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(
E95G, K121R, A152T, Y177F, H179D, W238T) or with the C-ter-
Results and Discussion
minal extension (Y177F, G251S, M252V) of neighboring sub-
units in the tetrameric assembly.
Sequence analysis of improved HheC variants
HheC was tailored by Fox and co-workers for the improved
conversion of (S)-4 to (R)-6 under industrial process condi-
Biochemical characterization of HheC2360 and HheC2656
[
25]
tions. As a result, a set of 1151 unique HheC sequences was
described, with a maximum of 42 substitutions per se-
Synthetic genes for HheC2360 and HheC2656 were expressed
in a recombinant fashion in E. coli MC1061 from a pBAD-based
expression vector at similar levels as HheC. After purification of
the enzymes, yielding samples that gave single bands on SDS-
PAGE, catalytic activities for dehalogenation and cyanolysis
were measured. Most data were compared with those for the
variant HheC-C153S, which is wild-type HheC containing
a single mutation that makes the enzyme less susceptible to
[
26]
quence.
To obtain insight into the sequence variation responsible for
the improved catalytic properties, the substitutions occurring
in the reported sequence population were analyzed. This anal-
ysis revealed that 153 of the 254 HheC residues are mutated in
less than 1% of the cases (i.e., in 11 or fewer of the sequences)
and that a further 49 residues are mutated in less than 5% of
the reported HheC sequences. Of the remaining HheC residues,
two subsets of 25 and 27 positions were classified either as fre-
quently mutated (in >66% of the sequences) or moderately
mutated (altered in 5–37% of the sequences), respectively. An
overview of the observed substitutions is given in a multiple
sequence alignment in Figure S1 in the Supporting Informa-
tion. It was expected that the set of most frequent mutations
would include the substitutions that are of major importance
for the relevant changes in properties of HheC, such as the
reported improvement of at least 1.5-fold in the conversion of
[4,10]
oxidative damage, but otherwise has similar properties.
The dehalogenation of the chloroalcohol ester (S)-4, which
yields the corresponding (S)-5 as an intermediate, is the first
HheC-catalyzed reaction in the production of (R)-6. The catalyt-
ic constants for this dehalogenation reaction with (R)- and (S)-
4, as well as those with several other haloalcohols, were
obtained by measuring initial halide release rates. Data were
measured with both mutants and were compared with data
for HheC-C153S (Table 1). The HheC-C153S enzyme exhibited
steady-state parameters similar to previously determined con-
[
26]
(
S)-4 to (R)-6.
The 25 most frequent mutations (Q37H, K38Q, K52I, Y70L,
stants for the wild-type enzyme. The k values were in the
cat
À1
range of 2.7 to 35.1 s per active site for all tested substrates.
Q72H, F82A, A83P, P84V, F86W, Q87R, G99D, A100T, R107K,
T134A, T146S, C153S, T154A, G174A, E181G, F186Y, T189S,
N195S, K203R, A222T, M245V) map to different parts of the
enzyme structure. Only two mutations target buried residues
For achiral 1,3-dichloropropan-2-ol (7) and 1,3-dibromopropan-
2-ol (8), as well as for (R)-4, the K values were lower than the
M
lowest tested haloalcohol concentration of 0.05 mm. For the
other haloalcohols, the KM values were in the 0.16 to 4 mm
range. Because of the very low KM values with the two achiral
dihalopropanols 7 and 8 and with (R)-4, the highest catalytic
efficiencies were found with these substrates, whereas the
lowest catalytic efficiency was observed for rac-3-chloropro-
pane-1,2-diol (rac-11), which has the highest KM value. Surpris-
(
A100T, G174A), whereas the other 23 affected residues each
2
have a solvent-exposed area of ꢀ5 ꢁ . Furthermore, of these
frequent substitutions, only F186Y is among the six first-shell
residues within a 5 ꢁ distance from the catalytic triad Y145-OH.
Another 34 residues lie within 5–10 ꢁ of the catalytic triad
Y145-OH (second shell), and eight of these are frequent substi-
tutions (F82A, A83P, P84V, F86W, A100T, T134A, T146S, G174A).
The most frequently occurring mutations were analyzed fur-
ther to obtain insight into the structural basis of the properties
of the enzyme variants under study.
ingly, both enantiomers of 4 showed low K values; this means
M
that the wild type already recognizes both enantiomers of this
substrate quite well. Previously, the HheC wild type was ap-
plied in the conversion of the methyl ester of 1 with stoichio-
[34]
metric yields but no catalytic constants were reported.
HheC2360 and HheC2656 were selected out of the reported
sequences for detailed investigation because they each contain
at least 24 of the mutations strongly enriched in the ProSAR
procedure, but originate from different branches of the diversi-
In conclusion, both HheC mutants exhibited elevated KM
values relative to HheC-C153S for all tested haloalcohols
(Table 1). These elevated KM values are not surprising because
the mutants were obtained after screening for conversion of
substrate concentrations as high as 20 mm (first and second
tier) and >600 mm (third tier) (S)-4. The screening condi-
tions thus lacked selective pressure for the evolution of HheC
mutants with high affinities.
[
25,26]
ty that was generated.
Each of these variants contains 37
[25]
mutations in total. Variant HheC2360 contains all of the fre-
quent mutations mentioned above with the exception of two
different amino acid substitutions (A100M, T146A), It also con-
tains 12 less frequent mutations (A60V, V75I, V112A, K121R,
P135S, Y166H, Y177G, L178V, H179D, V201W, V205Y, I246V).
Variant HheC2656 differs from HheC2360 in its sequence at 18
positions (K10L, E95G, A100, V112, P135, T146S, A152T, Y166,
S180T, A152T, Y177F, L178, S180T, H201, V205, W238T, G251S,
M252V). As in HheC2360, the majority of the less frequently oc-
curring mutations in HheC2656 are positioned in or flank struc-
tural regions that interact either with neighboring monomers
Mutant HheC2360 exhibited the highest increase in kcat (3.1-
fold) for the target haloalcohol substrate (S)-4, whereas for the
opposite enantiomer (R)-4 it showed only a 1.6-fold increase in
kcat. This increase results in a kcat value for (S)-4 similar to those
found with other tested haloalcohols [7, 8, and rac-3-bromo-
propane-1,2-diol (rac-12)], the kcat values of which were little
affected (0.8- to 1.2-fold changes). For rac-1-chloropropan-2-ol
(rac-9), 1-chloro-2-methylpropan-2-ol (10), and rac-2-chloro-1-
ꢀ
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