Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

Accepted Article

Title: Targeted Enzyme Engineering Unveiled Unexpected Patterns of

Halogenase Stabilization

Authors: Hannah Minges, Christian Schnepel, Dominique Böttcher,

Martin S. Weiß, Jens Sproß, Uwe T. Bornscheuer, and

Norbert Sewald

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To be cited as: ChemCatChem 10.1002/cctc.201901827

Link to VoR: http://dx.doi.org/10.1002/cctc.201901827

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10.1002/cctc.201901827

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Targeted Enzyme Engineering Unveiled Unexpected Patterns of

Halogenase Stabilization

Hannah Minges^[a], Christian Schnepel^[a], Dominique Böttcher^[b], Martin S. Weiß^[b], Jens Sproß^[c],

Uwe T. Bornscheuer^[b], and Norbert Sewald*^[a]

Abstract: Halogenases are valuable biocatalysts for selective C–H

activation, but despite recent efforts to broaden their application

scope by means of protein engineering, improvement of thermosta-

bility and catalytic efficiency is still desired. A directed evolution

campaign aimed at generating a thermostable flavin-dependent

tryptophan 6-halogenase with reasonable activity suitable for che-

moenzymatic purposes. These characteristics were tackled by com-

bining successive rounds of epPCR along with semi-rational muta-

genesis leading to a triple mutant (Thal-GLV) with substantially

increased thermostability (∆T_M= 23.5 K) and higher activity at 25 °C

than the wild type enzyme. Moreover, an active-site mutation has a

striking impact on thermostability but also on enantioselectivity. Our

data contribute to a detailed understanding of biohalogenation and

provide a profound basis for future engineering strategies to facilitate

chemoenzymatic application of these attractive biocatalysts.

an increasing demand for selective C-H activation catalysts we

felt encouraged to improve halogenase performance by means

of protein engineering to qualify for their application in biotech-

nology.

The synthetic interest in halogenated compounds lies in their

ability to act as building blocks and starting material for further

modifications, particularly as they provide access to a large

range of valuable products for metal catalyzed cross-coupling

reactions. In pharmaceutical and agrochemical chemistry halo-

genation often confers unique biological activities that qualifies

this synthetic methodology as an indispensable tool in synthetic

chemistry.^[5,6]As more than 5000 organohalogen compounds are

known from terrestrial and marine organisms, it seems obvious

to study and improve the elaborate repertoire of halogenating

enzymes.^[7]In general, the introduction of halogens into organic

scaffolds can be carried out on aliphatic carbons, olefins as well

as aromatic and heterocyclic rings.^[8]Electrophilic aromatic sub-

stitution is a widely-applied reaction type for arene functionaliza-

tion that is of major importance for the synthesis of bulk and fine

chemicals. However, an immense obstacle is that conventional

approaches for the introduction of halogens require harsh condi-

tions, while addressing electronically unfavored positions re-

mains challenging. As a consequence, mixtures of regioisomers

may be obtained resulting in a low overall yield of the desired

product, undesired by-products and hazardous waste.^[9]Con-

versely, enzymatic halogenation provides a safe alternative for

the synthesis of haloarenes enabling the regioselective halo-

genation of organic scaffolds under benign reaction conditions.

