Improving the stability and catalyst lifetime of the halogenase RebH by directed evolution

Article abstract of DOI:10.1002/cbic.201300780

We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3-LR, with a T_opt over 5-°C higher than that of wild-type, and 3-LSR, with a T_m 18-°C higher than that of wild-type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non-natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution. Evolving halos: We have used directed evolution to engineer an RebH halogenase variant with a T_opt more than 5-°C higher than that of wild-type RebH, and a second variant with a T_m 18-°C higher. These enzymes provided significantly improved conversion for halogenation of tryptophan and several non-natural substrates.

Full text of DOI:10.1002/cbic.201300780

CHEMBIOCHEM
COMMUNICATIONS
DOI: 10.1002/cbic.201300780
Improving the Stability and Catalyst Lifetime of the
Halogenase RebH By Directed Evolution
[
a]
Catherine B. Poor, Mary C. Andorfer, and Jared C. Lewis*
We previously reported that the halogenase RebH catalyzes
selective halogenation of several heterocycles and carbocycles,
but product yields were limited by enzyme instability. Here, we
use directed evolution to engineer an RebH variant, 3-LR, with
a Topt over 58C higher than that of wild-type, and 3-LSR, with
a Tm 188C higher than that of wild-type. These enzymes pro-
vided significantly improved conversion (up to fourfold) for
halogenation of tryptophan and several non-natural substrates.
This initial evolution of RebH not only provides improved en-
zymes for immediate synthetic applications, but also estab-
lishes a robust protocol for further halogenase evolution.
ieve synthetically useful product yields hinder the practicability
of RebH. During preparative-scale bioconversions in our labora-
tory, extensive RebH precipitation was observed after several
hours of reaction (well after the window in which kinetic data
were acquired); this suggests that significant improvements in
product yield might be possible by increasing the stability of
this enzyme.
Stability is an important property of all enzymes, particularly
those exposed to the reaction conditions encountered in in-
[12,13]
dustrial processes or for laboratory evolution experiments.
Improving enzyme thermostability has many benefits, includ-
ing prolonging catalyst lifetime, increasing enzyme tolerance
to stress (such as proteolysis and organic solvents), and ena-
bling reactions to be conducted at higher temperatures, which
Halogenated organic compounds pervade chemistry and play
important roles in industrial, agrochemical, pharmaceutical,
and materials products, as well as functioning as essential
[14–16]
can increase reaction rates.
Stable enzymes can also better
tolerate mutations introduced to alter other properties, such
as substrate scope and specific activity, as random mutations
[1–3]
building blocks and intermediates in organic synthesis.
[17]
Halogenated arenes comprise a particularly important class of
compounds, but conventional approaches to arene halogena-
tion by electrophilic aromatic substitution require harsh chemi-
are generally destabilizing. To the best of our knowledge, no
halogenase from a thermophilic organism has been character-
ized. Here, we describe the first use of directed evolution to
increase both the thermostability and the optimal operating
temperature of RebH.
[4,5]
cal oxidants and often suffer from poor regioselectivity.
In
nature, selective arene halogenation is catalyzed by flavin-
[
6]
dependent halogenases, which employ halide salts and air as
the halogen source and terminal oxidant, respectively
To improve thermostability without losing catalytic activity,
we employed a screen of RebH mutant libraries by incubating
at elevated temperatures and examining reaction conversions
(Scheme 1). Several groups have used halogenases, (including
[18]
at room temperature. Error-prone PCR was used to generate
a library of RebH variants with an average of two residue mu-
tations. The library was expressed in Escherichia coli in 96-well
plates; the cells were lysed, and the supernatants were trans-
ferred to microtiter plates for heat treatment. Tryptophan halo-
genation reactions were conducted overnight, and reaction
conversions were determined by HPLC analysis.
The first-generation mutant library was constructed from
wild-type (WT) RebH as the parent, and 1365 colonies were
screened following incubation at 428C for 2 h. Mutants show-
ing twice the conversion of WT were identified (confirmed fol-
lowing purification and incubation at 498C for 2 h). In addition,
Scheme 1. RebH- or PrnA-catalyzed 7-chlorination of tryptophan.
