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

TY - JOUR

T1 - 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

AU - McGregor, Lindsay

AU - Steiner, Roberto

PY - 2020/10/9

Y1 - 2020/10/9

N2 - Cofactor-independent urate oxidase (UOX) is a ~137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidised 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, we have integrated high-resolution neutron/X-ray crystallographic analysis at room temperature with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated A. 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 stabilised by T57 and N254 on different UOX protomers as well as by an O-H/interaction with 8AZA. The active site K10-T57 dyad features a charged K10-NH3+ side chain engaged in a strong H-bond with T57OG1, while the T57OG1-HG1 bond is rotationally dynamic and oriented toward the system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with T57HG1 central to a K10-assisted proton relay system. Room temperature crystallography and simulations also reveal conformational heterogeneity for N254 that modulates W1 stability in the peroxo hole. We propose this can be an active mechanism to facilitate W1/O2 exchange during catalysis.

AB - Cofactor-independent urate oxidase (UOX) is a ~137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidised 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, we have integrated high-resolution neutron/X-ray crystallographic analysis at room temperature with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated A. 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 stabilised by T57 and N254 on different UOX protomers as well as by an O-H/interaction with 8AZA. The active site K10-T57 dyad features a charged K10-NH3+ side chain engaged in a strong H-bond with T57OG1, while the T57OG1-HG1 bond is rotationally dynamic and oriented toward the system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with T57HG1 central to a K10-assisted proton relay system. Room temperature crystallography and simulations also reveal conformational heterogeneity for N254 that modulates W1 stability in the peroxo hole. We propose this can be an active mechanism to facilitate W1/O2 exchange during catalysis.

M3 - Article

SN - 2052-2525

JO - IUCrJ

JF - IUCrJ

ER -