Among the halogenating enzymes, flavin-dependent halogenas-

es currently provide the best candidates with regard to che-

moenzymatic utility. These biocatalysts merely require a halide

salt, molecular oxygen, and FADH₂as stoichiometric compo-

nents. Owing to their convenient handling at room temperature

and pH 7.4 without needing activating or protecting groups,

enzymatic halogenation emerged as a versatile tool for organic

chemistry, as it may help to overcome drawbacks of chemical

halogenation.^[10]Tryptophan halogenases hitherto constitute the

most examined members of this class, as they catalyze halo-

Introduction

Synthetic functionalization of non-activated C-H moieties usually

requires noble metal catalysts, toxic reagents, or extreme condi-

tions, generating considerable amounts of undesired by-

products and waste. Engineering new enzymes via directed

evolution as a shortcut of Darwinian evolution by combining

random mutagenesis with a suitable selection process is appre-

ciated as a promising approach to replace synthetic catalysts

and to improve reaction pathways suffering from low sustainabil-

ity. Evolved enzymes can be generated at comparatively low

cost and their incorporation into metabolic pathways paves the

way to develop cellular factories dedicated to selective fine

chemical production.^[1,2]In ground-breaking evolution campaigns

it was demonstrated by Arnold et al. that tailor-made enzymes

could be generated to catalyze new-to-nature reactions like

cyclopropanation or aziridination using engineered monooxy-

genases.^[3,4]Intrigued by the power of evolution and recognizing

[a]

H. Minges, Dr. C. Schnepel, Prof. Dr. N. Sewald

Organic and Bioorganic Chemistry, Department of Chemistry

Bielefeld University

genation of L-tryptophan as a free substrate, not requiring any

carrier protein. Regeneration of the cofactor is achieved in situ

with the NADH-dependent flavin reductase PrnF and, conversely,

NADH regeneration with an alcohol dehydrogenase or any other

enzyme catalyzing NAD⁺reduction (Scheme 1).^[11]

In 2000 van Pée and coworkers isolated and characterized PrnA,

the first member of the tryptophan halogenase family, from

Pseudomonas fluorescens.^[12]Further FAD-dependent trypto-

phan ha-logenases, such as the Trp 7-halogenase RebH from

Lechevalieria aerocolonigenes,^[13]the Trp 6-halogenase Thal

from Streptomyces albogriseolus,^[14]and the Trp 5-halogenase

PyrH from Streptomyces rugosporus^[15]were identified and

characterized as biocatalysts.^[16,17]

Universitätsstraße 25, 33615 Bielefeld, Germany

E-mail: norbert.sewald@uni-bielefeld.de

Dr. D. Böttcher, Dr. M. S. Weiß, Prof. Dr. U. T. Bornscheuer

Institute of Biochemistry, Department of Biotechnology and Enzyme

Catalysis, Greifswald University

[b]

[c]

Felix-Hausdorff-Str.4, 17489 Greifswald, Germany

Dr. J. Sproß

Industrial Organic Chemistry and Biotechnology

Department of Chemistry

Bielefeld University

Universitätsstraße 25, 33615 Bielefeld, Germany

Supporting information for this article is given via a link at the end of

the document.

10.1002/cctc.201901827

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mology.^[25]Studies by Andorfer et al. demonstrated that the

regioselectivity of the tryptophan 7-halogenase RebH could be

altered by combining random and targeted mutagenesis. Aiming

to engineer RebH into a C5- or C6-regioselective biocatalyst led

to the generation of two new complementary halogenase vari-

ants, catalyzing the chlorination of tryptamine with high regiose-

lectivity for positions C5 and C6, respectively.^[26]Based on the

active site of Trp 7-halogenase PrnA van Pée’s group generated

a single mutant with relaxed regioselectivity, yielding a 2:1 mix-

ture of 5- and 7-brominated Trp.^[27]Recently, structure-based

protein engineering performed by Moritzer et al. enabled a pro-

nounced alteration of regioselectivity that was exemplified for the

Trp 6-halogenase Thal. Exchange of merely five active-site

residues of Thal into the corresponding counterparts of RebH

generated a quintuple variant exhibiting a nearly complete

switch from C6- to C7-halogenation with >95% selectivity.^[28]

X

Trp 6-halogenase

Thal

X

Na , O₂, aqueous buffer

pH 7.4, 25 °C

NH

OH

NH

OH

Flavin

reductase

PrnF

FADH₂

FAD

H₂N

O

L

-tryptophan

-6-halotryptophan

NADH + H⁺

NAD⁺

NADH-regenerating enzyme

RR-ADH

Co-substrate

Oxidized

product

Scheme 1. Thal-catalyzed halogenation of

L-tryptophan leading to chlorination

or bromination at the C6-position of the indole moiety at pH 7.4 and 25 °C. The

cofactor FADH₂is regenerated by the flavin reductase PrnF and NADH is

provided by an alcohol dehydrogenase (RR-ADH) or another enzyme compo-

nent. X = Cl, Br

Engineering towards increased thermostability

Multiple examples of thermal enzyme stabilization obtained by

directed evolution are reported in literature. Kitaoka et al.

