Crystal structure of D-hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: Insights into the molecular basis of enzyme thermostability

Abstract

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 (HYDBp) at a 2.7-Å resolution. The structure of HYDBp consists of a core (α/β)8 triose phosphate isomerase barrel fold and a β-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYDBp shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYDTsp) and Bacillus stearothermophilus (HYDBst), 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 HYDTsp contains more aromatic residues in the interior of the structure than HYDBp and HYDBst. Changes of large aromatic residues in HYDTsp to smaller residues in HYDBp or HYDBst decrease the hydrophobicity and create cavities inside the structure. HYDTsp has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYDBp and HYDBst. Besides, HYDTsp 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.