Site-Directed Mutagenesis Studies on the Toluene Dioxygenase Enzymatic System: Role of Phenylalanine 366, Threonine 365 and Isoleucine 324 in the Chemo-, Regio-, and Stereoselectivity

Article abstract of DOI:10.1002/adsc.201700444

Toluene Dioxygenase (TDO) enzymatic complex has been widely used as a biocatalyst for the regio- and enantioselective preparation of cis-cyclohexadienediols, which are very important starting materials for organic synthesis. However, the lack of regio- and stereodiversity of the dioxygenation process by TDO and related dioxygenases constitutes a clear drawback when planning the use of these diols in synthetic schemes. In this work, we developed three TDO mutants in residues phenylalanine 366, threonine 365 and isoleucine 324, with the aim to alter the chemo-, regio- and stereoselectivity of the biotransformation of arenes. While no changes in the regioselectivity of the process were observed, dramatic variations in the chemo- and enantioselectivity were found for mutants I324F, T365N and F366 V in a substrate-dependent manner. (Figure presented.).

Full text of DOI:10.1002/adsc.201700444

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DOI: 10.1002/adsc.201700444
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Site-Directed Mutagenesis Studies on the Toluene Dioxygenase
Enzymatic System: Role of Phenylalanine 366, Threonine 365 and
Isoleucine 324 in the Chemo-, Regio-, and Stereoselectivity
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Marıa Agustina Vila, Diego Umpierrez, Nicolas Veiga, Gustavo Seoane,
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Ignacio Carrera, * and Sonia Rodrıguez Giordano *
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Laboratorio de Biocatalisis y Biotransformaciones, Departamento de Quımica Organica–Departamento de Biociencias,
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Facultad de Quımica, Universidad de la Republica, Montevideo, Uruguay
Tel: +59829244543
Fax: +59829241906; e-mail: icarrera@fq.edu.uy; soniar@fq.edu.uy
Quꢀmica Inorgꢁnica, Departamento Estrella Campos, Facultad de Quꢀmica, Universidad de la Repfflblica, Montevideo,
Uruguay
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Received: April 7, 2017; Revised: April 29, 2017; Published online: && &&, 0000
Supporting information for this article is available on the WWW under https://doi.org/10.1002/adsc.201700444
and efficient synthetic schemes. This has been high-
lighted in several reviews, which show the importance
Abstract: Toluene Dioxygenase (TDO) enzymatic
complex has been widely used as a biocatalyst for
the regio- and enantioselective preparation of cis-
cyclohexadienediols, which are very important
starting materials for organic synthesis. However,
the lack of regio- and stereodiversity of the
dioxygenation process by TDO and related dioxy-
genases constitutes a clear drawback when planning
the use of these diols in synthetic schemes. In this
work, we developed three TDO mutants in residues
phenylalanine 366, threonine 365 and isoleucine
324, with the aim to alter the chemo-, regio- and
stereoselectivity of the biotransformation of arenes.
While no changes in the regioselectivity of the
process were observed, dramatic variations in the
chemo- and enantioselectivity were found for
mutants I324F, T365N and F366 V in a substrate-
dependent manner.
of these dienediols for enantioselective organic syn-
thesis.[1] More than 400 cis-cyclohexadienediols have
been reported so far, with the methyl and halogen
substituted ones (1, X=CH3 or Cl, Br, I) being the
most used for synthetic preparations.[2]
Keywords: Toluene dioxygenase; cis-cyclohexadie-
nediols; Rieske dioxygenases; site-directed muta-
genesis
Scheme 1. (A) Use of cis-cyclohexadienediols in enantiose-
lective organic synthesis. (B) Regio- and stereoselectivity
exhibited by wild type dioxygenases (1 and 2) and by the
proposed mutants in this work.