achieved an impressive improvement in thermostability of a

phosphorylase from Bifidobacterium longum JM1217 by creating

a double mutant exhibiting a 20 °C higher thermostability than

the wild type that paved the way to an industrial process for

oligosaccharide production.^[29]Even though, several studies are

reported where increased thermostability was not detrimental to

enzyme activity,^[30,31]in a series of evolution campaigns targeting

increased thermostability, decreased catalytic efficiency of the

thermostable variants was noted.^[32–34]For instance, a thermo-

stable amylase mutant from Thermus sp. strain IM6501 (ThMA)

containing seven individual mutations resulted in a 15 °C in-

crease of the optimal reaction temperature. Still, this significant

gain in thermostability was achieved at the expense of de-

creased enzyme activity due to introduction of mutation

M376T.^[34]Likewise, Singh and coworkers performed a directed

evolution campaign to improve the thermostability of a xylanase

(XynA) from Thermomyces lanuginosus. Evolving a biocatalyst

suitable for harsh conditions of an industrial application revealed

a compromise between stability and activity for most of the

obtained mutants.^[35]Recent efforts also focused on improving

the thermostability of Trp halogenases. As reported by Poor

et al., three rounds of mutagenesis resulted in a thermostable

RebH variant containing eight randomly introduced mutations

leading to an increased melting temperature of 18 K. In addition

to an increased conversion of tryptophan, improved conversion

was also detectable for unnatural substrates. However, despite

these improvements its catalytic efficiency was reduced.^[36]In

general, low activity towards substrates and notable instability

cannot be neglected, as these deficiencies hinder an application

in industrial processes. Thermostable enzymes serve as promis-

ing starting points for further engineering amenable to address

other enzyme properties as these biocatalysts often show a

higher tolerance towards random mutations.^[37]Due to a current

lack of elaborate engineering campaigns on stabilizing Trp halo-

genases, we aimed at modifying and optimizing the tryptophan

6-halogenase Thal. Particularly, we embarked on unveiling

Enzymatic halogenation introduces a hotspot for further one-pot

aryl functionalization in chemocatalytic transformations. There-

fore, we and other groups sought to combine biotransformations

such as enzymatic halogenation with Suzuki-Miyaura cross-

coupling.^[18–21]As disclosed in a publication by the Gröger group,

one-pot feasibility of a Pd/Cu-catalyzed Wacker oxidation and an

enzymatic ketone reduction was achieved via compartmentaliza-

tion of the reactions employing a polydimethylsiloxane (PDMS)

thimble.^[22]Based on these findings, Micklefield et al. contributed

progress on the combination of biohalogenation and chemoca-

talysis into a sequential one-pot process. Here, membrane com-

partmentalization enabled the combination of FAD-dependent

halogenases with palladium-catalyzed cross-coupling in a one-

pot manner.^[23]Recently Goss et al. developed an in vivo ap-

proach for the generation of a brominated natural product ana-

logue and its subsequent cross-coupling for diversification of

halogenated natural products however, without mentioning

turnover or efficiency.^[24]Despite substantial efforts to improve in

vitro compatibility of halogenases, their application still suffers

from severe shortcomings such as low activity, enzyme stability

and their limitation to act on relatively electron rich aromatic

compounds.

Engineering of halogenases

Several approaches have been developed in recent years to

overcome limitations of tryptophan halogenases by means of

directed evolution. A comprehensive survey by Lewis et al.

focussed on profiling of the substrate spectrum of several FAD-

dependent halogenases towards a broad set of arenes with

different steric and functional groups. Examination of substrate-

activity profiles revealed that the substrate scope of FAD-

dependent halogenases is less strict than previously assumed.

