Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate

Article abstract of DOI:10.1002/anie.201606722

Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5′-chloro-5′-deoxyadenosine (5′-ClDA) into 5′-fluoro-5′-deoxyadenosine (5′-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5′-[¹⁸F]FDA, with overall radiochemical conversion (RCC) more than 3-fold higher than wild-type FlA1. Kinetic studies of the two-step reaction revealed that the variants show a significantly improved k_catvalue in the conversion of 5′-ClDA into S-adenosyl-l-methionine (SAM) but a reduced k_catvalue in the conversion of SAM into 5′-FDA.

Full text of DOI:10.1002/anie.201606722

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201606722
German Edition: DOI: 10.1002/ange.201606722
Biocatalysis
Directed Evolution of a Fluorinase for Improved Fluorination
Efficiency with a Non-native Substrate
+
+
+
Huihua Sun , Wan Lin Yeo , Yee Hwee Lim , Xinying Chew, Derek John Smith, Bo Xue,
Kok Ping Chan, Robert C. Robinson, Edward G. Robins, Huimin Zhao,* and Ee Lui Ang*
Abstract: Fluorinases offer an environmentally friendly alter-
native for selective fluorination under mild conditions. How-
ever, their diversity is limited in nature and they have yet to be
engineered through directed evolution. Herein, we report the
directed evolution of the fluorinase FlA1 for improved
conversion of the non-native substrate 5’-chloro-5’-deoxyade-
nosine (5’-ClDA) into 5’-fluoro-5’-deoxyadenosine (5’-FDA).
The evolved variants, fah2081 (A279Y) and fah2114 (F213Y,
A279L), were successfully applied in the radiosynthesis of 5’-
deoxyadenosine (5’-FDA) and l-methionine (l-Met) through
[10]
a nucleophilic substitution (S 2) mechanism.
Pioneering
N
work by OꢀHaganꢀs group has demonstrated the application
1
8
[11]
of the fluorinase FlA in the radiosynthesis of 5’-[ F]FDA,
as well as the coupling of FlA with additional enzymes to
1
8
[12]
18
produce a range of F-labeled nucleosides
labeled sugar
and a F-
[
11b,13]
[14]
for PET applications.
To explore the substrate promiscuity for broader applica-
tions, FlA has been reported to accept 5’-chloro-5’-deoxy-
1
8
[15]
[16]
[
F]FDA, with overall radiochemical conversion (RCC) more
adenosine (5’-ClDA),
Met analogues,
2’-deoxyadenosine substrates,
l-
5’-
[
17]
[18]
than 3-fold higher than wild-type FlA1. Kinetic studies of the
two-step reaction revealed that the variants show a significantly
improved kcat value in the conversion of 5’-ClDA into S-
adenosyl-l-methionine (SAM) but a reduced kcat value in the
conversion of SAM into 5’-FDA.
a methylaza analogue of SAM,
chlorodeoxy-2-ethynyladenosine (ClDEA),
highly complex di- and tetra-cyclic peptide conjugates of
ClDEA. It is noteworthy that the catalytic efficiency of wild-
type fluorinases with non-native substrates is often re-
[19]
and even
[20]
[17–19]
duced.
as directed evolution
Powerful enzyme engineering approaches such
[21]
[22]
O
rganofluorine compounds play important roles in phar-
and rational protein design
can
[
1]
[2]
[3]
maceuticals,
agrochemicals,
materials,
and positron
potentially restore the impaired activity. Thomsen et al.
identified a FlA mutant with increased activity for the ethyl
analogue of l-Met through a series of rationally designed
[
4]
emission tomography (PET) imaging. Many advances
[
5]
have been made in CÀF bond formation, while selective
fluorination under mild conditions remains challenging. The
discovery and characterization of four naturally occurring
fluorinase enzymes (FlA, FlA1, FlA3 and NobA
offers an attractive prospect for using biocatalysts to produce
[
17]
point mutations. However, directed evolution has yet to be
employed to engineer fluorinases. Herein, we report directed
evolution of the fluorinase FlA1 to improve the fluorination
efficiency for the non-native substrate 5’-ClDA.
[
6]
[7]
[7]
[7–8]
)
[
9]
fluorinated compounds. These fluorinases convert S-adeno-
syl-l-methionine (SAM) and fluoride ions into 5’-fluoro-5’-
5’-ClDA is an attractive substrate that could be fluori-
[
15]
nated in the presence of l-Met (Scheme 1).