Introduction
52 Enantiopure cis-cyclohexadienediols of type (1) have
53 been extensively used as starting materials for organic
These compounds are prepared from the corre-
54 synthesis of bioactive compounds and natural products sponding arenes using Rieske non-heme iron depend-
55 (Scheme 1A). The useful array of functional groups ent dioxygenases, which are responsible for the first
56 present in (1) allows the use of regio- and stereo- step in the microbial degradation of aromatic arenes.[3]
57 controlled organic transformations resulting in short To date, no other non-biocatalytic chemical method
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for their production at a preparative scale is known.[4] tive scale is highly desirable. In addition, for certain
Rieske non-heme iron dependent dioxygenases are substrates (as mono-substituted alkyl benzenes, indene
multicomponent enzymes constituted by a reductase, and styrene) TDO presents poor chemoselectivity,
an oxygenase and in some cases a ferredoxin, and are since it can catalyze hydroxylations on the aromatic
well known for their ability to activate molecular ring and in the side chain, rendering a mixture of
oxygen to oxidize organic compounds.[3d] Besides the compounds (see Scheme 2, compounds 7a–b, 8a–b and
dioxygen addition to aromatic rings, Rieske dioxyge- 9a–b)
nases are also able to catalyse other reactions, such as
sulfoxidation, benzylic oxidation, dehydrogenation,
10 and O- and N-dealkylations.[5] The oxygen activation
11 process occurs in the oxygenase component that
12 contains a Rieske-type [2Fe–2S] cluster and a mono-
13 nuclear non-heme iron in the active site.[3c] Rieske
14 dioxygenases are classified considering the homology
15 between the alpha subunits of the oxygenase compo-
16 nent, as well as the scope of arenes that are accepted
17 for the dioxygenation process. Five classes are
18 described: 1. Phthalate, 2. Benzoate, 3. Naphthalene,
19 4. Toluene and Bi-Phenyl and 5. Salicylate Dioxyge-
20 nases.[6]
21
Toluene Dioxygenase (TDO) is the enzymatic
22 complex that has been used to produce the vast
23 majority of the cis-cyclohexadienediols used for
24 synthetic applications.[1a,d] The biotransformation of
25 arenes using this enzyme is carried out using whole
26 cell biocatalysis with mutant organisms (P.putida F39/
27 D, P.putida UV4) or recombinant strains overexpress-
28 ing the TDO genes.[1a] A recent publication by our
29 group describes the biotechnological procedure to
30 obtain 35 g/L of cis-3-bromocyclo-3,5-hexadiene-1,2-
31 diol using the recombinant E. coli JM109
32 (pDTG601).[7]
Scheme 2. Substrates 5–9 and biotransformation products
5a–9b using wild type TDO. Unless stated, ee are >99%
(data from literature for wild type TDO, see supporting
information for specific references).
In this work, we propose to use site-directed
mutagenesis in selected amino acids of the active site
33
The TDO enzymatic system admits a high struc- pocket of TDO to alter the chemo-, regio- and
34 tural diversity of substrates, and the dioxygenation stereoselectivity of the dioxygenation of arenes. To the
35 reaction is highly regio- and stereoselective.[1a] Re- best of our knowledge, there are no previous studies
36 garding monosubstituted benzene derivatives, dioxy- about mutagenesis of TDO with the aim to alter the
37 genation always takes place at C2-C3 in a stereo- selectivity of the dioxygenation process. Previous
38 defined manner to afford diols of type (1) (except for reports about TDO mutants have used site-directed
39 fluorobenzene, where trace amounts of the regioiso- mutagenesis for determining the iron ligands in the
40 meric diol at C3-C4 are also obtained) (Scheme 1B).[8] active site, or directed evolution to improve the
41 This remarkable regio- and stereoselectivity for the bioconversion of indene or to accept 4-picoline as a
42 aromatic dioxygenation is shared by most Rieske substrate.[9]
43 dioxygenases except for Benzoate dioxygenase
44 (BzDO), which renders the dienediols of benzoic acids
45 with the opposite stereochemistry and with the diol Results and Discussion
46 moiety in the position 1,2 (ipso addition) (see
Mutant’s Selection and Design
47 Scheme 1B, compound 2).[1g]
48
The lack of regio- and stereodiversity of the To select the amino acid residues to be mutated in the
49 dioxygenation process by TDO and related dioxyge- active site of TDO, we used previous findings on the
50 nases constitutes a clear drawback for the synthetic regio- and stereoselectivity of the dihydroxylation of
51 potential of the cis-cyclohexadienediols in organic arenes using Naphthalene (NDO) and Biphenyl
52 synthesis, since the ready access to other regioisomers Dioxygenases (BPDO), which share a high degree of
53 (e.g. dioxygenation product at C3-C4) or the enan- homology with TDO.[6] Although these enzymes have
54 tiomer of (1) (see Scheme 1B, compounds 3 and 4), is not been greatly used for synthetic purposes, they
55 precluded. Although synthetic protocols have been were deeply studied in the past for their potential
56 developed to obtain regio- and stereoisomers of (1),[1a] application in bioremediation processes. In addition,
57 a biocatalytic approach to produce them in a prepara- NDO crystal structure has been early resolved,[10]
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which allowed for an in-depth study of its active site.