Moreover, the study unveiled that notable differences of the

substrate profile exist among these enzymes despite high ho-

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crucial motifs to improve halogenase performance in order to

obtain a catalyst with increased thermostability and activity at

elevated temperatures.

Table 1. Directed evolution of Thal-WT showing the evolved variants and their

identified mutations as well as thermostability parameters T₅₀and T_M.

Thal variant

mutation

T₅₀

T_M

/ °C

Results and Discussion

Thal-WT

--

47.0

58.6

59.7

63.0

65.5

68.6

64.3

66.1

70.0

47.7

63.8

65.6

71.2

72.2

n.d.

/

Thal-GR

S359G-K374R

Identification of thermostable Thal variants applying a high-

throughput fluorescence assay

Thal-GRV

S359G-K374R-I393V

S359G-K374L-I393V

Thal-GLV

A quantitative halogenase high-throughput fluorescence assay

applicable in microtiter plates, previously developed by us,

served to reduce the screening effort of large mutant libraries.^[38]

This robust halogenase screening method utilizes Suzuki-

Miyaura cross-coupling (SMC) with a fluorescence readout

enabling screening in cell lysate. Among a range of arylboronic

acids tested a priori, coupling between 3-aminophenylboronic

acid and the brominated amino acid turned out to be most effi-

cient to monitor quantitative formation of bromotryptophan due

to superior fluorescence properties. This activity assay in combi-

nation with a high-throughput strategy previously enabled identi-

fication of the thermostable variant Thal-GR comprising muta-

tions S359G / K374R, which exhibits a significantly increased

thermostability and 2.5-fold improved activity.^[38]

Based on prior achievements obtained from the first round of

Thal evolution we now embarked on a more comprehensive

evolution campaign to further improve its thermostability and

activity. Fluorogenic high-throughput screening previously estab-

lished served to identify improved halogenases from random

mutant libraries.

Starting from a modified evolution procedure, libraries of Thal-

GR mutants were constructed via epPCR and MEGAWHOP

cloning. Promising variants exhibiting increased thermostability

were subsequently detected by fluorogenic cross-coupling (Fig-

ure 1). For this purpose, the mutated Thal-GR colonies were

picked and cultivated in microtiter plates for halogenase expres-

sion and screening. Upon lysis, the libraries were treated for

20 min at 59 °C to select variants with enhanced thermostability.

The Thal-GR template served as a reference and possessed

significantly decreased activity under these screening conditions

providing selection pressure for enhanced thermostability.

Thal-GWV

Thal-GRV-N

Thal-GRV-T

Thal-GRV-MT

Thal-GRV-NT

S359G-K374W-I393V

S359G-K374R-I393V-D378N

S359G-K374R-I393V-A476T

S359G-K374R-I393V-L290M-A476T

S359G-K374R-I393V-D378N-A476T

n.d.

66.0

(n.d. = not determined, / = no reliable data obtained)

With this setup in hand, high-throughput screening via SMC to

monitor residual conversion of tryptophan revealed the beneficial

mutation I393V (Thal-GRV). To confirm increased enzyme sta-

bility, purified protein samples of Thal-GRV were incubated

between 47.0 and 67.0 °C for 20 min and subsequently assayed

for bromination activity at 25 °C. Determination of the half-

maximal conversion temperature (T₅₀) from these data revealed

an increase of 1.1 K compared to Thal-GR. Likewise, the melting

point (T_M) determined by nanoDSF was increased to 1.8 K that

is 18 K higher than the parameters of wild type protein (Table 1,

Figure 2A).

