Download Chap 7. Detection of Intermediates in Enzymatic Reactions

Chap 15. Protein Engineering
1. Dissection of the structure and activity of existing proteins
2. Production of novel proteins: enzymes and antibodies
Protein Engineering for Altering Enzyme
Reaction Specificity
1.
2.
3.
4.
Directed evolution
Sequence comparison
Structure comparison
Altering mechanism (Modification of the catalytic machinery
for a non-related catalytic activity)
5. Introduction of the whole catalytic machinery
6. Non-catalytic protein template (Introduction of catalytic
activity into non-catalytic protein)
Berglund, P.; Park, S. Current Organic Chemistry, 2005, 9, 325-336
Evolution of New Enzyme Activity in Nature
Divergent evolution
(a) Gerlt, J.A.; Babbitt, P.C. Curr. Opin. Chem. Biol.,
1998, 2, 607-612. (b) Babbitt, P.C.; Gerlt, J.A. J. Biol.
Chem., 1997, 272, 30591-30594.
Convergent evolution
Todd, A.E.; Orengo, C.A.; Thornton, J.M. Trends
Biochem. Sci., 2002, 27, 419-426.
1. Oxydosqualene cyclization produces different
steroids according to the site of deprotonation
by different enzymes
H
One mutant of cycloartenol synthase can produce lanosterol
H
cycloartenol
HO
- H at C-19
H
H
H
19 11 H
- H at C-8
8
HO
O
oxydosqualene
lanosterol
HO
H
lanosteryl cation
H
- H at C-11
cycloartenol synthase (EC 5.4.99.8)
lanosterol synthase (EC 5.4.99.7)
H
H
parkeol
HO
H
Selection: Lanostrerol is an intermediate of ergosterol that is an essential
fungal membrane component
Hart, E. A.; Hua, L.; Darr, L. B.; Wilson, W. K.; Pang, J.; Matsuda, S. P. T.
J. Am. Chem. Soc., 1999, 121, 9887-9888.
2. Substitutions of several residues interchange
the catalytic activity between a desaturase
and a hydroxylase
hydroxylase
OH
12
linoleic acid
O
OH
oleic acid
O
desaturase
HO
OH
oleate hydroxylase (EC 1.14.13.26)
ricinoleic acid
O
oleate desaturase (EC 1.3.1.35)
Six amino acid substitutions converted oleate hydroxylase to a desaturase.
Four amino acid substitutions converted oleate desaturase to a hydroxylase.
Broun, P.; Shanklin, J.; Whittle, E.; Somerville, C. Science, 1998, 282, 1315-1317.
2. A rationally designed mutant of glutathione
transferase A1-1 shows Michael addition activity
glutathione transferase (GST, EC 2.5.1.18)
4 aa substitutions in A1-1 + Cterminus from A4-4 into A1-1
gave 300-fold increase in
Michael activity and 10-fold
decrease in subst activity.
Nilsson, L.; Gustafsson, A.; Mannervik, B. Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 9408-9412.
3. The muconate lactonizing enzyme subgroup
catalyze three different reactions through
deprotonation of an α-proton
muconate lactonizing enzymes (EC 5.5.1.1)
CO2
O
O
O
O
O H
N
H
O
O
O
HN
Ala
AEE
Ala
O
O
H
O
O H
H
O
H
O
O
O
H
O
O
O
H
HO
HO
O2C
H O
HN
Ala
O
MLE II
OSBS
CO2
CO2
O
O
O
O H
O
O
O
O2C
O
O2C
O
racemization
Single-site mutants of
the L-Ala-D/L-Glu
epimerase and The
Ring opening muconate lactonizing
enzyme II show the osuccinyl-benzoatesynthase activity
b-elimination
Schmidt, D. M. Z.; Mundorff, E. C.; Dojka, M.; Bermudez, E.; Ness, J. E.; Govindarajan, S. G.;
Babbitt, P. C.; Minshull, J.; Gerlt, J. A. Biochemistry, 2003, 42, 8387-8393.
3. 2-Enoyl-CoA hydratase (Crotonase) and
4-CBA-CoA dehalogenase have similar active sites
but catalyze different reactions
O
crotonase
H2O
CoAS
O
OH
Michael addition
CoAS
Nucleophilic substitution
O
O
4-CBA-CoA dehalogenease
+ Cl
CoAS
CoAS
H2O
Cl
OH
Active site overlapping resulted in a seven-residue
substitution in 4-CBA-CoA dehalogenase which showed
crotonase activity.
Xiang, H.; Luo, L.; Taylor, K. L.; Dunaway-Mariano, D. Biochemistry, 1999, 38, 7638-7652.
