Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis

Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the rea...
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Autor*in:	Lindsay McGregor [verfasserIn] Tamás Földes [verfasserIn] Soi Bui [verfasserIn] Martine Moulin [verfasserIn] Nicolas Coquelle [verfasserIn] Matthew P. Blakeley [verfasserIn] Edina Rosta [verfasserIn] Roberto A. Steiner [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2021

Schlagwörter:	neutron/x-ray diffraction urate oxidase protein perdeuteration cofactor-independent oxidase biomolecular simulations

Übergeordnetes Werk:	In: IUCrJ - International Union of Crystallography, 2014, 8(2021), 1, Seite 46-59
Übergeordnetes Werk:	volume:8 ; year:2021 ; number:1 ; pages:46-59

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1107/S2052252520013615

Katalog-ID:	DOAJ087055597

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520			\|a Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.
650		4	\|a neutron/x-ray diffraction
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allfields	10.1107/S2052252520013615 doi (DE-627)DOAJ087055597 (DE-599)DOAJ9ffaa2f1f5434ce4923d9040711c415a DE-627 ger DE-627 rakwb eng QD901-999 Lindsay McGregor verfasserin aut Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis. neutron/x-ray diffraction urate oxidase protein perdeuteration cofactor-independent oxidase biomolecular simulations Crystallography Tamás Földes verfasserin aut Soi Bui verfasserin aut Martine Moulin verfasserin aut Nicolas Coquelle verfasserin aut Matthew P. Blakeley verfasserin aut Edina Rosta verfasserin aut Roberto A. Steiner verfasserin aut In IUCrJ International Union of Crystallography, 2014 8(2021), 1, Seite 46-59 (DE-627)777782758 (DE-600)2754953-7 20522525 nnns volume:8 year:2021 number:1 pages:46-59 https://doi.org/10.1107/S2052252520013615 kostenfrei https://doaj.org/article/9ffaa2f1f5434ce4923d9040711c415a kostenfrei http://scripts.iucr.org/cgi-bin/paper?S2052252520013615 kostenfrei https://doaj.org/toc/2052-2525 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2021 1 46-59
spelling	10.1107/S2052252520013615 doi (DE-627)DOAJ087055597 (DE-599)DOAJ9ffaa2f1f5434ce4923d9040711c415a DE-627 ger DE-627 rakwb eng QD901-999 Lindsay McGregor verfasserin aut Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis. neutron/x-ray diffraction urate oxidase protein perdeuteration cofactor-independent oxidase biomolecular simulations Crystallography Tamás Földes verfasserin aut Soi Bui verfasserin aut Martine Moulin verfasserin aut Nicolas Coquelle verfasserin aut Matthew P. Blakeley verfasserin aut Edina Rosta verfasserin aut Roberto A. Steiner verfasserin aut In IUCrJ International Union of Crystallography, 2014 8(2021), 1, Seite 46-59 (DE-627)777782758 (DE-600)2754953-7 20522525 nnns volume:8 year:2021 number:1 pages:46-59 https://doi.org/10.1107/S2052252520013615 kostenfrei https://doaj.org/article/9ffaa2f1f5434ce4923d9040711c415a kostenfrei http://scripts.iucr.org/cgi-bin/paper?S2052252520013615 kostenfrei https://doaj.org/toc/2052-2525 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2021 1 46-59
allfields_unstemmed	10.1107/S2052252520013615 doi (DE-627)DOAJ087055597 (DE-599)DOAJ9ffaa2f1f5434ce4923d9040711c415a DE-627 ger DE-627 rakwb eng QD901-999 Lindsay McGregor verfasserin aut Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis. neutron/x-ray diffraction urate oxidase protein perdeuteration cofactor-independent oxidase biomolecular simulations Crystallography Tamás Földes verfasserin aut Soi Bui verfasserin aut Martine Moulin verfasserin aut Nicolas Coquelle verfasserin aut Matthew P. Blakeley verfasserin aut Edina Rosta verfasserin aut Roberto A. Steiner verfasserin aut In IUCrJ International Union of Crystallography, 2014 8(2021), 1, Seite 46-59 (DE-627)777782758 (DE-600)2754953-7 20522525 nnns volume:8 year:2021 number:1 pages:46-59 https://doi.org/10.1107/S2052252520013615 kostenfrei https://doaj.org/article/9ffaa2f1f5434ce4923d9040711c415a kostenfrei http://scripts.iucr.org/cgi-bin/paper?S2052252520013615 kostenfrei https://doaj.org/toc/2052-2525 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2021 1 46-59
allfieldsGer	10.1107/S2052252520013615 doi (DE-627)DOAJ087055597 (DE-599)DOAJ9ffaa2f1f5434ce4923d9040711c415a DE-627 ger DE-627 rakwb eng QD901-999 Lindsay McGregor verfasserin aut Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis. neutron/x-ray diffraction urate oxidase protein perdeuteration cofactor-independent oxidase biomolecular simulations Crystallography Tamás Földes verfasserin aut Soi Bui verfasserin aut Martine Moulin verfasserin aut Nicolas Coquelle verfasserin aut Matthew P. Blakeley verfasserin aut Edina Rosta verfasserin aut Roberto A. Steiner verfasserin aut In IUCrJ International Union of Crystallography, 2014 8(2021), 1, Seite 46-59 (DE-627)777782758 (DE-600)2754953-7 20522525 nnns volume:8 year:2021 number:1 pages:46-59 https://doi.org/10.1107/S2052252520013615 kostenfrei https://doaj.org/article/9ffaa2f1f5434ce4923d9040711c415a kostenfrei http://scripts.iucr.org/cgi-bin/paper?S2052252520013615 kostenfrei https://doaj.org/toc/2052-2525 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2021 1 46-59
