Liu et al.
645
Table 1. Kinetic parameters of wild-type DERA and the Ser238Asp variant in the retroaldol reaction.
Enzymea
WT
Substrateb
kcat (s–1)
Km (mM)
0.64 ± 0.01
57 ± 7
kcat/Km (s–1 mM–1)
106 ± 2
0.0020 ± 0.0003
DERP
DER
68 ± 1
0.107 ± 0.005
Ser238Asp
DERP
DER
0.58 ± 0.05
0.21 ± 0.01
61 ± 11
39 ± 6
0.01 ± 0.001
0.005 ± 0.0009
Note: Enzymes and kinetic parameters were determined according to the procedures previously described (5)
aWT, wild-type DERA.
bDERP, D-2-deoxyribose-5-phosphate; DER, D-2-deoxyribose.
Scheme 3. (a) Acceptor model of wild-type DERA; (b) acceptor
model of the Ser238Asp variant.
strate specificity of an enzyme by protein engineering or
substrate design. Methyl pyranose 2a has been used as a key
synthon in the total synthesis of the anti-cancer agents
epothilones (6, 7).2 Work is in progress to further utilize the
structural information to design new substrates for DERA-
catalyzed aldol addition reactions.
O
Ser-238,239
Gly-205
A)
OH
OH
H
R1 R2
Hydrophilic
Hydrophobic
Acknowledgment
We thank the National Institutes of Research for support
of this research.
O
H
B)
O
Asp-238
R1 R2
Reference
O
R
1 = CH3,OCH3, CH2CH3,
H
Hydrophilic
Hydrophobic
1. For a recent special issue on this subject, see: Nature (Lon-
don), 409, 226 (2001).
R2 = OH, N3, H
2. J.B. Jones. Tetrahedron, 42, 3351 (1986); G. DeSantis and J.B.
Jones. Curr. Opin. Biotechnol. 10, 324 (1999).
3. R.N. Patel. In Stereoselective biocatalysis. Dekker, New York.
2000, and refs. cited therein.
4. T.D. Machajewski and C.-H. Wong. Angew. Chem. Int. Ed. 39,
1352 (2000), and refs. cited therein.
5. A. Heine, G. DeSantis, J.G. Luz, M. Mitchell, C.-H. Wong,
and I.A. Wilson. Science (London), 294, 369 (2001).
6. For details, see: J. Liu and C.-H. Wong. Angew. Chem. Int.
Ed. 41, 1404 (2002).
7. K.M. Koeller and C.-H. Wong. Nature (London), 409, 232
(2001).
binding pocket, but substrates with a negative charge in the
C-3 position would be disfavored. Indeed, the Ser238Asp-
mutant enzyme is 2.5 times more active (based on the
kcat/Km value of the retroaldol reaction) than the wild-type to
D-glyceraldehyde as a substrate, as indicated in Table 1. Mo-
lecular modeling indicates that the C-3 hydroxy hydrogen
forms a 2.9–3.2 Å hydrogen bond with the Asp-238 car-
boxylate, accounting for the increase in reactivity (Fig. 3).
In summary, this study has demonstrated that with a well-
defined enzyme structure, one can effectively alter the sub-
2 General enzymatic synthesis: To a 100 mL buffer solution (0.1 M KH2PO4, pH 7.5) containing 0.1 M acceptor aldehyde and 0.3 M donor
acetaldehyde was added 3000 units of DERA. The resulting solution was stirred in the dark for 3–6 days under argon. The reaction was
quenched by addition of 2 volumes of acetone. The mixture was then stirred at 0°C for 1 h and centrifuged to remove the precipitated en-
zyme. The aqueous phase was concentrated in vacuo, and the residue was purified by flash chromatography (silica, 1:2 to 4:1 EtOAc–hex-
ane) to yield 31% of 2c. Characterized by its lactone form: [a]D = 30.2 (c 0.7, CHCl3). IR (film) (cm–1): 3424.6, 2970.0, 1719.2, 1190.4,
1114.0, 1055.2. 1H NMR (600 MHz, CDCl3) d: 4.43 (dd, J = 4.4, 11.4 Hz, 1H), 3.97 (dd, J = 7.9, 11.4 Hz, 1H), 3.96 (m, 1H), 2.85 (dd, J =
5.7, 17.1 Hz, 1H), 2.74 (d, J = 3.9 Hz, 1H), 2.55 (dd, J = 6.2, 17.6 Hz, 1H), 1.79 (m, 1H), 1.64 (m, 1H), 1.35 (m, 1H), 1.01 (t, J = 7.1 Hz,
3H). 13C NMR (150 MHz, CDCl3) d: 171.07, 69.21, 67.81, 42.32, 38.03, 21.63, 11.23. HRMS m/z calcd. for C7H12O3 (M+): 144.0786;
1
found: 167.0678 ([M + Na]+). D-aldehyde product; 3% yield. Characterized by its lactone form: [a]D = –1.0 (c 0.6, CHCl3). H NMR
(600 MHz, CDCl3) d: 4.37 (t, J = 10.9 Hz, 1H), 4.25 (dd, J = 4.9, 11.0 Hz, 1H), 4.22 (br s, 1H), 2.73 (dd of AB, J = 3.1, 18.0 Hz, 1H), 2.68
(dd of AB, J = 4.4, 18.4 Hz, 1H), 2.37 (br s, 1H), 1.86 (m, 1H), 1.48 (m, 1H), 1.35 (m, 1H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz,
1
CDCl3) d: 170.30, 68.91, 64.50, 39.28, 39.18, 19.60, 11.25. 4a; 47% yield. H NMR (500 MHz, CDCl3) d major isomer): 5.14 (dt, J = 2.6,
7.7 Hz, 1H), 4.22 (m, 1H), 4.13 (dd, J = 10.1, 11.9 Hz, 1H), 3.71 (dd, J = 4.8, 11.8 Hz, 1H), 3.58 (ddd, J = 2.9, 4.8, 9.9 Hz, 1H), 2.93 (d,
J = 5.2 Hz, 1H), 2.10 (ddd, J = 2.6, 10.2, 19.3Hz, 1H), 1.92 (dt, J = 3.3, 16.8 Hz); minor isomer: 5.32 (q, J = 3.3 Hz, 1H), 4.24 (m, 1H),
4.10 (dd, J = 2.6, 12.5 Hz, 1H), 3.84 (dd, J = 4.8, 12.1 Hz, 1H), 3.77 (q, J = 3.3 Hz, 1H), 1.98 (ddd, J = 3.0, 9.9, 12.8 Hz), 1.88 (dt, J = 4.0,
13.2 Hz, 1H). 13C NMR (125 MHz, CDCl3) d major isomer: 92.28, 67.12, 58.97, 56.83, 35.14; minor isomer: 91.74, 64.92, 61.07, 60.63, 35.33.
© 2002 NRC Canada