1iqx: Difference between revisions

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CRYSTAL STRUCTURE OF COBALT-SUBSTITUTED AMINE OXIDASE FROM ARTHROBACTER GLOBIFORMIS

OverviewOverview

The role of the active site Cu(2+) of phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been studied by substitution with other divalent cations, where we were able to remove >99.5% of Cu(2+) from the active site. The enzymes reconstituted with Co(2+) and Ni(2+) (Co- and Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original Cu(2+)-enzyme (Cu-AGAO), but their K(m) values for amine substrate and dioxygen were comparable. X-ray crystal structures of the Co- and Ni-AGAO were solved at 2.0-1.8 A resolution. These structures revealed changes in the metal coordination environment when compared to that of Cu-AGAO. However, the hydrogen-bonding network around the active site involving metal-coordinating and noncoordinating water molecules was preserved. Upon anaerobic mixing of the Cu-, Co-, and Ni-AGAO with amine substrate, the 480 nm absorption band characteristic of the oxidized form of the topaquinone cofactor (TPQ(ox)) disappeared rapidly (< 6 ms), yielding the aminoresorcinol form of the reduced cofactor (TPQ(amr)). In contrast to the substrate-reduced Cu-AGAO, the semiquinone radical (TPQ(sq)) was not detected in Co- and Ni-AGAO. Further, in the latter, TPQ(amr) reacted reversibly with the product aldehyde to form a species with a lambda(max) at around 350 nm that was assigned as the neutral form of the product Schiff base (TPQ(pim)). Introduction of dioxygen to the substrate-reduced Co- and Ni-AGAO resulted in the formation of a TPQ-related intermediate absorbing at around 360 nm, which was assigned to the neutral iminoquinone form of the 2e(-)-oxidized cofactor (TPQ(imq)) and which decayed concomitantly with the generation of TPQ(ox). The rate of TPQ(imq) formation and its subsequent decay in Co- and Ni-AGAO was slow when compared to those of the corresponding reactions in Cu-AGAO. The low catalytic activities of the metal-substituted enzymes are due to the impaired efficiencies of the oxidative half-reaction in the catalytic cycle of amine oxidation. On the basis of these results, we propose that the native Cu(2+) ion has essential roles such as catalyzing the electron transfer between TPQ(amr) and dioxygen, in part by providing a binding site for 1e(-)- and 2e(-)-reduced dioxygen species to be efficiently protonated and released and also preventing the back reaction between the product aldehyde and TPQ(amr).

About this StructureAbout this Structure

1IQX is a Single protein structure of sequence from Arthrobacter globiformis with as ligand. Active as Amine oxidase (copper-containing), with EC number 1.4.3.6 Full crystallographic information is available from OCA.

ReferenceReference

Role of copper ion in bacterial copper amine oxidase: spectroscopic and crystallographic studies of metal-substituted enzymes., Kishishita S, Okajima T, Kim M, Yamaguchi H, Hirota S, Suzuki S, Kuroda S, Tanizawa K, Mure M, J Am Chem Soc. 2003 Jan 29;125(4):1041-55. PMID:12537504

