Characterization of the structure and function of Klebsiella pneumoniae allantoin racemase

Jarrod B. French, David B. Neau, Steven E. Ealick

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)447-460
Number of pages14
JournalJournal of Molecular Biology
Issue number3
StatePublished - Jul 15 2011
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health grant GM73220 to S.E.E. We thank the staff at the NE-CAT of the Advanced Photon Source, Argonne National Laboratory, for advice with data collection and processing. Data collection at the NE-CAT beamlines was supported by award RR-15301 from the N ational Center for Research Resources at the National Institutes of Health . Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under contract number DE-AC02-06CH11357 . We acknowledge Dr. Cynthia Kinsland of the Protein Production and Purification Facility in the Department of Chemistry and Chemical Biology for help with molecular biology and Ms. Leslie Kinsland for help with manuscript preparation. J.B.F. would like to acknowledge the Tri-Institutional Training Program in Chemical Biology for financial support.


  • X-ray crystallography
  • hydantoin
  • purine catabolism
  • racemization
  • ureide


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