Greatly reduced amino acid alphabets in directed evolution: Making the right choice for saturation mutagenesis at homologous enzyme positions

Article abstract of DOI:10.1039/b813388c

Enantioselective mutants of the thermally robust phenyl acetone monooxygenase (PAMO) as catalysts in Baeyer-Villiger reactions have been evolved by utilizing saturation mutagenesis in which drastically reduced amino acid alphabets are employed at homologo

Full text of DOI:10.1039/b813388c

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Greatly reduced amino acid alphabets in directed evolution: making the
right choice for saturation mutagenesis at homologous enzyme positionsw
Manfred T. Reetz* and Sheng Wu
Received (in Cambridge, UK) 1st August 2008, Accepted 12th September 2008
First published as an Advance Article on the web 2nd October 2008
DOI: 10.1039/b813388c
Enantioselective mutants of the thermally robust phenyl acetone
monooxygenase (PAMO) as catalysts in Baeyer–Villiger reac-
tions have been evolved by utilizing saturation mutagenesis in
which drastically reduced amino acid alphabets are employed at
homologous enzyme positions.
loop of the thermally less stable CHMO which was known to
accept a fairly wide variety of ketones,13 and indeed a mutant with
slightly broadened substrate scope was obtained.12 In the present
study we again considered the 441–444 loop, this time using a
different strategy.
We first aligned the sequences14 of WT PAMO and seven
other Baeyer–Villigerases, namely STMO, CPMO, CDMO,
CHMO, CHMO1, CHMO2 and CHMO313 (Fig. 1).
Directed evolution1 of enantioselectivity and/or substrate accep-
tance of enzymes as catalysts in organic chemistry has emerged as
a new approach to asymmetric catalysis.2 Our contribution to
making the method more efficient3 is the combinatorial active-site
saturation test (CAST),4 in which the sites around the binding
pocket of an enzyme are systematically considered for saturation
mutagenesis with the formation of focused libraries5 containing
enantioselective variants, a given site being composed of one or
more amino acid positions.4 We have employed algorithms6 to
analyze oversampling6,7 as a function of the % coverage of a
library.8 In a study involving an enzyme site comprised of three
residues, it was shown that the quality of an NDT-library encod-
ing a structurally balanced set of 12 amino acids is dramatically
higher than that of a conventional NNK-library encoding the
usual 20 amino acids (codon usage according to N: adenine/
cytosine/guanine/thymine; K: guanine/thymine; D: adenine/
guanine/thymine; T: thymine).8b Thus, considerably less screening
for enantioselectivity is required.8 However, in the case of a four
residue site, the use of NNK or NDT codon degeneracy requires
the screening of about 3.1 million or 162 000 transformants,
respectively, for 95% coverage,8 and when randomizing even
larger regions, comprehensive screening turns into a hopeless task.
In the present study we demonstrate how sequence alignment of
homologous enzymes can help in making an appropriate choice of
reduced amino acid alphabets,9 thereby minimizing the screening
effort.
At positions 441, 442, 443 and 444 a limited number of amino
acids appear, namely Ser/Ala, Ala/Val/Gly/Leu, Leu/Phe/Gly/Tyr
and Ser/Ala/Cys/Thr, respectively. Rather than utilizing designed
mixtures of oligonucleotides, we chose a more practical approach
by applying appropriate codon degeneracies6–8 which match as
well as possible the amino acids occurring at the four positions
while simultaneously introducing a limited number of additional
amino acids for slightly higher structural diversity (Table 1).
As the model reaction we chose the oxidative kinetic
resolution of rac-1a, which is hardly accepted by the WT
PAMO. In a very slow reaction, only small amounts of
product are formed after extended reaction times, the selectivity
factor E amounting to 1.2 in slight favor of (S)-2a. Increasing
the activity of the robust PAMO was thus the initial goal.
CHMO from Acinetobacter TD3 is known to catalyze the
reaction of rac-1a with an E-value of 4100 in favor of (R)-
2a,15 but this BVMO is far less thermostable than PAMO.
Other BVMOs have not been tested in these reactions thus far.
We chose phenyl acetone monooxygenase (PAMO) from
Thermobifida fusca as the enzyme to be engineered, which is the
first and only thermostable Baeyer–Villiger monooxygenase
(BVMO) identified thus far.10,11 Unfortunately, PAMO has a
narrow range of substrate acceptance, phenyl acetone and deriva-
tives thereof being the only ketones that react with reasonable
rates.10 In a previous study based on rational design, two of the
four residues in the loop spanning positions 441 to 444 next to the
binding pocket were deleted.12 They do not occur in the shorter
Using the codon degeneracies listed in Table 1, simulta-
neous randomization at all four positions in the 441–444 loop
Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1,
¨
45470 Mulheim/Ruhr, Germany.
¨
E-mail: reetz@mpi-muelheim.mpg.de; Fax: +49 208 306 2985;
Tel: +49 208 306 2000
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b813388c
Fig. 1 Sequence alignment of BVMOs (441–444 loop in red box).
Chem. Commun., 2008, 5499–5501 | 5499
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Table 1 Choice of codon degeneracies at each position in the 441–444 loop of PAMO. Degenerate codons: A (adenine); B (cytosine/guanine/
thymine); C (cytosine); G (guanine); S (cytosine/guanine); for the definition of K and N, see text
Amino acid positions
Codon degeneracy
Encoded amino acids
Codons
864
Oversampling for 95% coverage
2587
441
442
443
444
KCA
KBG
BGC
NSC
A, S
S, A, L, V, W, G
F, H, L, V, Y, G, D, R, C
S, A, P, T, R, G, C