Random and site-directed mutagenesis resulted in signifi-

cant increase of thermostability

Based on this increased thermostability Thal-GRV served as a

template to initiate the third generation of mutants. Screening of

merely 176 colonies led to identification of two new variants with

significantly higher thermostability, GRV-D378N (Thal GRV-N)

with T₅₀= 68.6 °C as well as Thal GRV-L290M/A476T (Thal-

Figure 1. High-throughput halogenase engineering assay based on Suzuki-Miyaura cross-coupling (SMC) for the generation of thermostable enzymes. Mutant

libraries of the Trp 6-halogenase Thal were constructed by epPCR and cultivated and expressed in 96-well plates. After lysis and thermal incubation promising

Thal variants were identified by HT-SMC screening for residual bromination activity at 25 °C.

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GRV-MT) (T₅₀= 66.1 °C). To exclude non-beneficial or detri-

mental mutations, we investigated whether both residues con-

tributed additively to the elevated thermostability. With the cur-

rent set of mutations in hand having a putative effect on thermo-

stability we laid our emphasis on evaluation and optimization of

residues identified by random evolution. Therefore, the variants

Thal-GRV-L290M and Thal-GRV-A476T were generated by site-

directed mutagenesis and analyzed as explained above. Inter-

estingly, Thal-GRV-L290M led to a negligible conversion of

tryptophan after the heat shock above 66 °C. Thus, this deleteri-

Site-saturation mutagenesis for improving catalytic proper-

ties

Despite their increased thermostability, the Thal variants identi-

fied in the third round of evolution (GRV-N; GRV-T; GRV-MT

and GRV-NT) exhibit significantly reduced kinetic performance

at elevated temperature. As we wished to retain activity, they

were discarded from further evolution. In addition, subsequently

identified Thal-GLV mutant exceeded those variants in terms of

T_Mand specific activity. Among the variants obtained so far,

ous mutation was rejected from the following evolution campaign. Thal-GRV was considered as the most promising mutant as it

In contrast, Thal-GRV-T confirmed its positive impact on ther-

mostability with T₅₀= 64.3 °C. Both beneficial mutations from the

third generation were combined finally providing the considera-

bly more thermostable and robust biocatalyst Thal-GRV-NT. T₅₀

increased further reaching 70 °C, whereas the T_Mof 66 °C was

slightly lower.

combined a significant increase in T₅₀and T_Mwith elevated

specific activity at 25 °C. We deduced that the amino acid posi-

tions 359, 374 and 393 proved to be crucial hotspots regarding

increased thermostability as well as activity.

Thal-GRV was subjected to site-saturation mutagenesis to fur-

ther elucidate the impact of the type of amino acid located at

these positions. Gly359, Arg374 and Val393 were individually

addressed by site-saturation PCR using NNK codon degeneracy

to cover the whole set of amino acids. As each of the three

addressed amino acids was randomized separately, screening

94 colonies allowed > 95% sequence coverage assuming a

negligible wild type background.^[39,40]Library quality was verified

by Sanger sequencing indicating the desired amino acid bias at

the selected position. Mutation of Gly359 or Val393 did not

reveal a more thermostable variant, thereby confirming that the

previously introduced residues Gly and Val were the most pre-

ferred ones. Interestingly, Arg374 seemed to be less beneficial

than Leu or Trp in variants Thal-GLV and Thal-GWV. It is worth

mentioning that both mutants were identified twice from the

Arg374 mutant library.

Kinetic characterization revealed stability-activity trade off

We wondered whether increased thermostability was evolved at

the expense of activity, as had been observed by others.^[34,35]

For this, specific activities were determined at 25 °C and 40 °C

(Figure 2B). To ensure sufficient thermostability of the auxiliary

enzyme PrnF responsible for FADH₂supply, its activity at 40 °C

was determined. For all Thal variants an increase of activity

compared to wild type was observed at 25 °C. At this stage of

evolution Thal-GR exhibited highest activity among the variants

obtained from random evolution exceeding the wild type by

factor two. Interestingly, Thal-GR was also significantly more

active than the wild type enzyme both at 25 °C and 40 °C. How-

ever, the specific activity decreased again upon further evolution

and introduction of additional mutations.