It can be
[
+]
[+]
[
*] Dr. H. Sun, W. L. Yeo, Prof. Dr. H. Zhao, Dr. E. L. Ang
Metabolic Engineering Research Laboratory (MERL)
Science and Engineering Institutes
Prof. R. C. Robinson
Department of Biochemistry, Yong Loo Lin School of Medicine
National University of Singapore, Singapore 117597 (Singapore)
and
Agency for Science, Technology, and Research (A*STAR)
31 Biopolis Way, Nanos #01-01, Singapore 138669 (Singapore)
NTU Institute of Structural Biology
E-mail: angel@merl.a-star.edu.sg
Nanyang Technological University (NTU)
5
and
9 Nanyang Drive, Singapore 636921 (Singapore)
Prof. Dr. H. Zhao
2
15 Roger Adams Laboratory, Box C3
University of Illinois at Urbana-Champaign
00 South Mathews Avenue, Urbana, IL 61801 (USA)
E-mail: zhao5@illinois.edu
School of Biological Sciences, NTU
60 Nanyang Drive, Singapore 637551 (Singapore)
6
and
[
+]
Lee Kong Chian School of Medicine
Dr. Y. H. Lim, X. Chew, Dr. K. P. Chan
50 Nanyang Avenue, Singapore 639798 (Singapore)
Institute of Chemical and Engineering Sciences (ICES), A*STAR
Dr. E. G. Robins
Singapore Bioimaging Consortium (SBIC), A*STAR
8
Biomedical Grove, Neuros #07-01/02/03, Singapore 138665
(
Singapore)
11 Biopolis way, #02-02, Singapore 138667 (Singapore)
Dr. D. J. Smith
Bioinformatics Institute, A*STAR
+
[
] These authors contributed equally to this work.
3
and
0 Biopolis Street, Matrix #07-01, Singapore 138671 (Singapore)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201606722.
Biotransformation Innovation Platform
61 Biopolis Drive, Proteos #04–14, Singapore 138673 (Singapore)
Dr. B. Xue, Prof. R. C. Robinson
Institute of Molecular and Cell Biology (IMCB), A*STAR
61 Biopolis Drive, Proteos #03–15, Singapore 138673 (Singapore)
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

Angewandte
Communications
Chemie
Scheme 1. The two-step conversion of 5’-ClDA into 5’-FDA by fluori-
nases.
readily synthesized and has good chemical stability. FlA1
from Streptomyces sp. MA37 was adopted as the parent
enzyme for directed evolution since FlA1 has been reported
[7]
to be the most efficient fluorinase for SAM and we have
found that FlA1 shows relatively higher activity with 5’-ClDA
among the four fluorinases (Figure S1 in the Supporting
Information). Saturation mutagenesis libraries were con-
structed by targeting 23 residues within 5 ꢁ from SAM in
a FlA1 homology model based on the structure of FlA
Figure 1. Comparison of the activity of FlA1 and the variants with 5’-
ClDA at 428C. Optimized conditions: 0.2 mm 5’-ClDA, 0.1 mm l-Met/
l-SeMet, 100 mm NaF, and 150 mm fluorinase. Yield=(concentration
of 5’-FDA generated/ concentration of the substrate 5’-ClDA)ꢀ100%.
(
PDB ID: 1RQP). The gene encoding nucleoside phosphor-
ylase (PNP) in Escherichia coli BL21 (DE3) was knocked out
in order to prevent 5’-FDA degradation by PNP in the cell
lysate. Then, the NNK libraries were cloned and expressed in
the BL21 (DE3) DPNP strain. A total of 1932 (23 ꢂ 84) clones
were screened in high-throughput assays.
Table 1: Comparison of the radiosynthesis efficiency of FlA1 and the
variants.
