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Related Experiment Videos

Structure and action of urocanase.

Dirk Kessler1, Janos Rétey, Georg E Schulz

  • 1Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstr. 21, 79104 Freiburg im Breisgau, Germany.

Journal of Molecular Biology
|August 18, 2004
PubMed
Summary

Pseudomonas putida urocanase was crystallized and its structure solved at 1.14 A resolution. The study details the enzyme

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Urocanase (EC 4.2.1.49) catalyzes urocanate hydration.
  • Understanding its structure-function relationship is crucial for biochemical studies.

Purpose of the Study:

  • To elucidate the crystal structure of Pseudomonas putida urocanase.
  • To propose a detailed catalytic mechanism based on the active site structure.

Main Methods:

  • Crystallization of urocanase after thiol group modification.
  • Structure determination using multiwavelength anomalous diffraction (MAD).
  • High-resolution (1.14 A) refinement of the crystal structure.

Main Results:

  • Determined the 3D structure of a Pseudomonas putida urocanase homodimer (2 x 557 residues).
  • Identified tightly bound NAD+ cofactors and the substrate urocanate in a sequestered active site cavity.
  • Proposed a catalytic mechanism involving substrate hydration by a water molecule within the active site cavity.

Conclusions:

  • The structure reveals a novel core domain fold and an NAD-binding domain acting as a lid.
  • The active site cavity likely opens for substrate passage, possibly by lifting the NAD domain.
  • Identified a potential misannotation in a related urocanase family member based on active site deviation.

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