A

B

Figure 2. Evolution of thermostability of Thal-WT and determination of catalyst properties. A) Data of half-maximal conversion of L-Trp indicate a stepwise in-

crease in T₅₀during evolution of Thal. Purified halogenase was incubated for 20 min applying a temperature gradient from 42-75 °C. Heat shocked enzyme was

employed for bromination of L-Trp at 25 °C. Final conversion of substrate was determined via RP-HPLC in order to calculate T₅₀from sigmoidal regression.

B) Determination of specific activity at 25 °C and 40 °C indicated an activity-stability trade-off during evolution of Thal. The subsequently engineered variant Thal-

GWV and -GLV exhibited improved thermostability and kinetic parameters compared to Thal-WT.

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Taken into consideration that Trp is only encoded by one base

triplet, the double identification of this variant is a strong indica-

tion for its positive effect on thermostability. Both Thal variants

have significantly higher T₅₀values than the wild type enzyme

(Thal-GLV: T₅₀= 63.0 °C; Thal-GWV: T₅₀= 65.5 °C) and the

melting temperature T_Mincreased likewise. Since Thal-GWV

exceeds the wild type specific activity at 25 °C and 40 °C, this

variant turns out to be endowed with substantially improved

catalytic properties and the best thermostability parameters

obtained during the evolution campaign. Likewise, variant Thal-

GLV, also containing a hydrophobic substitution in position 374,

exhibits high activity at 25 °C albeit having lower activity at 40 °C

compared to Thal-WT. As an elevated optimal reaction tempera-

ture (T_opt) could be expected for thermostable enzymes, we

wondered whether increased thermostability would permit the

use of higher reaction temperatures.^[36,41,42]Conversion-

temperature profiles of Thal-WT and the fittest variants revealed

a T_optranging from 25-30 °C (Figure S2). Hence, T_optwas not

altered notably despite significantly elevated T₅₀and T_Mob-

served for the evolved variants. However, it should be men-

tioned that biohalogenation requires an intricate reaction system

depending on many influencing factors e.g. cofactor regenera-

tion, oxygen supply etc. Noteworthy T_optdetermination does not

exclusively reflect the characteristics of Thal variants. Even

though we ensured that the flavin reductase PrnF remained

active after 30 min of incubation at 40 °C and also RR-ADH

withstands purification via heat-precipitation, it seems probable

that auxiliary enzymes and cofactors also suffer from long-term

incubation at higher temperatures during the reaction course.

Thus, cofactor supply opposes as a drawback of halogenation

reactions carried out at higher temperatures. These major bot-

tlenecks should be taken into account when determining optimal

reaction temperature of Thal variants. Nevertheless, a long-term

stability assay corroborates Thal-GLV’s higher robustness and

value as biocatalyst. Therefore, Thal-WT and Thal-GLV were

incubated at 40 °C for a variable time range and subsequently

Figure 3. Model of Thal-GLV shown in ribbon format based on the crystal

structure of Thal-WT in complex with

L-tryptophan (PDB code: 6H44).

Residues shown as brown stick model show mutational hotspots derived

from evolution of Thal (S359G, K374L, I393V). The conserved amino acid

residues K79 and E358 are highlighted as green stick model. The substrate

L-Trp is shown in grey. The picture was generated and further modified

using Pymol.

served initially could not be deduced from the exchange of

Lys374 to Arg374 since both basic residues are capable of

forming a salt bridge. Possibly, difference of pK_Avalues, chain

length and conformation of side chains influence interactions to

the dimer interface.^[44]

As Trp halogenases commonly form homodimers,^[45]this Arg

residue could favour a salt bridge to the second Thal monomer.

Previously Panja et al. disclosed that salt bridges occur more

frequently in thermophilic proteins, as these contribute to a more

rigid protein scaffold to withstand thermal stress.^[46]Surprisingly,

site-saturation mutagenesis unveiled that hydrophobic residues

are in some cases more favorable for halogenase stability and

lifetime. Probably van der Waals interactions and -stacking

support association of the monomers as evident from a Leu or

Trp residue at position 374 that was most preferred among all

types of amino acids. Similarly, Arnold et al. reported that in-

creased stability of an engineered thermostable hydrolase was

previously connected to stronger hydrophobic interactions and

an increase in hydrophobicity.^[30]

applied for bromination of L-Trp at 25 °C. As the auxiliary en-

zymes were added after thermal incubation of halogenase, the

concomitant cofactor regeneration should not suffer from any

limitation. Accordingly, Thal-GLV withstands 5 h incubation with

residual 8 % conversion of substrate, whereas the WT enzyme

was nearly inactive after 3 h incubation (Table S4).

