User:Adam Mirando/Sandbox 1: Difference between revisions

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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 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 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/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+] acceptor, creating NADH <ref name="thermo" />. Apart from NADH, XDH may also use O2 as a final electron acceptor. In contrast, conversion to the XO form precludes NAD+ from binding, permitting only the use of O2. Consequently, the reduction of O2 produces substantial amounts of H2O2 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>.

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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 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+/O2 binding site. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>C-terminal domain</scene> (purple, residues 590-1332) contains the Mo-pterin co-factor (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>.

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+/O2 binding site. The <scene name='User:Adam_Mirando/Sandbox_1/Xdh_domain/3'>C-terminal domain</scene> (purple, residues 590-1332) contains the Mo-pterin co-factor (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>.

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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 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" />.

The interactions of Arg880 and Glu802 appear to vary with analogous substrates and inhibitors, leading to the development of two different modes of substrate binding in the case of the xanthine oxidation mechanism. One mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Alloxanthine_bound/3'>scheme 1</scene>) suggests that Glu802 forms hydrogen bond interactions with the C6 carbonyl and N7 of xanthine while Arg880 forms hydrogen bonds with the carbonyl of C2 <ref name="gluarg" />. The second mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/3'>scheme 2</scene>) suggests an inverted orientation of the substrate allowing for hydrogen bond interactions between Arg880 and the C6 carbonyl of xanthine. In addition to the facilitation of substrate binding, this orientation would allow for a more catalytic role of Arg880, in that it would allow for stabilization of the enolate intermediate <ref name="gluarg" /><ref name="SubOri" />. Crystal structures involving the [http://en.wikipedia.org/wiki/Alloxanthine alloxanthine] inhibitor are indicative of scheme 1. However, crystal structures using the inactive, desulfinated enzyme in the presence of xanthine are supportive of scheme 2 <ref name="SubOri" />. In further support of scheme 2, Arg880 to methionine (R880M) mutants exhibit a complete loss xanthine activity. In contrast, Glu803 to valine (E802V) mutants show only a reduction in activity corresponding to an 8-fold increase in Km and a 92.6% reduction in kcat compared to the wild type enzyme <ref name="hypoxanthine" />. The hypoxanthine oxidation mechanism, however, shows an opposite response to the mutations. The E802V mutants exhibit a complete loss of activity while R880M show only a reduction (12-fold increase in Km and a 98.9% reduction in kcat). As such, a hypothetical binding arrangement in which Glu802 forms hydrogen bond interactions with the C6 carbonyl and N1 of hypoxanthine has been suggested <ref name="hypoxanthine" />.

===Xanthine Oxidation Mechanism===

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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 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 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/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>. Consequently, 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>.
@@ Line 11: / Line 11: @@
 <applet load='1FO4' size='300' frame='true' align='right' caption='Insert caption here' />
-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 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 Mo-pterin co-factor (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>.
+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 Mo-pterin co-factor (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>.
@@ Line 24: / Line 24: @@
 <applet load='3BDJ' size='300' frame='true' align='right' caption='Insert caption here' />
 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 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" />.
-	The interactions of Arg880 and Glu802 appear to vary with analogous substrates and inhibitors, leading to the development of two different modes of substrate binding in the case of the xanthine oxidation mechanism. One mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Alloxanthine_bound/3'>scheme 1</scene>) suggests that Glu802 forms hydrogen bond interactions with the C6 carbonyl and N7 of xanthine while Arg880 forms hydrogen bonds with the carbonyl of C2 <ref name="gluarg" />. The second mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/3'>scheme 2</scene>) suggests an inverted orientation of the substrate allowing for hydrogen bond interactions between Arg880 and the C6 carbonyl of xanthine. In addition to the facilitation of substrate binding, this orientation would allow for a more catalytic role of Arg880, in that it would allow for stabilization of the enolate intermediate <ref name="gluarg" /><ref name="SubOri" />. Crystal structures involving the [http://en.wikipedia.org/wiki/Alloxanthine alloxanthine] inhibitor are indicative of scheme 1. However, crystal structures using the inactive, desulfinated enzyme in the presence of xanthine are supportive of scheme 2 <ref name="SubOri" />. In further support of scheme 2, Arg880 to methionine (R880M) mutants exhibit a complete loss xanthine activity. In contrast, Glu803 to valine (E802V) mutants show only a reduction in activity corresponding to an 8-fold increase in K<sub>m</sub> and a 92.6% reduction in k<sub>cat</sub> compared to the wild type enzyme <ref name="hypoxanthine" />.  The hypoxanthine oxidation mechanism, however, shows an opposite response to the mutations. The E802V mutants exhibit a complete loss of activity while R880M show only a reduction (12-fold increase in K<sub>m</sub> and a 98.9% reduction in k<sub>cat</sub>). As such, a hypothetical binding arrangement in which Glu802 forms hydrogen bond interactions with the C6 carbonyl and N1 of hypoxanthine has been suggested <ref name="hypoxanthine" />.
+The interactions of Arg880 and Glu802 appear to vary with analogous substrates and inhibitors, leading to the development of two different modes of substrate binding in the case of the xanthine oxidation mechanism. One mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Alloxanthine_bound/3'>scheme 1</scene>) suggests that Glu802 forms hydrogen bond interactions with the C6 carbonyl and N7 of xanthine while Arg880 forms hydrogen bonds with the carbonyl of C2 <ref name="gluarg" />. The second mechanism (<scene name='User:Adam_Mirando/Sandbox_1/Active_site_residues/3'>scheme 2</scene>) suggests an inverted orientation of the substrate allowing for hydrogen bond interactions between Arg880 and the C6 carbonyl of xanthine. In addition to the facilitation of substrate binding, this orientation would allow for a more catalytic role of Arg880, in that it would allow for stabilization of the enolate intermediate <ref name="gluarg" /><ref name="SubOri" />. Crystal structures involving the [http://en.wikipedia.org/wiki/Alloxanthine alloxanthine] inhibitor are indicative of scheme 1. However, crystal structures using the inactive, desulfinated enzyme in the presence of xanthine are supportive of scheme 2 <ref name="SubOri" />. In further support of scheme 2, Arg880 to methionine (R880M) mutants exhibit a complete loss xanthine activity. In contrast, Glu803 to valine (E802V) mutants show only a reduction in activity corresponding to an 8-fold increase in K<sub>m</sub> and a 92.6% reduction in k<sub>cat</sub> compared to the wild type enzyme <ref name="hypoxanthine" />.  The hypoxanthine oxidation mechanism, however, shows an opposite response to the mutations. The E802V mutants exhibit a complete loss of activity while R880M show only a reduction (12-fold increase in K<sub>m</sub> and a 98.9% reduction in k<sub>cat</sub>). As such, a hypothetical binding arrangement in which Glu802 forms hydrogen bond interactions with the C6 carbonyl and N1 of hypoxanthine has been suggested <ref name="hypoxanthine" />.
 ===Xanthine Oxidation Mechanism===