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
(HYD
Bp
) at a 2.7-Å resolution. The structure of HYD
Bp
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 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.