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Crystal Structure of D-Hydantoinase from Burkholderia pickettii at a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability

Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031,¹ Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China,² Center for Advanced Biotechnology and Medicine and Rutgers University Department of Chemistry and Chemical Biology, Piscataway, New Jersey 08854-5638³

Received 17 January 2003/ Accepted 18 April 2003

D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD_Bp) at a 2.7-Å resolution. The structure of HYD_Bp consists of a core (/ß)₈ triose phosphate isomerase barrel fold and a ß-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYD_Bp shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYD_Tsp) and Bacillus stearothermophilus (HYD_Bst), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs. The more thermostable HYD_Tsp contains more aromatic residues in the interior of the structure than HYD_Bp and HYD_Bst. Changes of large aromatic residues in HYD_Tsp to smaller residues in HYD_Bp or HYD_Bst decrease the hydrophobicity and create cavities inside the structure. HYD_Tsp has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD_Bp and HYD_Bst. Besides, HYD_Tsp also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.

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