Amino acid Phe352 of NDO was shown to be crucial
for determining the regio- and stereoselectivity of the
dihydroxylation of biphenyl, anthracene and
phenanthrene.[11] Although the overall enzymatic
activity decreased in the studied mutants, changes in
the Phe352 for medium size amino acids promoted
biphenyl dioxygenation in the 3,4 position as the
major product (with variable ee%).[11c,12] Concerning
10 BPDO, residues Ile335 and Thr376 were determined
11 to alter the substrate scope and regioselectivity for the
12 dihydroxylation of biphenyl.[13] Previous studies using
13 Pseudomonas pseudoalcaligenes KF707 BPDO, dem-
14 onstrated that Ile335 was very important for determin-
15 ing substrate specificity and regioselectivity. The
16 substrate scope for a range of polychlorinated biphen-
17 yls (PCB) was strongly associated with the amino acid
18 side chain in this position. This was also in agreement
19 with the reported biotransformation ability of the
20 Burkholderia cepacia LB400 BPDO, which presents a
21 phenylalanine instead of an isoleucine residue in this
Figure 1. Active site of TDO loaded with O2 and toluene.
Selected amino acids I324, F366 and T376 for site-directed
mutagenesis are shown in yellow. Color code: C (grey), N
(blue), O (red), Fe (purple).
22 position.[14] The I335F substitution markedly affected described,[7] and cells were collected at stationary
23 substrate specificity and regioselectivity, promoting phase to perform resting cell assays (see experimental
24 dihydroxylation in the 3,4 position.[13e,14b]
section).
25
Regarding position 376 in BPDO, previous reports
26 have shown that both residues threonine and aspar-
27 agine are present at position 376 in different natural
Substrates
28 BPDO enzymes. All enzymes with broad substrate Five substrates were selected for characterization of
29 specificity contain asparagine at the 376 position, the different mutant enzymes: toluene, bromobenzene,
30 whereas the enzymes with narrow substrate specificity propylbenzene, styrene and indene (Scheme 2, 5–9).
31 contain threonine at this site.[15] Different mutations While all the selected substrates allowed us to detect
32 have been performed on this residue of BPDO. The changes in enzymatic activity and regio- and stereo-
33 introduction of amino acids smaller than Thr resulted selectivity of the different mutants, substrates (7–9)
34 in enzyme variants with reduced activity, while the also permitted the evaluation of the chemoselectivity,
35 inclusion of larger amino acids precluded substrate since oxidation can occur at two different positions for
36 binding.[13e]
An alignment performed among the abovemen- propylbenzene and indene renders the benzylic hy-
these three substrates (for the wild type TDO,
37
38 tioned BPDOs (B. xenovorans and P. pseudoalcali- droxylation products in good yields (7b, 9b), and
39 genes BPDO), NDO and TDO, allowed the identifica- styrene gives the dihydroxylation product in the side
40 tion of TDO residues equivalent to important chain olefin, (8b).
41 positions on NDO and BPDO (Figure S0 Supplemen-
42 tary Information). Particularly, Phe366 corresponds to
43 Phe352 in NDO, while Ile324 and Thr365 are equiv-
Biotransformation Results
44 alent to Ile335 and Thr376 from BPDO. Thus, these Results for the biotransformation of the mentioned
45 three residues were selected for site directed muta- substrates using the TDO mutants are described in
46 genesis and I324F, T365N and F366V mutations were Table 1. Regarding mutants I324F and T365N, a
47 performed. Since the crystal structure of TDO was similar outcome in the biotransformation of all
48 obtained in 2009,[16] we could identify the selected substrates was found except for indene (9). A general
49 residues in the active site pocket of this enzyme reduction in enzyme activity for bromobenzene (5)
50 (Figure 1).
and toluene (6) was observed for both mutants, with
51
Expression vectors for mutant enzymes I324F, almost complete loss of activity for (6), and a slight
52 T365N and F366V were obtained by quick change decrease in yield for (5). Interestingly, changes in
53 mutagenesis, using pDTG601 as template. The ob- chemoselectivity were observed with both substrates
54 tained expression vectors were transformed into E. (7) and (8). Hydroxylation of the benzylic position for
55 coli JM109 yielding the whole cell biocatalysts for propylbenzene (7) was not detected for both cases,
56 characterization. The different recombinant strains indicating that a change in substrate accommodation
57 were grown in batch fermentation as previously in the active site probably precludes this position from
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Table 1. Biotransformation results using TDO mutants and bromobenzene, toluene, propylbenzene, styrene and indene as
substrates.
3
4
5
TDO (wild type)
%
I324F
%
T365N
%
F366 V
%
Substrate
Product
ee%b
a:b c
ee%b
a:bc
ee%b
a:bc
ee%b
a:bc
–
a
a
a
a
5
6
5a
6a
7a
7b
8a
8b
9a
9b
57
19
31
10
8
1
5
7
99
–
–
–
49
nd
12
nd
3
5
nd
nd
99
–
>99
–
>99
54
–
–
–
55
3
18
nd
3
11
2
15
99
–
–
–
2
60
6
7
8
9
nd
nd
nd
nd
2
>99
90
>99
>99
33
>99
–
>99
>99
23
–
–
–
17
À9d
–
7
77:23
100:0
100:0
–
10
11
12
13
14
15
16
17
18
19
20
8
9
85:15
42:58
37:63
–
24:76
11:89
0:100
100:0
9
nd
29
–
53
[a] Overall biotransformation yield determined by 1H-NMR of the crude biotransformation mixture using 1,3-dinitrobenzene as
internal standard.