The screening identified fah2047 (A279L), fah2081
(
A279Y) and fah2019 (F213Y) as the top variants with
improved 5’-FDA yields (Table S1 in the Supporting Infor-
mation). The mutation F213Y was introduced to fah2047 to
give fah2114 (F213Y, A279L). FlA1 (FlA1 refers to wild-type
FlA1) and the variants (fah2047, fah2081, and fah2114) were
purified for validation through in vitro reactions. Since
purified fah2114 was found to be bound with SAM, which
cannot be removed (Figure S2), control reactions were run
without 5’-ClDA and l-Met to exclude the 5’-FDA produced
from endogenous SAM. At 378C, all of the purified variants
achieved higher 5’-FDA yields than FlA1 at both 4 h and 24 h
[a]
Fluorinase
T [8C]
RCC
(analytical) [%]
RCC
(overall) [%]
FlA1
FlA1
42
47
42
47
8Æ1
11Æ2
32Æ3
46Æ2
7Æ1
8Æ2
fah2081 (A279Y)
fah2114 (F213Y, A279L)
24Æ2
34Æ3
(
Figure S3).
To optimize the reaction conditions, we increased the
[
a] Overall RCC=% radioactivity in supernatantꢀ% RCC based on
radioHPLC. Typical % radioactivity in supernatant is in the range 73–
80%.
reaction temperature and found that the fluorinases were
more active at 428C (Figure S4). Enzyme concentrations and
substrate concentrations were also optimized for higher 5’-
FDA yields (data not shown). Under the optimized condi-
tions, we compared the reactions with either l-Met or l-
selenomethionine (l-SeMet; Figure 1). In the presence of l-
Met, fah2081 and fah2114 gave 2.4- to 2.7-fold higher yields
than FlA1 at 90 min (Table S2). The use of l-SeMet increased
the yield by around 2-fold for all of the fluorinases.
overall RCC for fah2114 is 4.3-fold higher than that for FlA1
at 478C. Thus, both evolved variants are at least 3-fold more
1
8
efficient than FlA1 in the radiosynthesis of 5’-[ F]FDA.
To understand how the variants improved the conversion
of 5’-ClDA into 5’-FDA, kinetic studies of the two-step
reaction (Scheme 1) were carried out for FlA1, fah2081, and
fah2114 (Tables 2 and 3 and Figure S7,S8). In the first step
(conversion of 5’-ClDA into SAM; Table 2), fah2081 and
fah2114 show faster turnover, with kcat values 4- and 29-fold
larger, respectively, than that of FlA1. On the other hand,
fah2081 and fah2114 are slower in the second step (conversion
of SAM into 5’-FDA; Table 3), with kcat values 2- and 9-fold
smaller, respectively, than that of FlA1. These kinetics data
reveal that only the first step of the reaction is significantly
accelerated by the directed evolution. The higher kcat value in
the first step is able to compensate for the slowing of the
second step, thereby resulting in an overall improved
conversion of 5’-ClDA into 5’-FDA.
Subsequently, the evolved fluorinases fah2081 and
1
8
fah2114 were used for the radiosynthesis of 5’-[ F]FDA.
The reaction conditions were optimized to achieve high
radiochemical conversion (RCC). We found that the RCC is
1
8
dependent on fluorinase concentration despite [ F]fluoride
being the limiting reagent (Figure S5). Moreover, the highest
conversion was observed when the concentration of 5’-ClDA
was approximately 4-fold that of fluorinase concentration
(
Figure S6). At 428C, the overall RCC for fah2081 is 3.4-fold
higher than that for FlA1 (Table 1). Since both fah2114 and
FlA1 are stable at 478C and higher temperatures may further
boost the RCC, we compared fah2114 with FlA1 at 478C. The
2
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
Table 2: Comparative kinetics data for FlA1 and the variants for the
[
a]
conversion of 5’-ClDA into SAM.
Fluorinase
Vmax
mmmin
5’-ClDA Km
[mm]
kcat
[min
[k /K ]
[mm min
cat
À1
m
À1
À1
À1
[
]
]
]
FlA1
fah2081
fah2114
0.67Æ0.04
2.66Æ0.22
19.28Æ0.44
8.36Æ0.82
59.26Æ4.44
9.45Æ2.22
0.13
0.53
3.86
16.08
8.96
407.91
[a] Assays contain 20 mm l-Met and various concentrations of 5’-ClDA.
Table 3: Comparative kinetic data of FlA1 and the variants for the
[
a]
conversion of SAM into 5’-FDA.