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-[[Image:1iqx.jpg|left|200px]]<br /><applet load="1iqx" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1iqx.jpg|left|200px]]<br /><applet load="1iqx" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1iqx, resolution 2.0&Aring;" />
 '''CRYSTAL STRUCTURE OF COBALT-SUBSTITUTED AMINE OXIDASE FROM ARTHROBACTER GLOBIFORMIS'''<br />
 ==Overview==
-The role of the active site Cu(2+) of phenylethylamine oxidase from, Arthrobacter globiformis (AGAO) has been studied by substitution with, other divalent cations, where we were able to remove &gt;99.5% of Cu(2+) from, the active site. The enzymes reconstituted with Co(2+) and Ni(2+) (Co- and, Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original, Cu(2+)-enzyme (Cu-AGAO), but their K(m) values for amine substrate and, dioxygen were comparable. X-ray crystal structures of the Co- and Ni-AGAO, were solved at 2.0-1.8 A resolution. These structures revealed changes in, the metal coordination environment when compared to that of Cu-AGAO., However, the hydrogen-bonding network around the active site involving, metal-coordinating and noncoordinating water molecules was preserved. Upon, anaerobic mixing of the Cu-, Co-, and Ni-AGAO with amine substrate, the, 480 nm absorption band characteristic of the oxidized form of the, topaquinone cofactor (TPQ(ox)) disappeared rapidly (&lt; 6 ms), yielding the, aminoresorcinol form of the reduced cofactor (TPQ(amr)). In contrast to, the substrate-reduced Cu-AGAO, the semiquinone radical (TPQ(sq)) was not, detected in Co- and Ni-AGAO. Further, in the latter, TPQ(amr) reacted, reversibly with the product aldehyde to form a species with a lambda(max), at around 350 nm that was assigned as the neutral form of the product, Schiff base (TPQ(pim)). Introduction of dioxygen to the substrate-reduced, Co- and Ni-AGAO resulted in the formation of a TPQ-related intermediate, absorbing at around 360 nm, which was assigned to the neutral iminoquinone, form of the 2e(-)-oxidized cofactor (TPQ(imq)) and which decayed, concomitantly with the generation of TPQ(ox). The rate of TPQ(imq), formation and its subsequent decay in Co- and Ni-AGAO was slow when, compared to those of the corresponding reactions in Cu-AGAO. The low, catalytic activities of the metal-substituted enzymes are due to the, impaired efficiencies of the oxidative half-reaction in the catalytic, cycle of amine oxidation. On the basis of these results, we propose that, the native Cu(2+) ion has essential roles such as catalyzing the electron, transfer between TPQ(amr) and dioxygen, in part by providing a binding, site for 1e(-)- and 2e(-)-reduced dioxygen species to be efficiently, protonated and released and also preventing the back reaction between the, product aldehyde and TPQ(amr).
+The role of the active site Cu(2+) of phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been studied by substitution with other divalent cations, where we were able to remove &gt;99.5% of Cu(2+) from the active site. The enzymes reconstituted with Co(2+) and Ni(2+) (Co- and Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original Cu(2+)-enzyme (Cu-AGAO), but their K(m) values for amine substrate and dioxygen were comparable. X-ray crystal structures of the Co- and Ni-AGAO were solved at 2.0-1.8 A resolution. These structures revealed changes in the metal coordination environment when compared to that of Cu-AGAO. However, the hydrogen-bonding network around the active site involving metal-coordinating and noncoordinating water molecules was preserved. Upon anaerobic mixing of the Cu-, Co-, and Ni-AGAO with amine substrate, the 480 nm absorption band characteristic of the oxidized form of the topaquinone cofactor (TPQ(ox)) disappeared rapidly (&lt; 6 ms), yielding the aminoresorcinol form of the reduced cofactor (TPQ(amr)). In contrast to the substrate-reduced Cu-AGAO, the semiquinone radical (TPQ(sq)) was not detected in Co- and Ni-AGAO. Further, in the latter, TPQ(amr) reacted reversibly with the product aldehyde to form a species with a lambda(max) at around 350 nm that was assigned as the neutral form of the product Schiff base (TPQ(pim)). Introduction of dioxygen to the substrate-reduced Co- and Ni-AGAO resulted in the formation of a TPQ-related intermediate absorbing at around 360 nm, which was assigned to the neutral iminoquinone form of the 2e(-)-oxidized cofactor (TPQ(imq)) and which decayed concomitantly with the generation of TPQ(ox). The rate of TPQ(imq) formation and its subsequent decay in Co- and Ni-AGAO was slow when compared to those of the corresponding reactions in Cu-AGAO. The low catalytic activities of the metal-substituted enzymes are due to the impaired efficiencies of the oxidative half-reaction in the catalytic cycle of amine oxidation. On the basis of these results, we propose that the native Cu(2+) ion has essential roles such as catalyzing the electron transfer between TPQ(amr) and dioxygen, in part by providing a binding site for 1e(-)- and 2e(-)-reduced dioxygen species to be efficiently protonated and released and also preventing the back reaction between the product aldehyde and TPQ(amr).
 ==About this Structure==
-IQX is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Arthrobacter_globiformis Arthrobacter globiformis] with CO as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Amine_oxidase_(copper-containing) Amine oxidase (copper-containing)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.4.3.6 1.4.3.6] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1IQX OCA].
+IQX is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Arthrobacter_globiformis Arthrobacter globiformis] with <scene name='pdbligand=CO:'>CO</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Amine_oxidase_(copper-containing) Amine oxidase (copper-containing)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.4.3.6 1.4.3.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1IQX OCA].
 ==Reference==
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 [[Category: tpq]]
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