Manipulation of the Active Site Loops of d-Hydantoinase, a (β/α)8-Barrel Protein, for Modulation of the Substrate Specificity

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Abstract
We previously proposed that the stereochemistry gate loops (SGLs) constituting the substrate binding pocket of d-hydantoinase, a (β/α)8-barrel enzyme, might be major structural determinants of the substrate specificity [Cheon, Y. H., et al. (2002) Biochemistry 41, 9410−9417]. To construct a mutant d-hydantoinase with favorable substrate specificity for the synthesis of commercially important non-natural amino acids, the SGL loops of the enzyme were rationally manipulated on the basis of the structural analysis and sequence alignment of three hydantoinases with distinct substrate specificities. In the SGLs of d-hydantoinase from Bacillus stearothermophilus SD1, mutations of hydrophobic and bulky residues Met 63, Leu 65, Phe 152, and Phe 159, which interact with the exocyclic substituent of the substrate, induced remarkable changes in the substrate specificities. In particular, the substrate specificity of mutant F159A toward aromatic substrate hydroxyphenylhydantoin (HPH) was enhanced by ∼200-fold compared with that of the wild-type enzyme. Saturation mutagenesis at position 159 revealed that kcat for aromatic substrates increased gradually as the size of the amino acid side chain decreased, and this seems to be due to reduced steric hindrance between the bulky exocyclic group of the substrate and the amino acid side chains. When site-directed random mutagenesis of residues 63 and 65 was conducted with the wild type and mutant F159A, the selected enzymes (M63F/L65V and L65F/F159A) exhibited ∼10-fold higher kcat values for HPH than the wild-type counterpart, which is likely to result from reorganization of the active site for efficient turnover. These results indicate that the amino acid residues of SGLs forming the substrate binding pocket are critical for the substrate specificity of d-hydantoinase, and the results also imply that substrate specificities of cyclic amidohydrolase family enzymes can be modulated by rational design of these SGLs.

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