Download Rational design_substrate specificity

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