5377-33-3Relevant academic research and scientific papers
Characterization of the structure and function of Klebsiella pneumoniae allantoin racemase
French, Jarrod B.,Neau, David B.,Ealick, Steven E.
experimental part, p. 447 - 460 (2012/06/18)
The oxidative catabolism of uric acid produces 5-hydroxyisourate (HIU), which is further degraded to (S)-allantoin by two enzymes, HIU hydrolase and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase. The intermediates of the latter two reactions, HIU and 2-oxo-4-hydroxy-4-carboxy-5- ureidoimidazoline, are unstable in solution and decay nonstereospecifically to allantoin. In addition, nonenzymatic racemization of allantoin has been shown to occur at physiological pH. Since the further breakdown of allantoin is catalyzed by allantoinase, an enzyme that is specific for (S)-allantoin, an allantoin racemase is necessary for complete and efficient catabolism of uric acid. In this work, we characterize the structure and activity of allantoin racemase from Klebsiella pneumoniae (KpHpxA). In addition to an unliganded structure solved using selenomethionyl single-wavelength anomalous dispersion, structures of C79S/C184S KpHpxA in complex with allantoin and with 5-acetylhydantoin are presented. These structures reveal several important features of the active site including an oxyanion hole and a polar binding pocket that interacts with the ureido tail of allantoin and serves to control the orientation of the hydantoin ring. The ability of KpHpxA to interconvert the (R)- and (S)-enantiomers of allantoin is demonstrated, and analysis of the steady-state kinetics of KpHpxA yielded a kcat/Km of 6.0 × 105 M- 1 s- 1. Mutation of either of the active-site cysteines, Cys79 or Cys184, to serine inactivates this enzyme. The data presented provide new insights into the activity and substrate specificity of this enzyme and enable us to propose a mechanism for catalysis that is consistent with the two-base mechanism observed in other members of the aspartate/glutamate family.
Absolute stereochemistry and preferred conformations of urate degradation intermediates from computed and experimental circular dichroism spectra
Pipolo, Silvio,Percudani, Riccardo,Cammi, Roberto
experimental part, p. 5149 - 5155 (2011/09/13)
The enzymatic oxidation of urate leads to the sequential formation of optically active intermediates with unknown stereochemistry: (-)-5-hydroxyisourate (HIU) and (-)-2-oxo-4-hydroxy-4-carboxy-5- ureidoimidazoline (OHCU). In accordance with the observation that a defect in HIU hydrolase causes hepatocarcinoma in mouse, a detoxification role has been proposed for the enzymes accelerating the conversion of HIU and OHCU into optically active (+)-allantoin. The enzymatic products of urate oxidation are normally not present in humans, but are formed in patients treated with urate oxidase. We used time-dependent density functional theory (TDDFT) to compute the electronic circular dichroism (ECD) spectra of the chiral compounds of urate degradation (HIU, OHCU, allantoin) and we compared the results with experimentally measured ECD spectra. The calculated ECD spectra for (S)-HIU and (S)-OHCU reproduced well the experimental spectra obtained through the enzymatic degradation of urate. Less conclusive results were obtained with allantoin, although the computed optical rotations in the transparent region supported the original assignment of the (+)-S configuration. These absolute configuration assignments can facilitate the study of the enzymes involved in urate metabolism and help us to understand the mechanism leading to the toxicity of urate oxidation products. The Royal Society of Chemistry 2011.