Fluorinase
Vmax
SAM Km
kcat
À1
[k /K ]
[mm min
cat
À1
m
Figure 3. Models of FlA1 mutants. A close-up of the SAM-binding sites
À1
À1
[mmmin
]
[mm]
[min
0.22
]
]
of FlA1 (green), fah2081 (cyan), and fah2114 (magenta), showing the
mutated positions at 213 and 279 as stick models. SAM is also shown
as a stick model (C yellow; O red; N blue).
FlA1
fah2081
fah2114
4.47Æ0.21
2.12Æ0.09
0.47Æ0.02
34.55Æ1.94
22.22Æ1.72
9.24Æ0.65
6.47
4.78
2.57
0.11
0.02
[a] Assays contain 200 mM NaF and various concentrations of SAM.
(Figure 3) were generated using our FlA1 crystal structure as
a template. Mutation at position 279 to bulkier residues may
restrict the movement of the 277–281 loop located above the
adenine ring, thereby leading to a tighter binding of the
adenine. Both mutations are close to the sidechain of Pro78,
and may participate in favorable hydrophobic contacts.
Leu279 of fah2114 is also within van der Waals contact of
an aromatic carbon in the adenine ring, thereby improving the
favorable hydrophobic environment. In addition, Tyr213 of
fah2114 is involved in polar interaction with Asp16, which
would act as a clamp between the protein monomers across
the ribose ring. Both of these monomers are also involved in
binding of the methionine moiety in SAM through 1) Ser269
and Arg270 on the monomer harboring Tyr213, and 2) Asp21
on the adjacent monomer harboring Asp16. Consequently,
tightening of the dimer interface through favorable interac-
tions along the binding site leads to further improved binding
of SAM.
[
7a]
The structure of FlA1 has been reported, but the data
[
7b]
were not initially publicly accessible. We crystallized the
wild-type FlA1 in space group P2 3 and solved the structure
to a resolution of 1.95 ꢁ (Figure 2; also see Figure S9 and
Table S3). FlA1 is composed of a dimer of trimers, similar to
that observed in the homologous FlA structures.
hexameric assembly was confirmed by size-exclusion chro-
matography (Figure S10). Adenosine and l-Met are bound at
the interface between two neighboring monomers. HPLC
analysis shows a much higher amount of adenosine bound to
purified FlA1 than the variants (Figure S2).
1
[
10b]
This
To gain structural insight into the mutations, homology
models of trimeric fah2081 and fah2114 complexed to SAM
The structural implications of stronger binding of SAM
explain the higher affinities of fah2081 and fah2114 for SAM
(
lower SAM K ) compared to FlA1 (Table 2). The very high
m
affinity of fah2114 for SAM is also consistent with the
observation that SAM is copurified with fah2114 (Figure S2).
Prior occupation of the pocket with SAM could impede the
À
binding of solvated F to the enzyme (since SAM is
[
10c]
À
a “competitive” inhibitor
of F binding). The binding of
À
solvated F to an unoccupied pocket is the initial step of
fluorination, which is followed by desolvation of F driven by
SAM binding and then the S 2 attack.
À
[10c]
Hence, the stronger
N
binding of SAM may cause the decreased kcat observed for
fah2081 and fah2114 in the conversion of SAM into 5’-FDA.
In conclusion, we have engineered the fluorinase FlA1
through directed evolution and identified variants with
improved activity for the non-native substrate 5’-ClDA. The
evolved fluorinases, fah2081 and fah2114, were used to
1
8
produce 5’-[ F]FDA, with overall RCC values 3.4- and 4.3-
fold higher, respectively, than FlA1. Kinetics studies indicated
that the variants improve the activity with 5’-ClDA mainly
through improvement of the kcat value in the first step of the
reaction (conversion of 5’-ClDA into SAM). This work
Figure 2. The hexameric structure of FlA1 (PDB ID: 5B6I) shown as
a ribbon diagram. Trimer A is formed by monomer A (purple), mono-
mer B (cyan), and monomer C (beige). Trimer B is formed by mono-
mer D (green), monomer E (pale purple), and monomer F (dark red).
Adenosine (yellow) and l-Met (red) are shown as spheres.
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

Angewandte
Communications
Chemie
represents the first demonstration that the catalytic efficiency
of a fluorinase on a non-native substrate can be improved
through directed evolution. Directed evolution is expected to
be widely employed to improve the catalytic efficiency of
fluorinases with a broad range of substrates for industrial
applications in the future. Endeavors in directed evolution
will hopefully lead to a collection of beneficial mutations for
specific substrates, which could offer deeper insight into the
structure–function relationships and in turn facilitate
rational-design-based fluorinase engineering.