[
7]
[8]
[9,10]
RebH, PrnA, and point mutants of these enzymes)
to
halogenate tryptophan and related small molecules on an ana-
lytical scale. We recently explored the substrate scope and se-
lectivity of RebH and showed that this enzyme can halogenate
a range of substituted indoles and naphthalenes on a prepara-
the melting temperature (T , the midpoint of the thermal un-
m
folding transition curve) of an improved mutant (the single
amino acid mutation S2P) was analyzed by circular dichroism
(CD) spectroscopy. The S2P mutant had a Tm 28C higher than
that of WT RebH, thus indicating increased stability. The bene-
ficial mutations identified in improved variants from the first
round were combined by using overlap extension PCR, and
the best variant (1-PVM: mutations S2P, M71V, and K145M)
from this library showed an almost 20-fold improvement in
conversion compared to WT (Figure 1A).
[
11]
tive scale. Although the scope, selectivity, and mild reaction
conditions we employed highlight the synthetic utility of enzy-
matic halogenation, the high enzyme loadings required to ach-
[a] Dr. C. B. Poor, M. C. Andorfer, Prof. J. C. Lewis
Department of Chemistry, University of Chicago
5
735 S. Ellis Ave., Chicago, IL 60637 (USA)
E-mail: jaredlewis@uchicago.edu
Mutant 1-PVM was used as the parent for a second-genera-
tion random mutagenesis library. Of the 1008 colonies
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300780.
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1286 – 1289 1286

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
Figure 1. Halogenation conversion after incubation at 498C for 2 h. Reac-
tions were performed on tryptophan with A) 2% and B) 0.5% enzyme load-
ing.
Scheme 2. General scheme for RebH-catalyzed arene halogenation, and sub-
strates used to examine enzyme scope. a) RebH, 0.2 equiv FAD, 40 equiv glu-
cose, cofactor regeneration system, 100 mm NaCl, 25 mm HEPES, 5% iPrOH,
pH 7.4.
screened following incubation at 518C for 2 h, variant 4G6
(
E423D and E461G) provided a further 2.5-fold increase in con-
version. The third-generation random mutagenesis library used
G6 as the template and comprised 1008 colonies. Each of the
4
[
a]
three best-performing variants from the third round of screen-
ing following incubation at 548C for 3 h contained a single
amino acid mutation. Following combination, the two best var-
iants were identified as 3-LR (S130L, Q494R) and 3-LSR (S130L,
N166S, Q494R; Figure 1B).
Table 1. Representative yields for preparative 3-L(S)R-catalyzed halo-
genation reactions and comparisons to WT RebH-catalyzed reactions.
[a]
[b]
Product Enzyme ([mol%]) T [8C] t [h] Yield [%] Fold improvement
1
2
3
4
3-LR (0.4)
3-LSR (0.8)
3-LSR (1.0)
3-LSR (2.5)
40
21
40
21
16 69
30 62
36 56
48 67
2.8
2.3
4.1
1.7
The melting temperatures of the best mutants identified
through the rounds of genetic diversification, screening, and
recombination were analyzed to probe the relationship be-
tween halogenase conversion and thermostability (Figure 2A).
[
c]
[
a] Isolated yield of pure product. [b] Ratio of product concentrations rela-
tive to internal standard from HPLC analysis of crude reaction mixtures
[
19]
when using 3-L(S)R or WT RebH as specified. [c] 88:12 ratio of 5-/6-hal-
ogenation products.
3-LR at 408C afforded a 2.8-fold increase in the yield of 1 rela-
tive to the reaction with WT RebH at 358C (optimal reaction
[19]
conditions for both enzymes). Furthermore, a 69% isolated
yield of 1 was obtained when using only 0.4 mol% 3-LR load-
ing, compared to a 37% yield by the same loading of WT
RebH.
Improved conversions (1.7- to 4.1-fold relative to WT) of the
non-natural substrates 2-aminonaphthalene, 2-methyltrypta-
mine, and tryptoline (to 2–4, respectively) were also observed
with 3-LSR (Scheme 2, Table 1). Reactions with 3-LSR and WT
RebH were conducted at 218C and 408C for identical times
and enzyme loadings. In each case, the highest conversion was
observed at the same temperature for both 3-LSR and WT
RebH: 218C for the formation of 2 and 4, and 408C for 3. Inter-
estingly, the selectivity of tryptoline halogenation (at the 5 vs.
the 6 position) increased from 60% with RebH to 88% for 3-
LSR (no other changes in selectivity were observed). Bioconver-
sions with 3-LSR continued for 30 h at up to 408C, whereas WT
RebH began precipitating into inactive aggregates within
hours of initiating reactions, thus clearly illustrating the stabili-
Figure 2. A) Thermal denaturation curves obtained using CD at 222 nm.
B) Conversion–temperature profiles of RebH (0.4 mol% RebH).