of PAMO by saturation mutagenesis according to the Quik-
Change protocol16 provided a library of PAMO variants
which were subsequently evaluated as whole cell catalysts in
the oxidative kinetic resolution of rac-1. For 95% coverage,
only 2587 transformants would have to be screened.8 We
reduced the screening effort even more by arbitrarily restrict-
ing the search to 1700 clones, which were evaluated in the
oxidative kinetic resolution of rac-1a. About 145 transfor-
mants were found to be more active than the WT PAMO in
this transformation, which is a reflection of the high quality of
the library.8b Had we striven for 95% coverage, even more hits
would have been discovered. Seven of the best (R)-selective
mutants and six of the best (S)-selective variants were se-
quenced (Table 2).
The gross mechanistic features of BVMOs are well known.13
They are flavine-dependent enzymes in which molecular oxy-
gen reacts with the reduced form of the flavine to generate an
intermediate alkyl-hydroperoxide anion, which in turn adds
nucleophilically to the carbonyl O-atom with formation of the
short-lived Criegee-intermediate.13 As evident from the X-ray
structure of WT PAMO,11 the 441–444 loop is near Arg337,
the residue which has been postulated to stabilize the Criegee-
intermediate.11,12 The present results show that amino acid
substitutions in the loop, and not just ‘‘space-providing’’
deletions as demonstrated previously,12 influence substrate
acceptance and enantioselectivity dramatically. Loops are
generally considered to be flexible, making the interpretation
of point mutations problematic,1 but even in cases of reduced
flexibility sound interpretations require detailed QM/MM
studies, which we plan to perform for further insight.
The results are striking in several respects. Whereas both
(S)- and (R)-selective variants were identified, the latter
showed considerably higher E-values, variant 254-21 display-
ing the highest enantioselectivity (E = 70 in favor of (R)-2). It
is also noteworthy that such greatly reduced sets of amino
acids, used as ‘‘building blocks’’ in the saturation mutagenesis
experiment, lead to such different enantioselectivities. For
example, the (S)-selective variant 121-10 is characterized by
Ala/Leu/Val/Ser at positions 441/442/443/444, whereas the
(R)-selective variant 254-67 with Ala/Ala/Asp/Ser differs at
only two positions. Importantly, some of the few ‘‘additional’’
amino acids resulting from the choice of the codon degenera-
cies, beyond those occurring in the eight BVMOs as shown by
the sequence alignment, appear in all of the best (R)-selective
variants. Finally, some of the best mutants were isolated,
purified and checked for thermostability relative to that of the
robust WT PAMO. All of the variants tested (254-21, 254-60
and 254-67) showed half-lives at 50 1C comparable to that of
the WT PAMO.10,12 Thermostability is very high, e.g., there is
no loss of activity after a heat treatment at 50 1C for 40 h.
We also studied the oxidative kinetic resolution of substrate
rac-1b using some of the mutants identified earlier as catalysts
for the conversion of rac-1a. Table 3 shows that enantioselec-
tivity is even better in this case. The (S)-selective mutants in
Table 2 were not tested because of their low enantioselectivity.
Other ketones such as 2-[4-methylphenyl]cyclohexanone did
not react, possibly indicating an electronic effect.
In conclusion, the primary message of this model study is
the finding that enormously reduced amino acid alphabets can
be used successfully in saturation mutagenesis at multi-residue
sites, cases in which the conventional use of NNK codon
degeneracy encoding all 20 amino acids would lead to a
screening problem.8b This was illustrated by developing a
simple strategy for the rapid directed evolution of reaction
rate (substrate acceptance) and enantioselectivity of the ther-
mally stable Baeyer–Villiger monooxygenase PAMO based on
the use of reduced amino acid alphabets in saturation muta-
genesis at all four positions of a loop. These alphabets were
Table 2 Oxidative kinetic resolution of rac-1a in whole-cell catalysis (WT sequence: Ser/Ala/Leu/Ser; mutations are marked boldface)
Mutant
Conversion (%)a
Enantioselectivity
E-Value
Sequence at positions 441–444
132-10
122-10
132-18
121-02
121-10
120-20
254-21
122-02
254-58
254-60
254-67
254-94
254-95
a
38
36
45
41
32
50
46
48
15
34
53
45
44
(S)
(S)
(S)
(S)
(S)
(S)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
2.8
2.9
1.8
1.4
1.5
1.9
70
31
48
28
36
Ala/Leu/Phe/Ala
Ala/Leu/Val/Ala
Ala/Ser/Phe/Ala
Ser/Ser/Phe/Gly
Ala/Leu/Val/Ser
Ser/Ala/Phe/Ser
Ala/Trp/Tyr/Thr
Ser/Trp/Arg/Ser
Ser/Trp/Tyr/Ala
Ala/Ala/Asp/Gly
Ala/Ala/Asp/Ser
Ser/Ser/Asp/Ser
Ala/Ser/Asp/Ser
31
36
Reaction time 24 h. Conditions: ESI.w
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Table 3 Oxidative kinetic resolution of rac-1b with formation of (R)-2b
Variant Conversion (%)a E-value Sequence at positions 441–444
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254-21
254-58
254-60
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254-94
254-95
a
59
36
39
51
39
48
4200
4200
147
187
133
Ala/Trp/Tyr/Thr
Ser/Trp/Tyr/Ala
Ala/Ala/Asp/Gly
Ala/Ala/Asp/Ser
Ser/Ser/Asp/Ser
Ala/Ser/Asp/Ser
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defined by only two, six, nine and seven amino acids, respec-
tively. The choice of the amino acids as building blocks was
guided by sequence alignment of eight BVMOs focusing on a
loop region, and supplemented by a select few additional
amino acids as specified by appropriate degenerate codons.
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was achieved, even though many other hits potentially acces-
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