User:Adam Mirando/Sandbox 1: Difference between revisions

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===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+ as its final receptor. Following chemical modification (ie fluorodinitrobenzene) or oxidation (ie 4,4'-dithiopridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie 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 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+ from binding while opening a channel accessible for O2 in the now dispersed peptide cluster<ref name="gluarg" />. The oxidation of Cys1316 and 1324 also eliminates the NAD+ 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+. Oxidation of the cysteines changes the structure of this loop, preventing its insertion into the FAD domain<ref name="conver" />.

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+ as its final receptor. Following chemical modification (ie fluorodinitrobenzene) or oxidation (ie 4,4'-dithiopridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie 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 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+ from binding while opening a channel accessible for O2 in the now dispersed peptide cluster<ref name="gluarg" /> (<scene name='User:Adam_Mirando/Sandbox_1/Cluster_cys535_xdh/3'>XDH structure</scene> and <scene name='User:Adam_Mirando/Sandbox_1/Xo_cys_cluster/2'>XO structure</scene>: shown are Cys992 (red), Lys537 (cyan, Cys535 could not be solved), peptide cluster (teal), Asp529 (yellow), 422-432 loop (fuchsia), and FAD (silver). The oxidation of Cys1316 and 1324 also eliminates the NAD+ 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+. Oxidation of the cysteines changes the structure of this loop, preventing its insertion into the FAD domain<ref name="conver" />.

XDH can be irreversibly converted to XO via trypsin digestion after Lys551. The cleavage disrupts an interaction between Phe549 and Arg427 resulting in a shift of the highly charged loop between residues 423 and 433. The shift of this loop displaces Asp429 from its former location in contact with C6 of the flavin while introducing a new contact with Arg426. This new arrangement modifies the electrostatic potential of the flavin as well as prevents the binding of NAD+ to the flavin domain<ref name="structure" />.

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 ===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'-dithiopridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie 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 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" />. The oxidation of 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" />.
+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'-dithiopridine) the oxidase form is favored. Once oxidized, incubation with a reducing agent (ie 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 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" /> (<scene name='User:Adam_Mirando/Sandbox_1/Cluster_cys535_xdh/3'>XDH structure</scene> and <scene name='User:Adam_Mirando/Sandbox_1/Xo_cys_cluster/2'>XO structure</scene>: shown are Cys992 (red), Lys537 (cyan, Cys535 could not be solved), peptide cluster (teal), Asp529 (yellow), 422-432 loop (fuchsia), and FAD (silver). The oxidation of 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" />.
 XDH can be irreversibly converted to XO via trypsin digestion after Lys551. The cleavage disrupts an interaction between Phe549 and Arg427 resulting in a shift of the highly charged loop between residues 423 and 433. The shift of this loop displaces Asp429 from its former location in contact with C6 of the flavin while introducing a new contact with Arg426. This new arrangement modifies the electrostatic potential of the flavin as well as prevents the binding of NAD<sup>+</sup> to the flavin domain<ref name="structure" />.