[b] Enantiomeric excess was determined using chiral HPLC (see experimental section).
[c] a:b ratio was determined by H-NMR of the crude biotransformation mixtures.
1
[d] The minus sign indicates an inverted relation of enantiomers. nd=not detected.
21 being oxidised. We have recently reported a computa-
22 tional model for TDO that nicely explained and
23 predicted the outcome of the biotransformation for
24 mono and 1,4-disubstituted arenes.[17] This model
25 allowed us to gain reliable insights into the benzylic
26 hydroxylation of monosubstituted alkyl arenes, ex-
27 plaining the reasons behind propylbenzene being a
28 better substrate for benzylic oxidation than similar
29 compounds with longer or shorter substituents. Opti-
30 mal fit of this substrate in TDO active site, with its
31 side chain facing residues Leu272, Val309 and Ile324,
32 compels the benzylic carbon atom to be closer to the
33 coordinated O2 molecule. In the case of the mutant
34 I324F, our model shows that the change for phenyl-
35 alanine generates a steric hindrance for accommodat-
36 ing the substrate in the same position, and the benzylic
37 carbon atom moves 1.5 Š further away from the O2
38 molecule (Figure 2). This nicely explains the observed
39 result for I324F mutant, for which the benzylic
40 oxidation was not detected.
Figure 2. Propylbenzene docked into the active site of wild-
type (green) or I324F (grey) TDO. As it can be seen, the
benzylic position of propylbenzene is further away from the
O2 molecule for I324F (distances are shown in Š). Color
code: C (grey), N (blue), O (red), Fe (purple), S (yellow).
41
Mutants I324F and T365N also presented an
42 interesting behaviour with styrene (8). Although
43 dihydroxylation of the aromatic ring was also reduced
44 for this substrate for both mutants, dihydroxylation of
A docking study of styrene with TDO and I324F
45 the alkene on the side chain increased five folds for mutant, based on our model,[17] sheds light on the
46 I324F, and ten times for T365N compared to the wild observed difference in chemoselectivity (Figure 3). In
47 type enzyme. This implies a big change in chemo- the wild type enzyme, 85% of the formed diols
48 selectivity, favouring formation of the side chain diol corresponds to dihydroxylation of the aromatic ring.
49 while in the wild type the dioxygenation in the The docking studies revealed that styrene accommo-
50 aromatic ring is preferred. Regarding the enantiose- dates in the active site at an ideal distance from the
51 lectivity for the dihydroxylation of the side chain, both molecular O2, favouring dihydroxylation of the aro-
52 mutants displayed different results. In the case of matic ring in wild type TDO. The presence of a larger
53 I324F enantioselectivity was changed from >99% to residue at position 324, displaces styrene ring from its
54 54%, while for T365N it remained unchanged (> coplanar geometry with molecular O2, disfavouring its
55 99%). This indicates that Ile 324 is an important oxidation. Additionally, the styrene side chain comes
56 residue for determining the stereoselectivity of dihy- closer to the reactive O2 molecule which could
57 droxylation of some substrates.
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account for the observed increment in the yield of side agrees with previous results obtained with
chain dihydroxylation.
BPDO.[13c,e,14–15]
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The TDO F366V mutant presented a drastic
reduction in enzyme activity, larger than those
observed for any other mutant. Indeed, the F366V
variant was not capable of dihydroxylating the
aromatic ring for substrates (6), (7) and (8), showing
only 2% conversion for bromobenzene (5), the best
substrate for the wild type biocatalyst. This general
decrease in enzyme activity could be correlated,
according to our model, with a general increment in
the estimated binding DG, due to the decrease in
substrate-receptor lipophilic interactions (see Ta-
ble S4-S8). When the Phe366 residue is exchanged for
valine, the structural changes in the active site upon
mutation are significant (see for example Figure S7c),
dramatically increasing its size. In general, this can be
associated with more variation among the docking
solutions (Tables S4–S8; an exception to this trend is
toluene), which in turn may imply that substrates are
loosely associated to the active site, and reaction
become less likely to occur.
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14
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26
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Figure 3. Styrene docked into the active site of wildtype
(green) or I324F (grey) TDO. The benzylic carbon atom is
nearer to the O2 molecule for I324F (distances in Š). Color
code: C (grey), N (blue), O (red), Fe (purple), S (yellow).