WT RebH had a melting temperature of 52.48C; for the most
thermostable variant, 3-LSR, it was 70.08C. The 188C increase
in Tm indicates significant improvement in enzyme stability. To
determine whether improved thermostability enables the use
of higher reaction temperatures, conversion–temperature pro-
files of RebH variants were constructed (Figure 2B). With the
accumulation of beneficial mutations, the optimum tempera-
ture for halogenation (T ) of tryptophan based on total con-
opt
version to halogenated product (not initial rate) increased by
at least 58C, from 30–358C for WT RebH to 408C for 3-LR.
Mutant 3-LR produced 100% more 7-chlorotryptophan than
WT RebH on an analytical scale when at the respective Topt.
To establish the relevance of these thermostability improve-
ments to preparative-scale biocatalysis, halogenation by 3-LR
and 3-LSR of several substrates was examined (Scheme 2,
Table 1). Based on HPLC analysis, reaction of tryptophan with
[11]
ty of 3-LSR. Therefore, directed evolution can be used to
improve halogenase stability, lifetime, and selectivity; 3-L(S)R
should prove a good starting point for evolving RebH variants
[20]
with activity with a range of additional substrates.
To better illustrate the extended lifetimes of these enzymes,
halogenation of 2-methyltryptamine (10 mg) with 3-LSR and
WT RebH was monitored under optimal conditions for each
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1286 – 1289 1287

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
decreasing the flexibility of the polypeptide chain. Indeed, the
five other RebH crystal structures in the PDB start their models
[31,32]
at amino acid 2 or 3;
in 3-LSR, the electron density map
extends to amino acid one, thus indicating increased order at
the N terminus. The increased rigidity of the N terminus might
also help stabilize the protein by preventing it from acting as
[33]
a “fraying” point for thermal unfolding. Mutation K145M is
near the surface of the protein and in the area of two arginine
residues (Figure 4); WT RebH has higher positive charge densi-
Figure 3. Plots of conversion against time for halogenation of 2-methyltryp-
toamine by WT and 3-LSR RebH at 408C.
[
19]
enzyme at 408C. As noted above, maximum product yields
for both enzymes were observed at this temperature; however,
the reaction profiles (Figure 3) show that 3-LSR remained
active for significantly longer (~threefold), thereby affording an
approximately fourfold improvement in yield (Table 1, entry 3).
Steady-state kinetic analysis of the enzymes showed that WT
RebH has a k /K similar to that of 3-LSR at 408C (Table 2, en-
cat
m
Table 2. Kinetic data for halogenation of 2-methyltryptamine by RebH
and 3-LSR at 408C and 218C.
[
19]
Figure 4. The local environment of the K145M mutation. Overlay of WT
RebH (grey backbone carbons, cyan side-chain carbons, blue side-chain ni-
trogens; PDB ID: 2OAM) and 3-LSR (light blue backbone carbons, yellow
side-chain carbons, blue nitrogens, green sulfur; PDB ID: 4LU6).
À1
À1
À1
Enzyme
RebH
T [8C]
K
m
[mm]
k
cat [s
]
k
cat/K
m
[s mm
]
À4
40
40
21
21
280.1Æ18.4
202.5Æ12.7
16.8Æ3.8
0.25Æ0.0095
0.15Æ0.0093
0.060Æ0.0057
0.013Æ0.00036
8.8ꢂ10
7.9ꢂ10
3.6ꢂ10
3.3ꢂ10
À4
À3
À4
3
-LSR
RebH
-LSR
ty around this lysine, and the methionine substitution in 3-LSR
might yield stability by reducing the charge density at this po-
3
40.2Æ3.7
[34]
sition. Moreover, the side chain of methionine has a confor-
mation that enhances packing with neighboring residues; this
[35]
tries 1 and 2), but a significantly higher k /K at 218C (Table 2,
might enhance thermostability.
In our previous work with the tryptophan halogenase RebH,
cat
m
entries 3 and 4). These data are consistent with the notion that
stabilized enzymes have decreased conformational flexibility
and that whereas this decrease can be beneficial for stability
[11]
product yield was limited by enzyme instability. Even though
RebH has a melting temperature above 508C, improvements in
stability under our reaction conditions were desired, so a pro-
tein engineering approach was pursued. Three rounds of error-
prone PCR, recombination, and screening resulted in variants
3-LR (seven mutations) with a Topt more than 58C higher than
that of WT, and 3-LSR (eight mutations) with a Tm 188C higher
(
prolonging lifetime, or enabling reaction at high temperature),
[
21,22]
it can be detrimental to activity.