Hydrophobic interactions enhance enzyme stability

Mass spectrometric analysis revealed increased tendency

for dimer formation

The power of random evolution especially lies in the identifica-

tion and localization of unexpected mutations and new hotspots

within the protein scaffold that can be hardly predicted by a

rational approach. Investigating the location and impact of muta-

tions can support to provide a rationale for increased thermosta-

bility. The recently resolved crystal structure of Thal-WT served

to localize the mutations known in Thal-GWV-NT.^[28,43]As ob-

served for other thermostable enzyme variants such as RebH it

was not surprising that the majority of mutations is located on

the protein surface remote from the active site (Figure S3).^[36]An

increase of surface charge is known to impede protein aggrega-

tion. Therefore, the elevated thermostability of Thal-GR ob-

MS analysis served to provide insight into the influence of stabi-

lizing mutations on the association of monomers into the corre-

sponding homodimer. Native MS provides a model based on the

gas phase that does not exactly reflect the state of the protein in

its natural environment. However, it resembles the biological

status of the protein in solution, prior to the ionization event,

revealing structural information on the protein of interest.^[47]

Owing to the ability of native ESI-MS to preserve noncovalent

interactions, characterization of several protein classes has

successfully been applied.^[48–50]

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Figure 4. Analysis of thermostability and kinetic parameter for engineered Thal single mutants. A) HPLC-data correlating with the final conversion of L-Trp at

25 °C after incubation of purified halogenase at elevated temperatures for 20 min revealed a significant increase in T₅₀for Thal-S359G. B) Determination of

specific activity at 25 °C and 40 °C for Thal single mutants indicated elevated activity of Thal-S359G, -K374W and I393V.

It is known from previous reports that tryptophan halogenases

PyrH, RebH and PrnA tend to form dimers, as evident from gel

filtration and crystal structures.^[45,51,52]Likewise, Thal was found

as a dimer in the crystal structure whereas it exists as a mono-

mer in solution.^[28]ESI-MS measurement performed under native

conditions showed that Thal is present in solution both as a

homodimer and as a monomer. These findings corroborate that

Thal tends to dimer formation in solution similar to other halo-

genases. When analyzing the mutated isoforms, a higher

amount of homodimer in relation to the monomer compared to

the wild type enzyme was detected (Figure S4). These data

therefore support the hypothesis that elevated thermostability

and activity result from increased association of the halogenase

monomers. Furthermore, determination of the protein mass by

MS under denaturing conditions indirectly confirms the mutation

of the targeted amino acids in the Thal variants (Figure S5-S7).

electrophilicity of the halogenating species within the catalytic

cycle.^[54]We wondered whether the mutation S359G would

influence thermostability. Interestingly, the single mutation

S359G resulted in a considerably elevated T₅₀exceeding the

wild type by more than 7.8 K. This observation was also con-

firmed by an increase in T_Mof 10.9 K, while other single muta-

tions merely led to a slight improvement of the thermostability

parameters (Table 2, Figure 4A). Thus, the influence of S359G

was probably most decisive even though being located in the

active site (Figure 3). Interestingly, the mutation A476T resulted

in a less stable biocatalyst, where T_Mcould not even be deter-

mined

Table 2. Evolved Thal single mutants to study influence of individual mutations

identified by random mutagenesis of Thal with corresponding T₅₀and T_M

values.

