJ' size='300' frame='true' align='right' caption='Bovine Milk Xanthine Dehydrogenase' />
	Several active site residues have been implicated in the substrate binding and catalytic roles of xanthine oxidoreductase. The molybdenum center is accessible only through a 5 Ǻ x 3 Ǻ channel that is 5 Ǻ deep. The active site pocket itself is lined by several <scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/1'>conserved residues</scene>: Glu802, Leu873, Arg880, Phe914, Phe1009, and Glu1261 <ref name="SubOri" /><ref name="sequence">11796116</ref>. The several [http://en.wikipedia.org/wiki/Hydrophobic hydrophobic] residues, Leu873, Phe914, and Phe1009, serve to form the active site pocket<ref name="SubOri" /><ref name="sequence" />. The conserved Glu1261 <ref name="gluarg" /> is located near the molybdopterin co-factor (see “Xanthine Oxidation Mechanism” above”) and acts as a general base to extract a proton from the hydroxyl group of the molybdenum center. The complete loss of enzymatic activity following mutations of this residue confirms its important role in catalysis <ref name="hypoxanthine" />. Arg880 and Glu802 are thought to be involved in the mechanism through the formation of stabilizing interactions with the reaction intermediates <ref name="gluarg" /><ref name="SubOri" /><ref name="hypoxanthine" />.		Several active site residues have been implicated in the substrate binding and catalytic roles of xanthine oxidoreductase. The molybdenum center is accessible only through a 5 Ǻ x 3 Ǻ channel that is 5 Ǻ deep. The active site pocket itself is lined by several <scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/1'>conserved residues</scene>: Glu802, Leu873, Arg880, Phe914, Phe1009, and Glu1261 <ref name="SubOri" /><ref name="sequence">11796116</ref>. The several [http://en.wikipedia.org/wiki/Hydrophobic hydrophobic] residues, Leu873, Phe914, and Phe1009, serve to form the active site pocket<ref name="SubOri" /><ref name="sequence" />. The conserved Glu1261 <ref name="gluarg" /> is located near the molybdopterin co-factor (see “Xanthine Oxidation Mechanism” above”) and acts as a general base to extract a proton from the hydroxyl group of the molybdenum center. The complete loss of enzymatic activity following mutations of this residue confirms its important role in catalysis <ref name="hypoxanthine" />. Arg880 and Glu802 are thought to be involved in the mechanism through the formation of stabilizing interactions with the reaction intermediates <ref name="gluarg" /><ref name="SubOri" /><ref name="hypoxanthine" />.

Adam Mirando at 02:12, 29 April 2010

Adam Mirando — Thu, 29 Apr 2010 02:12:34 GMT

←Older revision		Revision as of 02:12, 29 April 2010
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	== Links ==		== Links ==

-	[1FO4] - Bovine xanthine dehydrogenase	+	[http://www.proteopedia.org/wiki/index.php/2ckj 2CKJ] - Human milk XOR
		+
		+	[http://www.proteopedia.org/wiki/index.php/1fo4 1FO4] - Bovine xanthine dehydrogenase
		+
	[http://www.proteopedia.org/wiki/index.php/1fiq 1FIQ] - Bovine xanthine oxidase		[http://www.proteopedia.org/wiki/index.php/1fiq 1FIQ] - Bovine xanthine oxidase
		+
		+	[http://www.proteopedia.org/wiki/index.php/1wyg 1WYG] - Rat XDH triple cysteine mutant(C535A, C992R and C1324S)
		+
		+	[http://www.proteopedia.org/wiki/index.php/2we7 2WE7] - XDH from ''Mycobacterium Smegmatis''
		+
	[http://www.proteopedia.org/wiki/index.php/1rm6 1RM6] - 4-hydroxybenzoyl-CoA reductase (xanthine oxidase family)		[http://www.proteopedia.org/wiki/index.php/1rm6 1RM6] - 4-hydroxybenzoyl-CoA reductase (xanthine oxidase family)
		+
	[http://www.proteopedia.org/wiki/index.php/1dgj 1DGJ] - Aldehyde Oxidoreductase		[http://www.proteopedia.org/wiki/index.php/1dgj 1DGJ] - Aldehyde Oxidoreductase