1. Rational design of an enzyme with high substrate specificity

2. 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

3. 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)

4. 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)

5. 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

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

7. 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)

8. Comparison with other hydantoinases
(60-73)
(93-100)
( )
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

9. Catalytic activity of single and double mutants

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

11. 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)

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

13. 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)

14. 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)

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

16. Binding energies of the mutants with HPH