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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 H [2] N H HO Use COOH O CH3 C CONH S CH3 H2N Building blocks for semi-synthetic antibiotics, antimicrobial & antiviral peptides, pesticides, pyrethroids, HO H COOH NH2 [1] COOH [3] O H R H C O C HN D-hydantoinase H R HO C O R C S [1]: D-p-Hdroxyphenylglycine NH2 N-carbamyl-D-amino acid [2]: Amoxicillin [3]: Cefadroxil Nitrous Acid or NH3 +CO2 N-carbamoylase H NH2 NH C O Spontaneous Racemaization C CONH C COOH NH D-5'monosubstituted hydantoin CH3 N O C HN NH C R H C COOH NH2 O L-5'-monosubstituted hydantoin D-amino acid Biomolecular Eng. Lab. 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 Lee et al. AMB (1997) Kim et al. AEM (2002) Cheon et al. Biochem (2003) R H C OH O C HN NH R= C O Biomolecular Eng. Lab. Substrate specificity and sequence homology of hydantoinases Relative activity (%) Substrate 1 D-Hydantoinase Bs HYD1 Bt HYD2* Ph HYD3** -Hydantoin 100 100 100 -Isopropyl(IPH) 5 11 230 -Phenyl(PH) 48 230 792 -Hydroxyphenyl(HPH) 12 82 889 Sequence homology (%) 100 92 75 from B. stearothermophilus SD1 from B. thermocatenulatus GH2 3 D-hydantoinase from E. coli 2 D-Hydantoinase * Park et al., Appl. Biochem. Biotech. (1999) ** Kim et al., J. Bacteriol. (2000) Biomolecular Eng. Lab. 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 Biomolecular Eng. Lab. Prediction of the critical residues by docking the target substrate (HPH) into the active site of the enzyme AutoGrid Autotors (HPH) AutoDock Biomolecular Eng. Lab. Analysis of the substrate binding pocket Identification of stereochemistry gate loops (SGLs) which determine the substrate specificity of the enzyme SGL1 SGL3 SGL3 SGL1 SGL2 SGL2 Cheon et al. Biochem (2004) Biomolecular Eng. Lab. Comparison with other hydantoinases SGL-1 (60-73) BstHyd BspHyd BpiHyd HLDMPLGGTVTKD HLDMPFGGTVTAD HVETVSFNTQSAD SGL-2*(93-100) CLTNKGEP CLTKKGES CQQDRGHS SGL-3*(150-162) XVFMAYKNVFQAD KVFMAYKNVFQAD XVFMAYRGMNMID 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 Green Blue Orange : BstHyd (1K1D) : BspHyd (1YNY); Bacillus sp. AR9 : BpiHyd (1NFG); Burkholderia picketti Catalytic activity of single and double mutants Systematic mutational analysis Relative activity (%) 600 450 300 Increasing size 150 Thr Ser Ala 0 Ala Ile Met 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 100 374 ± 59 540 ± 4 Structure Relative activity for HPH (%) 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 Mutations M63/F159 M63H/F159N M63H/F159S 100 450 ± 59 353 ± 62 Structure Relative activity for HPH (%) 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 KM HPH kcat/KM kcat/KM (M-1 s-1) (kcat/KM)HPH/ (kcat/KM)Hyd Fold increase 4.9 3.6×103 4.8 1 62 5.1 1.2×104 89 19 59 2.9 2.1×104 952 198 Kcat (s-1) kcat KM (s-1) (mM) (mM) (M-1 s-1) Wild-type 59 78 760 18 M63H/F159N 57 420 140 M63I/F159S 8 380 22 Lee et al. Enzyme Microbiol Tech (2010) Transition-state modeling Ground state HPH Transition state HPH in tetrahedral form Binding energies of the mutants with HPH