Similarly contrasting sta-
bility and kinetic data have been reported for other stabilized
[
23,24]
enzymes that provided increased product yields,
but stabi-
[
25,26]
lized enzymes with essentially unchanged
or even in-
[
27,28]
creased
A variety of structural features can impart stability to pro-
k /K have also been reported.
than that of WT. That different mutants had the highest T and
cat
m
m
Topt values indicates that thermostability and halogenase con-
version are not strictly coupled. A common belief accounting
for this divergence is that increased rigidity helps stability but
[
29]
teins; to gain insight into these features, we solved the crys-
tal structure of 3-LSR and compared it with the WT RebH struc-
ture. Phases of 3-LSR were obtained by molecular replacement
by using WT RebH (PDB ID: 2OAM) as the search model. The
[21,22]
hinders activity.
Ultimately, however, 3-LSR did provide
100% improvement in halogenase conversion at its T , and
opt
crystal structure was refined to 3.05 ꢁ (final Rwork =18%, Rfree
=
this improvement held for several non-natural substrates. This
initial demonstration of RebH evolution not only provides im-
proved enzymes for immediate synthetic applications, but also
establishes a robust protocol for further halogenase optimiza-
tion.
24%). WT RebH and 3-LSR are similar overall with a backbone
root mean square deviation (RMSD) of 0.32 ꢁ. The differences
in the structures are at the eight changed amino acids.
Investigating the location and nature of the mutations in
the structure of 3-LSR might provide a molecular basis for the
increased thermostability and T . Mutation Q494R is on the
opt
protein surface (neutral side chain of glutamine, positively Acknowledgements
charged arginine). Increasing surface charge deters protein ag-
[
30]
gregation. For the serine-to-proline mutation of S2P (N ter-
minus), proline residues generally increase protein rigidity by
This work was supported by an NIH Pathways to Independence
Award (5R00GM087551-03) and a Searle Scholar Award (11-SSP-
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1286 – 1289 1288

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
2
02) to J.C.L. and an NSF predoctoral fellowship to M.C.A. We
[16] H. Zhao, F. H. Arnold, Protein Eng. 1999, 12, 47–53.
[
[
17] A. A. Pakula, R. T. Sauer, Annu. Rev. Genet. 1989, 23, 289–310.
thank Prof. Phoebe Rice for the use of crystallography software,
Dr. Elena Solomaha for assistance with CD spectroscopy, and
James Payne for optimization of protein expression and lysis con-
ditions in 96-well plate format. This work is based on research
conducted at the APS on the NE-CAT beamlines, which are sup-
ported by a grant from the NIGMS (P41 GM103403) from the
NIH. Use of the APS, an Office of Science User Facility operated
for the U.S. DOE Office of Science by Argonne National Laborato-
ry, was supported by the U.S. DOE under Contract No. DE-AC02-
18] P. C. Cirino, R. Georgescu in Methods in Molecular Biology, Vol. 230: Di-
rected Enzyme Evolution: Screening and Selection Methods (Eds.: F. H.
Arnold, G. Georgiou), Humana, Totowa, 2003, pp. 117–125.
[19] In preparative-scale reactions, we found that 3-LSR provided the high-
est conversions without agitation, whereas WT RebH provided the
highest conversions with agitation. So, these conditions were used for
each enzyme, respectively, in preparative scale reactions. Steady-state
kinetics for both enzymes were measured for reactions that were
shaken to achieve reproducible results.
[
[
[
[
[
[
[
[
[
20] J. C. Lewis, S. M. Mantovani, Y. Fu, C. D. Snow, R. S. Komor, C.-H. Wong,
F. H. Arnold, ChemBioChem 2010, 11, 2502–2505.
21] P. Zꢄvodszky, J. Kardos, ꢅ. Svingor, G. A. Petsko, Proc. Natl. Acad. Sci.
USA 1998, 95, 7406–7411.
22] M. Wolf-Watz, V. Thai, K. Henzler-Wildman, G. Hadjipavlou, E. Z. Eisen-
messer, D. Kern, Nat. Struct. Mol. Biol. 2004, 11, 945–949.
23] X.-Q. Pei, Z.-L. Yi, C.-G. Tang, Z.-L. Wu, Bioresour. Technol. 2011, 102,
3337–3342.
06CH11357. We thank all beamline staff at NE-CAT for their assis-
tance.
