Rational design of an enzyme with high substrate specificity

D-Hydantoinse for non-natural D-amino acids D-Alanin, D-Serine, D-Tyrosine, D-Valine, D-Phenylalanine, D-,L-Homophenylalanine, D-Phenylglycine, D-p-Hydroxyphenylglycine Use Building blocks for semi-synthetic antibiotics, antimicrobial & antiviral peptides, pesticides, pyrethroids, [1]: D-p-Hdroxyphenylglycine [2]: Amoxicillin [3]: Cefadroxil

Design of the enzyme with high substrate specificity for D-Hydantoinase from B. stearothermophilus SD1 (β/α)8-barrel fold, Homo-tetramer (52 kDa) Metallo-hydrolase requiring Mn2+ for catalysis Useful properties for practical application - Strict D-specific and high catalytic activity - Easy over-expression in E. coli - Highly thermostable Low specific activity for the substrate with a bulky aromatic group at 5’-position Design of the enzyme with high substrate specificity for synthesis of important D-amino acids (i.e., D-HPG, D-PG) Target substrate : p- Hydroxyphenylhydantoin C H N O R R= Lee et al. AMB (1997) Kim et al. AEM (2002) Cheon et al. Biochem (2003)

Substrate specificity and sequence homology of hydantoinases Relative activity (%) Bs HYD1 Bt HYD2* Ph HYD3** -Hydantoin 100 -Isopropyl- (IPH) 5 11 230 -Phenyl- (PH) 48 792 -Hydroxyphenyl- (HPH) Sequence homology (%) 12 82 92 889 75 1 D-Hydantoinase from B. stearothermophilus SD1 2 D-Hydantoinase from B. thermocatenulatus GH2 3 D-hydantoinase from E. coli * Park et al., Appl. Biochem. Biotech. (1999) ** Kim et al., J. Bacteriol. (2000)

Procedure for designing the substrate specificity 1) Analyze the substrate binding pocket based on 3-D structure - Comparison with the homologous enzymes if available 2) Predict the critical loops or residues interacting with a target substrate (cf: TIM barrel fold) 3) Mutagenesis to determine the critical loops or residues If the critical loops or residues are confirmed, go to the next step Otherwise, go to the step 1 and repeat the procedure 4) Saturation mutagenesis at the critical loops or residues to generate the best mutant based on the size and hydrophobicity/hydrophilicity of amino acids

Prediction of the critical residues by docking the target substrate (HPH) into the active site of the enzyme Autotors (HPH) AutoGrid AutoDock

Analysis of the substrate binding pocket Identification of stereochemistry gate loops (SGLs) which determine the substrate specificity of the enzyme SGL1 SGL2 SGL3 SGL1 SGL3 SGL2 Cheon et al. Biochem (2004)

Comparison with other hydantoinases (60-73) (93-100) (150-162) Identification of critical residues at each loop by docking analysis - SGL 1: H60 (metal coordination), M63 - SGL 2: L94 (not effective) - SGL 3: K150 (metal coordination), F152, Y155 (catalytic residue), F159 Hydrophobicity design 먼저 설명, Comparison 설명 Green : BstHyd (1K1D) Blue : BspHyd (1YNY); Bacillus sp. AR9 Orange : BpiHyd (1NFG); Burkholderia picketti

Catalytic activity of single and double mutants

Systematic mutational analysis Relative activity (%) Increasing size 159th residue 63rd residue Increasing size M63I/F159S mutant is the optimal combination.

Mutation based on the size of amino acid residues Mutations M63/F159 M63I/F159A M63I/F159S Structure Relative activity for HPH (%) 100 374 ± 59 540 ± 4 Combination of amino acids with different sizes significantly increased the catalytic activity for the target substrate Lee et al. Enzyme Microbiol Tech (2010)

Hydrophilic amino acid and hydrogen bond The hydropathy index of an amino acid: a number representing the hydrophobic or hydrophilic properties of its sidechain

Mutation based on hydrophobicity M63/F159 M63H/F159N M63H/F159S Mutations Structure Relative activity for HPH (%) 100 450 ± 59 353 ± 62 Interaction with a hydroxyl group of the substrate is critical N: H2NC(=0)CH2 S: H0CH2 Lee et al. Enzyme Microbiol Tech (2010)

Kinetics analysis of the designed mutants Hydantoin HPH Kcat (s-1) KM (mM) kcat/KM (M-1 s-1) kcat (kcat/KM)HPH/ (kcat/KM)Hyd Fold increase Wild-type 59 78 760 18 4.9 3.6×103 4.8 1 M63H/F159N 57 420 140 62 5.1 1.2×104 89 19 M63I/F159S 8 380 22 2.9 2.1×104 952 198 Lee et al. Enzyme Microbiol Tech (2010)

Transition state HPH in tetrahedral form Transition-state modeling Ground state HPH Transition state HPH in tetrahedral form

Binding energies of the mutants with HPH