allfieldsSound	10.1107/S2052252520013615 doi (DE-627)DOAJ087055597 (DE-599)DOAJ9ffaa2f1f5434ce4923d9040711c415a DE-627 ger DE-627 rakwb eng QD901-999 Lindsay McGregor verfasserin aut Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis. neutron/x-ray diffraction urate oxidase protein perdeuteration cofactor-independent oxidase biomolecular simulations Crystallography Tamás Földes verfasserin aut Soi Bui verfasserin aut Martine Moulin verfasserin aut Nicolas Coquelle verfasserin aut Matthew P. Blakeley verfasserin aut Edina Rosta verfasserin aut Roberto A. Steiner verfasserin aut In IUCrJ International Union of Crystallography, 2014 8(2021), 1, Seite 46-59 (DE-627)777782758 (DE-600)2754953-7 20522525 nnns volume:8 year:2021 number:1 pages:46-59 https://doi.org/10.1107/S2052252520013615 kostenfrei https://doaj.org/article/9ffaa2f1f5434ce4923d9040711c415a kostenfrei http://scripts.iucr.org/cgi-bin/paper?S2052252520013615 kostenfrei https://doaj.org/toc/2052-2525 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2021 1 46-59
language	English
source	In IUCrJ 8(2021), 1, Seite 46-59 volume:8 year:2021 number:1 pages:46-59
sourceStr	In IUCrJ 8(2021), 1, Seite 46-59 volume:8 year:2021 number:1 pages:46-59
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topic_facet	neutron/x-ray diffraction urate oxidase protein perdeuteration cofactor-independent oxidase biomolecular simulations Crystallography
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authorswithroles_txt_mv	Lindsay McGregor @@aut@@ Tamás Földes @@aut@@ Soi Bui @@aut@@ Martine Moulin @@aut@@ Nicolas Coquelle @@aut@@ Matthew P. Blakeley @@aut@@ Edina Rosta @@aut@@ Roberto A. Steiner @@aut@@
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callnumber-first	Q - Science
author	Lindsay McGregor
spellingShingle	Lindsay McGregor misc QD901-999 misc neutron/x-ray diffraction misc urate oxidase misc protein perdeuteration misc cofactor-independent oxidase misc biomolecular simulations misc Crystallography Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis
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topic_title	QD901-999 Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis neutron/x-ray diffraction urate oxidase protein perdeuteration cofactor-independent oxidase biomolecular simulations
topic	misc QD901-999 misc neutron/x-ray diffraction misc urate oxidase misc protein perdeuteration misc cofactor-independent oxidase misc biomolecular simulations misc Crystallography
topic_unstemmed	misc QD901-999 misc neutron/x-ray diffraction misc urate oxidase misc protein perdeuteration misc cofactor-independent oxidase misc biomolecular simulations misc Crystallography
topic_browse	misc QD901-999 misc neutron/x-ray diffraction misc urate oxidase misc protein perdeuteration misc cofactor-independent oxidase misc biomolecular simulations misc Crystallography
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title	Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis
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title_full	Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis
author_sort	Lindsay McGregor
journal	IUCrJ
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author_browse	Lindsay McGregor Tamás Földes Soi Bui Martine Moulin Nicolas Coquelle Matthew P. Blakeley Edina Rosta Roberto A. Steiner
container_volume	8
class	QD901-999
format_se	Elektronische Aufsätze
author-letter	Lindsay McGregor
doi_str_mv	10.1107/S2052252520013615
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title_sort	joint neutron/x-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis
callnumber	QD901-999
title_auth	Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis
abstract	Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.
abstractGer	Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.
abstract_unstemmed	Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3+ side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.
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title_short	Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis
url	https://doi.org/10.1107/S2052252520013615 https://doaj.org/article/9ffaa2f1f5434ce4923d9040711c415a http://scripts.iucr.org/cgi-bin/paper?S2052252520013615 https://doaj.org/toc/2052-2525
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author2	Tamás Földes Soi Bui Martine Moulin Nicolas Coquelle Matthew P. Blakeley Edina Rosta Roberto A. Steiner
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up_date	2024-07-04T00:03:24.811Z
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