It is noteworthy that this mutation drastically
affected the enantioselectivity of the enzyme, lowering
the enantiomeric excess for the formation of (5a)
from >99% to 60% (Table 1). Our model offers some
As mentioned before, the main difference between answers concerning this change in the F366V enantio-
28 I324F and T365N was found in the biotransformation selectivity during bromobenzene dihydroxylation. Ac-
29 of indene (9). While I324F showed no activity for this cording to the docking results shown in Figure S4b,
30 substrate, T365N doubled the production of (9b), different orientations of the substrate into the active
31 while reducing by half the production of (9a). In the site were found, which place the bromine atom almost
32 case of I324F, our model shows some trends that could in the opposite direction from the usual orientation,
33 explain the lack of reaction of indene with this mutant. and can in principle give rise to the respective
34 The introduction of a phenylalanine bulky residue at enantiomer. Remarkably enough, the calculated enan-
35 position 324 (Figure 1) destabilizes the binding posi- tiomeric excess is 100% for the wildtype TDO and
36 tion for indene favouring different orientations (see 65% for F366V mutant (Table S5), in excellent agree-
37 table S4 supporting info). This suggests that indene is ment with the experimental values (99 and 60%,
38 probably loosely bound into the active site of I324F, respectively). The change in enantioselectivity for
39 which can explain the loss of enzymatic activity.
F366V was also observed for the preparation of (8b),
40
The outcome for the biotransformation of (7), (8) (9a) and (9b). This mutant can then provide an
41 or (9) with the mutant T365N cannot be explained by interesting foundation to build up an enzyme with
42 the model. The mutation at position 365 does not opposite stereoselectivity, using saturation mutagene-
43 produce a significant change nearby the substrate (see sis in phenylalanine 366 or combining the F366V
44 Figure 1), and since the protein backbone is fixed mutation with others that anchor the substrate in the
45 during the docking runs, our model does not consider opposite direction.
46 any protein structural or electronic changes that may
Intriguingly, while this mutant presented almost
47 be involved. In order to rationalize this phenomenon, null activity for the hydroxylation of the aromatic
48 we are now carrying out molecular mechanics and ring, that was not the case for the oxidation of
49 DFT optimization calculations on mutant T365N. substrates (8) and (9) in non-aromatic positions.
50 These results will be published in due course.
Production of (8b) and (9a) were doubled with this
51
Overall, although we did not detect any change in mutant, relative to the wild type TDO. These results
52 regioselectivity for TDO I324F and T365N when indicate that the F366V TDO mutant did not lose the
53 tested with substrates (5–9), we found that these hydroxylation capability, but drastically changed its
54 residues are indeed very important for determining chemo and enantioselectivity.
55 chemoselectivity of the enzyme. Both amino acids also
When styrene is docked into the active site of
56 modulate the enantioselectivity for some substrates. F366V, new solutions are found that place the benzylic
57 The importance of these residues in enzyme activity carbon near the O2 molecule (Table S8 and Fig-
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ure S7c). This phenomenon is much more pronounced the monohydroxylation at the benzylic position takes
than for the wild type TDO, and explains the better place. This complex scenario can only be satisfactorily
yield formation of (8b).
modelled by a higher-level computational model
In the case of indene, the computational model based on molecular mechanics or electronic structure
indicates that the orientation compatible with the methods. We are now starting to work on this issue,
formation of (9b) for the wildtype enzyme, was not trying to rationalize the TDO mechanism of action.
longer found for the F366V mutant (Table S4). This
suggests that the indene monohydroxylation ability of
TDO can be suppressed by the F366V mutation, in
Conclusion
10 agreement with the experimental findings. Several Three different TDO mutants have been developed
11 efforts have been oriented to the development of through site directed mutagenesis, and the effects of
12 TDO mutants that favoured formation of indene diol the different mutations have been analysed for the
13 (9a) over the monooxygenation product (9b).[18] biotransformation of substrates (5–9) using our pre-
14 Interest in this product relates to its use as a precursor viously reported computational model. None of the
15 in the synthesis of the antiviral agent Indinavir. developed mutants showed a variation in the regiose-
16 Previous efforts have developed a TDO variant that lectivity of arene dihydroxylation. However, chemo-
17 accumulated five different mutations and reduced the and enantioselectivity were largely affected in a
18 formation of 1-indenol from 60% to 20% (showing a substrate dependent manner.
