Communication
ChemComm
Table 3 Baeyer–Villiger oxidation of ketones 1a–1f by WT CHMOs
and variants
and D. Hilvert, Annu. Rev. Biochem., 2018, 87, 131; (e) G. Qu, A. Li,
C. G. Acevedo-Rocha, Z. Sun and M. T. Reetz, Angew. Chem., Int. Ed.,
2020, DOI: 10.1002/anie.201901491.
CHMOs
Enzyme 1a
1b
1c
1d
1e
1f
2 (a) W. P. Dijkman, C. Binda, M. W. Fraaije and A. Mattevi, ACS
Catal., 2015, 5, 1833; (b) X. D. Kong, Q. Ma, J. Zhou, B. B. Zeng and
J. H. Xu, Angew. Chem., Int. Ed., 2014, 53, 6641; (c) A. O. Magnusson,
M. Takwa, A. Hamberg and K. Hult, Angew. Chem., Int. Ed., 2005,
CHMOThermo WT
51(R) 99(S) 99(S) 94(S) 34(À) 89(S)
98(R) 99(S) 99(S) 97(S) 99(À) 98(S)
91(S) 99(S) 88(S) 98(R) 94(+) 98(R)
94(S) 99(S) 74(S) 96(R) 93(+) 98(R)
95(S) 99(S) 50(S) 98(R) 94(+) 98(R)
34(R) 99(S) 98(S) 74(S) 21(+) 15(S)
89(R) 99(S) 99(S) 98(S) 99(À) 99(S)
97(S) 98(S) 47(S) 98(R) 99(+) 99(R)
96(S) 97(S) 34(S) 94(R) 99(+) 98(R)
95(S) 98(S) 20(S) 94(R) 99(+) 98(R)
56(R) 99(S) 99(S) 98(S) 33(À) 95(S)
98(R) 99(S) 99(S) 99(S) 99(À) 99(S)
91(S) 94(S) 80(R) 99(R) 99(+) 94(R)
94(S) 94(S) 84(R) 99(R) 99(+) 96(R)
88(S) 94(S) 73(R) 99(R) 99(+) 84(R)
F279W
F279P
F279V
F279I
WT
F279W
F279P
F279V
F279I
4
4, 4582; (d) J. Xu, Y. Cen, W. Singh, J. Fan, L. Wu, X. Lin, J. Zhou,
M. Huang, M. T. Reetz and Q. Wu, J. Am. Chem. Soc., 2019, 141, 7934.
3
4
(a) M. T. Reetz and S. Wu, J. Am. Chem. Soc., 2009, 131, 15424;
CHMORhodo
(
b) S. Wu, J. P. Acevedo and M. T. Reetz, Proc. Natl. Acad. Sci. U. S. A.,
010, 107, 2775.
(a) K. Balke, A. Beier and U. T. Bornscheuer, Biotechnol. Adv., 2018,
6, 247; (b) M. J. F u¨ rst, A. Gran-Scheuch, F. S. Aalbers and M. W. Fraaije,
2
3
ACS Catal., 2019, 9, 11207; (c) J. Dong, E. Fernandez-Fueyo, F. Hollmann,
C. E. Paul, M. Pesic, S. Schmidt, Y. Wang, S. Younes and W. Zhang,
Angew. Chem., Int. Ed., 2018, 57, 9238; (d) D. Holtmann, M. W. Fraaije,
I. W. Arends, D. J. Opperman and F. Hollmann, Chem. Commun., 2014,
CHMORhodo2 WT
F282W
F282P
F282V
F282I
5
0, 13180; (e) S. Schmidt, K. Castiglione and R. Kourist, Chemistry, 2018,
4, 1755; ( f ) J. B. Wang, G. Li and M. T. Reetz, Chem. Commun., 2017,
3, 3916.
D. Sheng, D. P. Ballou and V. Massey, Biochemistry, 2001, 40, 11156.
2
5
5
small-sized substrates by mutating only a single site, such as 1b
and 1c. Remarkably, for 1c with a small ethyl group, we found the
reversal of enantioselectivity was achieved by CHMORhodo2 from
an ee value of 99% (S) (WT) to 84% (R), other CHMO variants from
different species also gave an increased ratio of (R)-products in
comparison to WT. In the future, the mutation of F277 combined
with other known active sites (e.g., L143, P431–L435 and F505)
would be implemented to verify if there are any cooperative or
additive effects for the reversal of enantioselectivity toward sub-
strates with small substituents.
In conclusion, the ‘‘designed’’ directed evolution of enzymes
is usually focused on the mutation of ligand-binding sites,
especially the sites in direct contact with the substituents of
ligands. Here, we discovered that a ‘‘second sphere’’ site of
enzymes acts as a stereo- and regiocontrolled switch, mutagen-
esis of which may induce the reshaping of active sites so as to
tune the preference of chiral products. Furthermore, the knowl-
edge obtained from engineering CHMOAcineto could be effi-
ciently transferred to other CHMOs.
