5138 J. Am. Chem. Soc., Vol. 120, No. 21, 1998
Tripp et al.
Table 1. Kinetic Parameters for NADP+ for Wild-Type and S39T
SADH
secondary alcohol dehydrogenases (SADH), which have been
isolated from a number of anaerobic bacteria, are Zn-containing
NADP -dependent tetramers that have high activity toward
+
parameter
wild-type SADH
S39T SADH
secondary alcohols. The SADH from the anaerobic thermophile,
cat (s-1
k
)
49.3 ( 2.7
7.4 ( 0.7
6.7 ( 0.7
106.6 ( 6.3
8.9 ( 1.0
Thermoanaerobacter ethanolicus, is stable at temperatures up
-6
K
m
(×10 M)
3
(×10 M s-1
6
-1
to 80 °C, and exhibits high activity in the enantioselective
kcat/K
m
)
12.0 ( 1.5
reduction of cyclic and acyclic secondary alcohols, as well as
ketoesters.4 Because of its thermostability, resistance to
organic cosolvents, and reactivity for a wide variety of
substrates, SADH is a potentially useful biocatalyst for synthetic
,5
Table 2.
m
kcat/K Values for Oxidation of Ethanol, 1-Propanol, and
2-Propanol at 50 °C by Wild-Type and S39T SADH
(M-1 s-1
)
k
cat/K
m
applications. The SADH from Thermoanaerobium brockii
substrate
ethanol
-propanol
-propanol
wild-type
222 ( 36
273 ( 35
S39T
6
(TBADH) has been applied in several chiral syntheses.
43 ( 5
Previously, a temperature-dependent reversal of the enan-
1
2
206 ( 25
(3.0 × 10 ) ( (0.7 × 10 )
7
tiospecificity of SADH was documented by Pham and Phillips.
5
4
4
4
(1.0 × 10 ) ( (2 × 10 )
(S)-2-Butanol is the preferred substrate at temperatures below
2
2
6 °C, while (R)-2-butanol is preferred at temperatures above
6 °C. Similarly, 2-butanone is reduced to (R)-2-butanol by
SADH, while ketones with longer alkyl chains give predomi-
nantly (S)-alcohols. This indicates that SADH has the capabil-
ity, albeit limited, of (R)-alcohol production. If it is possible
to enhance this characteristic, then ketone reduction with SADH
could become a successful method of producing a wide variety
of (R)-alcohols. This is significant, since ADHs typically obey
Prelog’s rule and are hence specific for production of (S)-
enantiomers of secondary alcohols.8 Since little is known about
the molecular basis for the stereochemical preferences of SADH,
the purpose of this work is to initiate the study of SADH active
site residues that govern binding and catalysis of enantiomeric
substrates and products. We have examined the effect of
mutation of Ser39 to threonine on the regiospecificity and
enantiospecificity of SADH. The results show that a threonine
residue at position 39 of SADH increases the (R)-specificity
for 2-butanol and 2-pentanol.
Figure 1. Temperature dependence of enantiospecificity of wild-type
and S39T SADH for 2-butanol. Open circles: Wild-type SADH. Open
squares: S39T SADH. Filled circle: Data from reduction of 2-butanone
by wild-type SADH. Filled square: Data from reduction of 2-butanone
by S39T SADH.
Results
Activity of Wild-Type of S39T SADH. The mutation of
Ser39 to threonine increases the specific activity of purified
SADH from 77.5 units/mg to 118.3 units/mg under standard
assay conditions, using 2-propanol. This result is consistent
values were compared for ethanol, 1-propanol, and 2-propanol
(Table 2). Wild-type SADH is observed to have a 370-fold
specificity ratio for 2-propanol relative to 1-propanol. For S39T
SADH, a 145-fold specificity ratio is seen for 2-propanol relative
to 1-propanol, mainly due to the reduction of kcat/Km for
9
with the report by Sakoda and Imanaka of a Thr f Ser mutation
in the active site of Bacillus stearothermophilus primary ADH
that diminished activity by a comparable amount. To determine
if these effects on activity are related to substrate specificity or
2
-propanol. Additionally, the S39T mutation has the effect of
diminishing ethanol specificity. These results show that al-
though there are some effects on primary versus secondary
alcohol specificities by this mutation, they are not significant
enough to regard Ser39 as a key residue governing the
regiospecificity of SADH.