19 decreased overall catalytic activity). The F366V
Drastic changes in chemoselectivity were observed
20 mutant reported here, renders exclusively the diol for the biotransformation of propylbenzene and
21 (9a) in an increased yield in contrast to the wild type styrene with I324F and T365N mutants, indicating that
22 enzyme (showing a better overall effect than the these positions are important for determining the
23 reported accumulation of five mutations mentioned orientation of the substrate in the active site and the
24 before). In agreement with this previously reported overall outcome of the biotransformation. In the case
25 TDO mutant, the F366V enzyme variant also affected of I324F mutation, the inserted phenylalanine residue
26 the stereoselectivity of the reaction favouring the is much bulkier than the isoleucine amino acid. In this
27 opposite enantiomer of (9a) compared to that pro- regard, the substrates bearing large and flexible side
28 duced using the wild type enzyme. These results show chains (propylbenzene) can only accommodate them
29 the importance of Phe366 on the search of an as in Figure 2, making the carbon atom at the benzylic
30 adequate biocatalyst for the synthesis of Indinavir and position further away from the coordinated O2 mole-
31 can be used as a foundation for further studies.
cule, and preventing the monohydroxylation from
32
The effect of F366V mutation on the enantioselec- taking place. When the chain is short and rigid
33 tivity is much larger than the one observed for the (styrene), the Phe324 residue pushes and tilts the
34 F352V substitution in NDO. Additionally, Phe352 substrate towards the O2 molecule, favouring the
35 position in NDO was shown to have an important oxidation of the olefin against the dioxygen addition
36 effect on the regioselectivity of this enzyme. Both to the aromatic ring (Figure 3).
37 NDO mutants, F352V and F352L, favoured dihydrox-
The most dramatic changes in enzyme activity
38 ylation in the 3,4 position for biphenyl and were observed for the F366V variant. This mutation
39 phenanthrene. The authors argued that stronger largely affected both the yield and the enantioselectiv-
40 effects were associated to the bulkier substrates since ity of the enzyme, lowering the enantiomeric excess
41 they are likely to come in closer contact with all amino for the formation of (5a) from >98% to 60%. The
42 acids at the active site. This could also be the case for calculated enantiomeric excesses are in excellent
43 our mutants, where larger changes in chemoselectivity agreement with these values, which adds further
44 are associated to bulkier substrates (8) and (9); evidence on the satisfactory performance of the
45 however, changes in regioselectivity were not ob- developed computational model. This mutant can then
46 served with any of the tested substrates.
provide an interesting foundation to build up an
47
The possibility of having different substrate orien- enzyme with opposite stereoselectivity, which will
48 tations in the active site could explain why a mixture constitute a nice tool in organic synthesis. Further-
49 of stereoisomers is observed during some of the more, F366V presented a switch in chemoselectivity
50 biotransformations, rather than an enantiomerically for the oxidation of indene, yielding (9a) as the only
51 pure product. It could also explain the switch on product. This is extremely interesting for the biocata-
52 chemoselectivity. Even though our docking model lytic preparation of Indinavir.
53 gave us some answers, the computational assessment
Overall, our results shed light on the importance of
54 of this phenomenon for the most flexible substrates is Ile324, Thr365 and Phe366 residues in determining the
55 not straightforward. It is highly possible that the side chemo- and enantioselectivity of toluene dioxygenase.
56 chain of substrates (7) and (8) can be rearranged The reported results constitute an interesting basis for
57 before the dihydroxylation at the C=C double bond or further studies in the field.
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England Biolabs protocol. Mutations were confirmed by
sequencing in Macrogen, Korea.
1
2
3
Experimental Section
Strains and Growth Conditions
E. coli JM109 was acquired from New England BioLabs
(Ipswich, MA, USA), and E. coli JM109 (pDTG601) was
generously donated by Prof. David T. Gibson (1938–2014).
All his strain collections are currently managed by Prof.
Rebecca Parales (University of California, Davis). E. coli
JM109, E. coli JM109 (pDTG601) and the three mutants E.
coli JM109 (pTDOM) were properly stored in cryovials at
À708C.
E. coli JM109 was routinely cultivated in LuriaÀBertani (LB)
broth or on LB agar plates at 378C. Recombinant strains E.
coli JM109 (pDTG601) and the three mutants E. coli JM109
(pTDOM) were grown in LB agar plates or LB broth
supplemented with ampicillin 200 mg/mL. Mineral Salts
Broth (MSB) was used as the culture media for growing E.
coli JM109 (pDTG601) and the three mutants E. coli JM109
(pTDOM) in batch fermentations.
4
5
6
7
8
9
Whole-Cell Biotransformation
All mutants and the wild type strain were grown in a 5 L
bioreactor (Sartorius Biostat A plus) using a modification of
our previously published procedure.[7] Fresh plates of E. coli
JM109 (pDTG601) and E. coli JM109 (pTDOM) were
streaked from frozen stocks, and a single colony was used to
inoculate 5 mL of LB-Amp which was incubated in orbital
shaker overnight (150 rpm, 378C). Two 500 mL Erlenmeyer
flasks containing 150 mL of MSB medium were inoculated
with 1.5 mL of the grown culture, and grown for 12 h in
orbital shaker at 378C, 150 rpm. Both entire cultures were
used to inoculate 2.5 L of defined mineral salt media in a
bioreactor (Sartorius Biostat A plus), and the system was set
to 500 rpm, 308C, and air flow rate of 4 L/min, and a pulse of
antifoam agents (Aldrich’s Antifoam Y: Silicone dispersion
in water 1:1) was added at the beginning of the run. The pH
value was controlled automatically to 6.8 by addition of
ammonium hydroxide during the whole process. A glucose
fed-batch started 6 hours after inoculation by adding glucose
(0.7 g/mL solution) from an initial rate of 0.08 mL/min to
0.54 mL/min in a 20 h period. IPTG was added to a final
concentration of 10 mg/L 12 hours after inoculation to induce
enzyme expression, and stirrer speed was set to 900 rpm.