6 (a) I. Polyak, M. T. Reetz and W. Thiel, J. Am. Chem. Soc., 2012,
1
34, 2732; (b) I. Polyak, M. T. Reetz and W. Thiel, J. Phys. Chem. B,
2013, 117, 4993.
7 (a) M. T. Reetz, B. Brunner, T. Schneider, F. Schulz, C. M. Clouthier and
M. M. Kayser, Angew. Chem., Int. Ed., 2004, 43, 4075; (b) M. D. Mihovilovic,
F. Rudroff, A. Winninger, T. Schneider, F. Schulz and M. T. Reetz, Org.
Lett., 2006, 8, 1221; (c) Z.-G. Zhang, G.-D. Roiban, J. P. Acevedo, I. Polyak
and M. T. Reetz, Adv. Synth. Catal., 2013, 355, 99; (d) C. M. Clouthier,
M. M. Kayser and M. T. Reetz, J. Org. Chem., 2006, 71, 8431; (e) Y. Hu,
J. Wang, Y. Cen, H. Zheng, M. Huang, X. Lin and Q. Wu, Chem. Commun.,
2019, 55, 2198; ( f ) M. T. Reetz, F. Daligault, B. Brunner, H. Hinrichs and
A. Deege, Angew. Chem., Int. Ed., 2004, 43, 4078.
8 (a) G. Li, M. Furst, H. R. Mansouri, A. K. Ressmann, A. Ilie,
F. Rudroff, M. D. Mihovilovic, M. W. Fraaije and M. T. Reetz, Org.
Biomol. Chem., 2017, 15, 9824; (b) G. Li, M. Garcia-Borras, M. Furst,
A. Ilie, M. W. Fraaije, K. N. Houk and M. T. Reetz, J. Am. Chem. Soc.,
2018, 140, 10464; (c) M. J. L. J. F u¨ rst, E. Romero, J. R. G o´ mez
Castellanos, M. W. Fraaije and A. Mattevi, ACS Catal., 2018, 8, 11648.
9
(a) K. Balke, M. Baumgen and U. T. Bornscheuer, ChemBioChem,
017, 18, 1627; (b) K. Balke, S. Schmidt, M. Genz and U. T.
Bornscheuer, ACS Chem. Biol., 2016, 11, 38.
0 (a) H. L. van Beek, E. Romero and M. W. Fraaije, ACS Chem. Biol.,
017, 12, 291; (b) Y. Zhang, Y.-Q. Wu, N. Xu, Q. Zhao, H.-L. Yu and
J.-H. Xu, ACS Sustainable Chem. Eng., 2019, 7, 7218.
11 C. T. Walsh and Y.-C. J. Chen, Angew. Chem., Int. Ed. Engl., 1988,
7, 333.
2
1
2
2
The financial support from the National Natural Science
Foundation of China (91956128) and the Natural Science
Foundation of Zhejiang Province (LY19B020014) are gratefully
acknowledged.
1
2 (a) E. Romero, J. R. G. Castellanos, A. Mattevi and M. W. Fraaije,
Angew. Chem., Int. Ed., 2016, 55, 15852; (b) I. A. Mirza, B. J. Yachnin,
S. Wang, S. Grosse, H. Bergeron, A. Imura, H. Iwaki, Y. Hasegawa,
P. C. K. Lau and A. M. Berghuis, J. Am. Chem. Soc., 2009, 131, 8848;
(
c) B. J. Yachnin, M. B. McEvoy, R. J. D. MacCuish, K. L. Morley,
P. C. K. Lau and A. M. Berghuis, ACS Chem. Biol., 2014, 9, 2843;
d) R. Orru, H. M. Dudek, C. Martinoli, D. E. Torres Pazmino,
A. Royant, M. Weik, M. W. Fraaije and A. Mattevi, J. Biol. Chem.,
011, 286, 29284–29291.
(
Conflicts of interest
2
There are no conflicts to declare.
13 Z. G. Zhang, R. Lonsdale, J. Sanchis and M. T. Reetz, J. Am. Chem.
Soc., 2014, 136, 17262.
Notes and references
14 (a) L. Zhou, X. Liu, J. Ji, Y. Zhang, X. Hu, L. Lin and X. Feng, J. Am.
Chem. Soc., 2012, 134, 17023; (b) M. J. Taschner, D. J. Black and
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(d) M. Ogasawara, Y. Tseng, S. Arae, T. Morita, T. Nakaya, W. Wu
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1
Selected reviews of directed evolution: (a) A. Currin, N. Swainston,
P. J. Day and D. B. Kell, Chem. Soc. Rev., 2015, 44, 1172; (b) H.
Renata, Z. J. Wang and F. H. Arnold, Angew. Chem., Int. Ed., 2015,
5
4, 3351; (c) N. J. Turner, Nat. Chem. Biol., 2009, 5, 567; (d) C. Zeymer
Chem. Commun.
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