3. The reaction specificity of alanine racemase
altered into that of an aminotransferase
by a double active-site mutation
alanine
racemase
H2N
H2N
CO2
L-alanine
CO2
O
D-alanine
D-amino acid
aminotranferase
CO2
pyruvate
Arg219Glu resulted in 103-fold decrease in racemase activity and
a 5.4-fold increase in transaminase activity. Tyr265Ala eliminated
racemase activity.
(a) Watanabe, A.; Yoshimura, T.; Mikami, B.; Hayashi, H.; Kagamiyama, H.; Esaki, N.
J. Biol. Chem., 2002, 277, 19166-19172. (b) Yow, G.-Y.; Watanabe, A.; Yoshimura, T.; Esaki, N.
J. Mol. Catal. B: Enzym., 2003, 23, 311-319.
3. Conversion of ala racemase into an
aldolase with a single-point mutation
Alanine racemase from Geobacillus stearothermophilus (EC 5.1.1.1)
L-threonine aldolase (EC 4.1.2.5)
base
H2N
CO2
O
O P O
O
OH
D-(2R,3S)-phenylserine
CO2
HO
H
N
N
H
H
O
CH3
b-phenylserine-PLP aldimine
O
H2N
CO2
+
glycine
retro-aldol reaction
Tyr265Ala mutant: 3  103 fold reduced racemase activity and 2.3  105
fold increased aldolase activity with D-(2R,3S)-phenylserine, compared
to the wild type enzyme. Highly enantioselective.
Seebeck, F.P.; Hilvert, D. J. Am. Chem. Soc., 2003, 125, 10158-10159.
H
4. Hydrolysis of nitriles by a papain mutant
R2
R2
H
N
CN
R1
S Enzyme
R1
a thioimidate
Wild type
nitrile hydratase (EC 4.2.1.84)
nitrilases (EC 3.5.5.X)
papain (EC 3.4.22.2)
NH
H
N
X
Papain
Gln19Glu
O2C Glu19
H
R2
H
N
N
H
R2
S Enzyme
R1
O
H
N
NH2
R2
O
H
N
R1
H2O
Dufour, E.; Storer, A.C.; Ménard, R. Biochemistry, 1995, 34, 16382-16388.
OH + NH3
R1
4. Aldol addition activity of C. antarctica lipase B
O
O
R
H
R
C. antarctica
lipase B
Ser105Ala HO
Ala105
CH3 H
O
H
R
Asp187
O
H
N
N
O
H
H
Oxyanion
hole
R
His224
Substrate docking
250 ps dynamics
O
H
3-methyl-butanal
Asp187
Gln106
Ala105
Thr40
His224
Critical H-bond distance
Trp104
(a) Branneby, C.; Carlqvist, P.; Magnusson, A.; Hult, K.; Brinck, T.; Berglund, P.
J. Am. Chem. Soc., 2003, 125, 874-875.
(b) Branneby, C.; Carlqvist, P.; Hult, K.; Brinck, T.; Berglund, P.
J. Mol. Catal. B: Enzym., 2004, 31, 123-128.
5. Conversion of cyclophilin into
a proline-specific endopeptidase (hydrolase)
Cyclophilin (EC 5.2.1.8)
O
cyclophilin
N
OH
O
O
N
OH
R
O
R
H2N
cis X-Pro
Cis-trans isomerization
NH2
trans X-Pro
1. Several candidate sites for the serine evaluated
2. Rest of triad introduced in best serine mutant
New hydrolytic activity was 8  108-fold over the uncatalyzed reaction
Quéméneur, E.; Moutiez, M.; Charbonnier, J.-B.; Ménez, A. Nature, 1998, 391, 301-304.
6. Introduction of catalytic activity into
non-catalytic proteins
Scytalone dehydratase activity (EC 4.2.1.94) into nuclear transport factor 2
OH O
HO
OH OH
- H2O
OH
a mutant
of NTF2
HO
8 mutations caused 150-fold
activity improvement
Nixon, A. E.; Firestine, S. M.; Salinas, F. G.; Benkovic, S. J. Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 3568-3571.
6. Introduction of catalytic activity
into non-catalytic proteins
Triose phosphate isomerase (TIM, EC 5.3.1.1) activity into ribose binding protein
The most active mutant (NovoTim 1.2) of RBP contains 13-amino acid
substitutions and shows 105-fold rate improvement over the uncatalyzed
reaction.
(a) Looger, L. L.; Dwyer, M. A.; Smith, J. J.; Hellinga, H. W. Nature, 2003, 423, 185-190.
(b) Dwyer, M. A.; Looger, L. L.; Hellinga, H. W. Science, 2004, 304, 1967-1971