After the culture reached the stationary phase (c.a.26 h, 30 g/
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Chemicals and Molecular Biology Reagents
Biotransformation substrates 5 to 9 were purchased from
Sigma Aldrich (St Louis, MO, USA) and were used without
further purification. Molecular biology reagents were pur-
chased from New England Biolabs (Ipswich, MA, USA) and
Thermo Fisher Scientific (Waltham, MA, USA). Solvents for
product purification were destilled prior to use. HPLC n-
hexane was acquired from Pharmco-Aaper and HPLC 2-
propanol was acquired from Carlo Erba Reagents.
L
cdw aprox) cells were harvested by centrifugation
(4000rpm, 20 min, 48C), and the pellet was resuspended in
isotonic phosphate buffer (KH2PO4 3 g/L, NaHPO4 12 g/L,
NaCl 0.7 g/L) to a final OD600 =30. Biotransformations were
performed using resting cells, thus, 100 mL of this cell
suspension were placed in 500 mL Erlenmeyer flasks and
supplemented with glucose to a final concentration of 5 g/L.
Neat substrates were added to a final concentration of
10 mM and incubated overnight at 288C, 150 rpm. Resting
cell cultures were centrifuged at 4000 rpm, 48C, 20 min, the
supernatant was collected and the cells were properly
disposed. The supernatant was lyophilized, and the solid was
washed with 33 100 mL ethyl acetate. The organic solvent
was evaporated in vacuo to afford a reaction crude that was
analysed by 1H-NMR for yield determination (vide infra).
Products were properly purified by preparative thin layer
chromatography (silica gel 60F-254 plates and visualized by
UV light 254 nm) or flash column chromatography using
silica gel (Kieselgel 60, EM reagent, 230–400 mesh). Pure
products were identified by -1H-NMR and compared with
literature data (vide infra)
DNA Manipulation
Standard procedures were used for DNA manipulation.[19]
Plasmid DNA was purified using commercial chromatogra-
phy kits (Thermo Fisher Scientific). Phusionꢃ High-fidelity
DNA polymerase (New England Biolabs) was used accord-
ing to the manufacturers’ instructions. PCR amplifications
were performed in a GeneAMP PCR system 2400 (Perkin
Elmer) using adequate cycling periods and DNA samples
were routinely analyzed by agarose gel electrophoresis
(Sambrook 2001). Transformations of electrocompetent cells
were performed on a BioRad MicroPulserTM Electroporator.
Generation of TDO Variants (TDOM): I324F, T365N,
F366V
Mutant variants of TDO were constructed using a modifica-
tion of Quikchangeꢃ method (Agilent Technologies). Ampli-
fications with Phusion High Fidelity DNA polymerase (New
England Biolabs) were performed on a GeneAMP PCR
system 2400 (Perkin Elmer) using plasmid pDTG601 as a
template and the primers listed in Table S1 (see supporting
info).
Proton chemical shifts (d) for the products obtained for the
wild type biotransformation are described below:
5a.
Cis-(1S,2S)-3-bromocyclohexa-3,5-diene-1,2-diol[20]
1H-NMR (400 MHz, CDCl3): d 6.37 (d, J=6 Hz, 1H), 5.88
(dd, J=9, 3 Hz, 1H), 5.79 (ddd, J=9, 6, 3 Hz, 1H), 4.47 (dt,
7, 3, 3 Hz 1H), 4.34 (d, J=7 Hz, 1H).
After amplification, samples were digested with DpnI
(Fermentas) for 3 h at 378C. DNA was precipitated with
ethanol and stored at À208C for at least 1 hour. DNA was
recovered in water and used to transform E. coli JM109
electrocompetent cells prepared in our lab according to New
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used to separate the enantiomers of 5a, 7a, 7b, 8a and 9a
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2
3
4
5
6
7
8
9
7a.
Cis-(1S,2R)-3-propylcyclohexa-3,5-diene-1,2-diol[21]
(see supporting information for the detailed retention times
for each enantiomer). Analytical conditions for separation of
1-phenyl-1,2-ethanediol and 1,2-indandiol were optimized by
analysis of the chemically synthetized racemic mixture.[26]
The typical injection volume was 20 ml and chromatograms
were monitored at maximum absorption wavelength for each
product (see supporting info). Assignment of the absolute
configuration was based on comparison with previously
reported results for wild type TDO.