Keywords: biocatalysis · directed evolution · halogenases ·
RebH · thermostability
24] A. McDaniel, E. Fuchs, Y. Liu, C. Ford, Microb. Biotechnol. 2008, 1, 523–
531.
[
[
[
1] J. Fauvarque, Pure Appl. Chem. 1996, 68, 1713–1720.
2] P. Jeschke, Pest Manage. Sci. 2010, 66, 10–27.
3] M. Z. Hernandes, S. M. T. Cavalcanti, D. R. M. Moreira, W. F. de Azeve-
do, Jr., A. C. L. Leite, Curr. Drug Targets 2010, 11, 303–314.
4] K. Smith, G. A. El-Hiti, Curr. Org. Synth. 2004, 1, 253–274.
5] A. Podgor sˇ ek, M. Zupan, J. Iskra, Angew. Chem. Int. Ed. 2009, 48, 8424–
25] D. Zhang, F. Zhu, W. Fan, R. Tao, H. Yu, Y. Yang, W. Jiang, S. Yang, Appl.
Microbiol. Biotechnol. 2011, 90, 1361–1371.
26] Y. Wang, E. Fuchs, R. da Silva, A. McDaniel, J. Seibel, C. Ford, Starch/
Staerke 2006, 58, 501–508.
[
[
27] R. Kumar, M. Sharma, R. Singh, J. Kaur, Mol. Cell. Biochem. 2013, 373,
149–159.
8
450; Angew. Chem. 2009, 121, 8576–8603.
6] K.-H. van Pꢃe, E. P. Patallo, Appl. Microbiol. Biotechnol. 2006, 70, 631–
41.
7] E. Yeh, S. Garneau, C. T. Walsh, Proc. Natl. Acad. Sci. USA 2005, 102,
960–3965.
8] M. Hçlzer, W. Burd, H.-U. Reißig, K.-H. van Peꢃ, Adv. Synth. Catal. 2001,
43, 591–595.
9] W. S. Glenn, E. Nims, S. E. O’Connor, J. Am. Chem. Soc. 2011, 133,
9346–19349.
28] D. Matꢃ, C. Garcꢆa-Burgos, E. Garcꢆa-Ruiz, A. O. Ballesteros, S. Camarero,
M. Alcalde, Chem. Biol. 2010, 17, 1030–1041.
[
[
[
[
6
[
[
29] G. A. Petsko, Methods Enzymol. 2001, 334, 469–478.
30] M. S. Lawrence, K. J. Phillips, D. R. Liu, J. Am. Chem. Soc. 2007, 129,
3
1
0110–10112.
31] E. Yeh, L. C. Blasiak, A. Koglin, C. L. Drennan, C. T. Walsh, Biochemistry
007, 46, 1284–1292.
32] E. Bitto, Y. Huang, C. A. Bingman, S. Singh, J. S. Thorson, G. N. Phil-
lips, Jr., Proteins Struct. Funct. Bioinf. 2008, 70, 289–293.
[
[
[
3
2
1
[10] A. Lang, S. Polnick, T. Nicke, P. William, E. P. Patallo, J. H. Naismith, K.-H.
33] P. R. Blake, J.-B. Park, F. O. Bryant, S. Aono, J. K. Magnuson, E. Eccleston,
J. B. Howard, M. F. Summers, M. W. W. Adams, Biochemistry 1991, 30,
van Pꢃe, Angew. Chem. Int. Ed. 2011, 50, 2951–2953; Angew. Chem.
2
011, 123, 3007–3010.
11] J. T. Payne, M. C. Andorfer, J. C. Lewis, Angew. Chem. Int. Ed. 2013, 52,
271–5274; Angew. Chem. 2013, 125, 5379–5382.
12] M. Adamczak, S. H. Krishna, Food Technol. Biotechnol. 2004, 42, 251–
64.
10885–10895.
[
[
[
[
[
34] A. Martin, V. Sieber, F. X. Schmid, J. Mol. Biol. 2001, 309, 717–726.
35] E. Querol, J. A. Perez-Pons, A. Mozo-Villarias, Protein Eng. 1996, 9, 265–
5
271.
2
13] J. D. Bloom, S. T. Labthavikul, C. R. Otey, F. H. Arnold, Proc. Natl. Acad.
Sci. USA 2006, 103, 5869–5874.
[
[
14] I. Wu, F. H. Arnold, Biotechnol. Bioeng. 2013, 110, 1874–1883.
15] H. H. Liao, Enzyme Microb. Technol. 1993, 15, 286–292.
Received: December 13, 2013
Published online on May 21, 2014
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1286 – 1289 1289