2016 (PDB ID: 5LMZ), after the submission of this communi-
cation.
8] Y. Wang, Z. Deng, X. Qu, F1000Res. 2014, 3, 61.
9] D. OꢀHagan, H. Deng, Chem. Rev. 2015, 115, 634 – 649.
10] a) C. D. Cadicamo, J. Courtieu, H. Deng, A. Meddour, D.
OꢀHagan, ChemBioChem 2004, 5, 685 – 690; b) C. J. Dong, F. L.
Huang, H. Deng, C. Schaffrath, J. B. Spencer, D. OꢀHagan, J. H.
Naismith, Nature 2004, 427, 561 – 565; c) X. Zhu, D. A. Rob-
inson, A. R. McEwan, D. OꢀHagan, J. H. Naismith, J. Am. Chem.
Soc. 2007, 129, 14597 – 14604.
[
[
[
[
11] a) L. Martarello, C. Schaffrath, H. Deng, A. D. Gee, A. Lock-
hart, D. OꢀHagan, J. Labelled Compd. Radiopharm. 2003, 46,
1181 – 1189; b) H. Deng, S. L. Cobb, A. D. Gee, A. Lockhart, L.
Martarello, R. P. McGlinchey, D. OꢀHagan, M. Onega, Chem.
Commun. 2006, 652 – 654.
12] M. Winkler, J. Domarkas, L. F. Schweiger, D. OꢀHagan, Angew.
Chem. Int. Ed. 2008, 47, 10141 – 10143; Angew. Chem. 2008, 120,
Acknowledgements
[
[
This work was funded by the GlaxoSmithKline—Singapore
Economic Development Board partnership for green and
sustainable manufacturing, A*STAR ICES, and A*STAR
IMCB. We thank members of MERL for their comments and
Saei Weng Chia from SBIC for his assistance during the
radiolabeling work.
10295 – 10297.
13] M. Onega, J. Domarkas, H. Deng, L. F. Schweiger, T. A. Smith,
A. E. Welch, C. Plisson, A. D. Gee, D. OꢀHagan, Chem.
Commun. 2010, 46, 139 – 141.
14] M. Onega, M. Winkler, D. OꢀHagan, Future Med. Chem. 2009, 1,
865 – 873.
[
[
15] H. Deng, S. L. Cobb, A. R. McEwan, R. P. McGlinchey, J. H.
Naismith, D. OꢀHagan, D. A. Robinson, J. B. Spencer, Angew.
Chem. Int. Ed. 2006, 45, 759 – 762; Angew. Chem. 2006, 118, 773 –
Keywords: biocatalysis · directed evolution · fluorinases ·
halogenation · radiosynthesis
776.
[
16] S. L. Cobb, H. Deng, A. R. McEwan, J. H. Naismith, D.
OꢀHagan, D. A. Robinson, Org. Biomol. Chem. 2006, 4, 1458 –
1460.
[
[
[
17] M. Thomsen, S. B. Vogensen, J. Buchardt, M. D. Burkart, R. P.
Clausen, Org. Biomol. Chem. 2013, 11, 7606 – 7610.
18] M. E. Sergeev, F. Morgia, M. R. Javed, M. Doi, P. Y. Keng, J.
Mol. Catal. B 2013, 97, 74 – 79.
19] a) S. Thompson, Q. Zhang, M. Onega, S. McMahon, I. Fleming,
S. Ashworth, J. H. Naismith, J. Passchier, D. OꢀHagan, Angew.
Chem. Int. Ed. 2014, 53, 8913 – 8918; Angew. Chem. 2014, 126,
[
1] a) K. Muller, C. Faeh, F. Diederich, Science 2007, 317, 1881 –
886; b) S. Purser, P. R. Moore, S. Swallow, V. Gouverneur,
1
Chem. Soc. Rev. 2008, 37, 320 – 330; c) S. Swallow, Prog. Med.
Chem. 2015, 54, 65 – 133; d) E. P. Gillis, K. J. Eastman, M. D.
Hill, D. J. Donnelly, N. A. Meanwell, J. Med. Chem. 2015, 58,
8315 – 8359.