1H-NMR (400 MHz, CD3COCD3): d 5.85 (ddd, J=10, 5,
2 Hz, 1H), 5.71–5.66 (m, 2H), 4.22 (s, 1H), 3.95 (t, J=5 Hz,
1H), 3.81 (s, 1H), 3.59 (s, 1H), 2.30–2.23 (m, 1H), 2.17–2.06
(m, 1H), 1.63–1.43 (m, 2H), 0.93 (t, J=7 Hz, 3H).
7b. Cis-(1S,2R)3-[(R)1’-hydroxyethyl]
cyclohexa-3,5-diene-1,2-diol[21]
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11
12
1H-NMR (400 MHz, CD3COCD3): d 5.90–5.87 (m, 2H),
5.73–5.70 (m, 1H), 4.22 (m, 1H), 4.17 (m, 1H), 3.80 (brs, 1H),
Docking Calculations
13 3.50 (brs, 1H), 1.75 (m, 1H), 1.58 (m, 1H), 0.93 (m, 3H).
14
Molecular-docking experiments were carried out according
to Vila et al[17] by using the GOLD docking program (v
4.1.2).[27] Various rotamers were considered for some of the
amino acids in the binding site: Glu 215 (8), Phe 216 (3), Asp
219 (4), Met 220 (4), Ile 276 (4), Val 309 (3), His 311 (4), Leu
321 (3), Ile 324 (3), Phe 366 (2). The mutated residues and
those in contact with them and with the substrate at the
same time were set free to move during the docking runs.
Each substrate was docked 40 times, and the docking modes
that achieved the highest Goldscore fitness were retained
and compared. The most stable docking poses were selected
considering the values of the ChemScore and Goldscore
scoring functions.[20,28] Other miscellaneous parameters were
assigned the default values given by the GOLD program.
The amino acid mutations and the rendering of the results
were done with Discovery Studio Visualizer software.[29]
15
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17
8a. Cis-2,3-dihydroxy-1-vinylcyclohexa-4,6-diene[22]
1H-NMR (400 MHz, CD3COCD3): d 6.42 (dd, J=16, 8 Hz
1H), 5.97–5.91 (m, 2H), 5.80 (d, J=9, 1H), 5.50 (d, J=18 Hz,
1H), 5.12 (d, J=11 Hz, 1H), 4.41–4.37 (m, 1H), 4.32–4.28 (m,
1H), 3.98 (d, J=8 Hz, 1H), 3.63–3.61 (m, 1H).
18
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8b. (R)1-Phenyl-1,2-ethanediol[23]
1H-NMR (400 MHz, CD3COCD3): d 7.43–7.37 (m, 2H),
7.35–7.29 (m, 2H), 7,28–7.21 (m, 1H), 4.72 (dd, J=8, 4 Hz,
1H), 4.34 (d, J=4 Hz, 1H), 3.85 (dd, J=7, 5 Hz, 1H), 3.66–
3.62 (m, 1H), 3.57–3.51 (m, 1H).
9a. Cis-(1S,2R)-indandiol[24]
1H-NMR (400 MHz, CDCl3): d 7.51–4.43 (m, 1H), 7.32–7.25
(m, 3H), 5.00 (s, 1H), 4.50 (s, 1H), 3.14 (dd, J=16, 6 Hz, 1H),
2.98 (dd, J=16, 4 Hz, 1H), 2.80 (brs, 1H), 2.69 (brs, 1H).
Acknowledgements
Authors would like to thank the Agencia Nacional de
´
´
Investigacion e Innovacion (ANII, Project Fondo Clemente
Estable 103485), CSIC Grupos I+D 981 (UdelaR) and
9b. (1S)-Indenol[24]
1H-NMR (400 MHz, CDCl3): d 7.54 (d, J=7, 1H), 7.46–7.44
(m, 4H), 6.76 (d, J=6 Hz, 1H), 6.43 (dd, J=6, 2 Hz, 1H),
5.21 (s, 1H).
´
PEDECIBA Quımica for financial support. M. A. Vila
´
´
thanks UdelaR-Comision Academica de Posgrado for a
Ph.D. scholarship. The authors would like to thank Daniel
´
Galdran (visiting student from Universitat de Valencia-Spain)
for the preparation of the racemic diols (8b) and (9a).
Analytical Methods
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Site-Directed Mutagenesis Studies on the Toluene Diox-
ygenase Enzymatic System: Role of Phenylalanine 366,
Threonine 365 and Isoleucine 324 in the Chemo-, Regio-,
and Stereoselectivity
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M. A. Vila, D. Umpierrez, N. Veiga, G. Seoane, I.
´
Carrera*, S. Rodrıguez Giordano*