+
cofactor specificity, steady state kinetic parameters for NADP
specificity were measured for each protein (Table 1). Both
+
enzymes exhibit very similar Km values for NADP , but kcat
and kcat/Km values are higher for S39T SADH.
Enantiospecificity of Wild-Type and S39T SADH. Values
Regiospecificity of Wild-Type and S39T SADH. To
determine whether the S39T mutation has any effect on
specificity for primary or secondary alcohol substrates, kcat/Km
7
of kcat/Km were measured by the procedure of Pham et al. for
the (R)- and (S)-enantiomers of 2-butanol and 2-pentanol at
temperatures between 288 and 328 K for wild-type and S39T
mutant SADH. The recombinant wild-type T. ethanolicus
SADH exhibits temperature-dependent stereospecificity for
(
3) Bryant, F. O.; Weigel, J.; Ljungdahl, L. G. Appl. EnViron. Microbiol.
988, 460-465.
4) (a) Lamed, R. J.; Keinan, E.; Zeikus, J. G. Enzyme Microb. Technol.
981, 3, 144-148. (b) Keinan, E.; Hafeli, F. V.; Seth, K. K.; Lamed, R. J.
1
1
(
2
-butanol identical to the enzyme isolated from T. ethanolicus
7
Am. Chem. Soc. 1986, 108, 162-169.
used in previous experiments (Figure 1, open circles). Analysis
of the data collected for S39T SADH (Figure 1, open squares)
demonstrates that it is more specific for (R)-2-butanol than wild-
(
5) (a) Zheng, C.; Pham, V. T.; Phillips, R. S. Bioorg. Med. Chem. Lett.
1
1
992, 2, 619-622. (b) Zheng, C.; Pham, V. T.; Phillips, R. S. Catal. Today
994, 22, 607-620.
1
0
(6) (a) Keinan, E.; Seth, K. K.; Lamed, R. J. Am. Chem. Soc. 1986, 108,
type SADH, and its racemic temperature, Tr, decreases from
3
474-3480. (b) De Amici, M.; De Micheli, C.; Carrea, G.; Spezia, S. J.
297 K for wild-type SADH to 183 K (Table 3). Furthermore,
Org. Chem. 1989, 54, 2646-2650. (c) Keinan, E.; Sinha, S. C.; Singh, S.
P. Tetrahedron 1991, 47, 4631-4638.
(10) The enantiospecificity ratio, E ) (kcat/Km)R/(kcat/Km)S, is directly
related to the difference in free energy of activation between the (R)- and
(7) (a) Pham, V. T.; Phillips, R. S.; Ljungdahl, L. G. J. Am. Chem. Soc.
q
1
989, 111, 1935-1936. (b) Pham, V. T.; Phillips, R. S. J. Am. Chem. Soc.
(S)-alcohols. ∆∆G ) -RT ln E, from transition state theory. Separation
q
1
990, 112, 3629-3632.
of ∆∆G into entropic and enthalpic components is given by the expression
q
q
q
7
q
q
(8) Drauz, K.; Waldmann, H. Enzyme Catal. Org. Synth: A Compre-
∆∆G ) ∆∆H - T∆∆S . The racemic temperature, Tr ) ∆∆H /∆∆S ,
q
hensiVe Handbook 1995, 2, 600-616.
is the temperature at which ∆∆G ) 0, and no discrimination is made in
reaction or formation of enantiomers.
(9) Sakoda, H.; Imanaka, T. J. Bacteriol. 1992, 174, 1397-1402.