[
[
2] P. Jeschke, ChemBioChem 2004, 5, 571 – 589.
3] D. W. Grainger, C. W. Stewart in Fluorinated Surfaces, Coatings,
and Films, Vol. 787, American Chemical Society, Washington,
9
059 – 9064; b) S. Thompson, M. Onega, S. Ashworth, I. N.
Fleming, J. Passchier, D. OꢀHagan, Chem. Commun. 2015, 51,
3542 – 13545.
20] S. Thompson, I. N. Fleming, D. OꢀHagan, Org. Biomol. Chem.
016, 14, 3120 – 3129.
21] a) H. Tao, V. W. Cornish, Curr. Opin. Chem. Biol. 2002, 6, 858 –
1
2
001, pp. 1 – 14.
4] a) S. M. Ametamey, M. Honer, P. A. Schubiger, Chem. Rev.
008, 108, 1501 – 1516; b) M. M. Alauddin, Am. J. Nucl. Med.
[
[
[
[
2
2
Mol. Imaging 2012, 2, 55 – 76; c) O. Jacobson, D. O. Kiesewetter,
X. Chen, Bioconjugate Chem. 2015, 26, 1 – 18.
864; b) C. A. Denard, H. Ren, H. Zhao, Curr. Opin. Chem. Biol.
2015, 25, 55 – 64; c) J. L. Porter, R. A. Rusli, D. L. Ollis,
5] a) T. Furuya, A. S. Kamlet, T. Ritter, Nature 2011, 473, 470 – 477;
b) C. D. Murphy, G. Sandford, Expert Opin. Drug Metab.
Toxicol. 2015, 11, 589 – 599; c) M. G. Campbell, T. Ritter,
Chem. Rev. 2015, 115, 612 – 633; d) J.-W. Lee, M. T. Oliveira,
H. B. Jang, S. Lee, D. Y. Chi, D. W. Kim, C. E. Song, Chem. Soc.
Rev. 2016, 45, 4638 – 4650.
6] a) D. OꢀHagan, C. Schaffrath, S. L. Cobb, J. T. G. Hamilton,
C. D. Murphy, Nature 2002, 416, 279-279; b) C. Schaffrath, H.
Deng, D. OꢀHagan, FEBS Lett. 2003, 547, 111 – 114.
ChemBioChem 2016, 17, 197 – 203; d) P. Molina-Espeja, J.
Vina-Gonzalez, B. J. Gomez-Fernandez, J. Martin-Diaz, E.
Garcia-Ruiz, M. Alcalde, Biotechnol. Adv. 2016, 34, 754 – 767.
22] a) U. T. Bornscheuer, M. Pohl, Curr. Opin. Chem. Biol. 2001, 5,
[
137 – 143; b) S. Lutz, Curr. Opin. Biotechnol. 2010, 21, 734 – 743;
c) M. Kaushik, P. Sinha, P. Jaiswal, S. Mahendru, K. Roy, S.
Kukreti, J. Mol. Recognit. 2016, 29, 499 – 503.
[
[
7] a) H. Deng, L. Ma, N. Bandaranayaka, Z. Qin, G. Mann, K.
Kyeremeh, Y. Yu, T. Shepherd, J. H. Naismith, D. OꢀHagan,
ChemBioChem 2014, 15, 364 – 368; b) The crystal structure of
FlA1 was made public in the Protein Data Bank on 2 August
Received: July 21, 2016
Revised: August 24, 2016
Published online: && &&, &&&&
4
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Biocatalysis
Faster Fluorination: Fluorinases are chal-
lenging to engineer owing to high sub-
strate specificity. A fluorinase (structure
shown) was engineered through directed
evolution and the evolved variants
H. Sun, W. L. Yeo, Y. H. Lim, X. Chew,
D. J. Smith, B. Xue, K. P. Chan,
R. C. Robinson, E. G. Robins, H. Zhao,*
E. L. Ang*
&&&& — &&&&
showed improved activity with the non-
native substrate 5’-chloro-5’-deoxyadeno-
sine. Kinetics studies revealed a faster
first step of the reaction. Structural data
suggest that the mutations lead to tighter
binding of S-adenosyl-l-methionine.
Directed Evolution of a Fluorinase for
Improved Fluorination Efficiency with
a Non-native Substrate
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!