Thal variant

Thal-WT

T₅₀/ °C

47.0

54.8

49.8

51.5

51.1

50.0

51.8

52.0

T_M/ °C

47.7

58.6

48.4

49.7

48.5

46.3

50.0

/

Analysis of single mutations confirmed significance of

active site mutation and hydrophobic residues

Thal-S359G

Thal-K374R

Thal-K374L

Thal-K374W

Thal-I393V

Thal-D378N

Thal-A476T

During each round of Thal evolution the thermostability in-

creased as evident by rise of T₅₀and T_Mvalues. However, selec-

tion on improved thermostability does not automatically evolve

enzyme activity. As we observed an additive effect on thermo-

stability caused by the mutations D378N and A476T (Thal-GRV-

NT), we wished to dissect the individual contribution of each

residue to thermostability and catalytic activity. Therefore, single

mutants were generated to provide insights on the influence on

both parameters.

The serine residue S347 in PrnA is conserved among FAD-

dependent tryptophan halogenases and mutation causes se-

verely decreased activity. Even though its role remains elusive

yet, S347 was postulated to participate in an extended hydrogen

bonding network.^[53]Its counterpart S359 in Thal is in close

proximity to the essential Glu358, a highly conserved residue

among FAD-dependent Trp halogenases postulated to enhance

(/ = no reliable data obtained)

We suggest this mutation to result in decreased enzyme stability

associated with denaturation or misfolding. Even though residual

activity for Thal-A476T was determined, full conversion of sub-

strate was not achieved in bromination assays. These findings

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are in line to the reduced specific activity of GRV-T and GRV-NT, Brevundimonas sp. BAL3, exhibits a high preference of indole

finally proving the detrimental effect of mutation A476T (Fig-

ure 4B).

Comparison of enzymatic parameters and overall conver-

sion of generated Thal variants

bromination over chlorination. Conversely, Trp halogenases are

reported to favour incorporation of less bulky chloride.^[15,55]

Hence, we examined whether Thal mutagenesis also influences

halide selectivity. LC-MS analysis confirmed Thal-GWV’s ability

to incorporate either chloride or bromide into L-Trp with high

Regarding the specific activity of the Thal single mutants, three

mutations (Thal-S359G, Thal-K374W and Thal-I393V) came into

focus as they were the only sites significantly exceeding wild

type activity at 25 °C as well as at 40 °C. Here, Thal-K374W

exhibits the highest specific activity among the single mutants

and reaches full conversion of substrate (Figure 5). Like for the

WT enzyme full substrate turnover is observed after 60 min. The

progress curve reflects that the K374W mutant catalyzes halo-

genation faster than the WT already reaching >90% conversion

after 45 min.

efficiency. Hence, mutation S359G did not alter ion selectivity

(Figure S8). Though this evolution campaign focusses on in-

creasing thermostability and activity, examples are reported

where engineered halogenase variants exhibit improved halo-

genation of non-natural substrates.^[25,36]Most potent variants

Thal-GLV and S359G were chosen as representatives to inves-

tigate the mutation’s effect on a selected panel of different sub-

strates. Both variants show unaffected quantitative conversion of

indole and tryptamine, whereas methyl-pyrrole-3-carboxylate

was brominated to a lower extent compared to the WT enzyme

(Table S5). Apart from

tion of -Trp with comparable high efficiency. It was surprising to

observe a substantially lower turnover of -Trp for the evolved

variant Thal-GWV (Table 3). The conversion of -Trp to -6-Br-

L-Trp, Thal also catalyzes the halogena-

D

Trp dropped significantly from >99% bromination observed for

the wild type to merely 20% for Thal-GWV. It was assumed that

the active-site mutation S359G is responsible for this altered

preference. Accordingly, the single mutant Thal-S359G is signif-

icantly less active towards the

conversion probably caused by weaker substrate affinity. Like-

wise, chlorination of -Trp is also significantly reduced in case of

variants harbouring mutation S359G. In contrast, conversion of

-Trp is not affected for other Thal single mutants described

D-enantiomer reaching only 17%

D

herein, probably because the mutations are located distant to

the active site.

Table 3. Conversion of

Thal variant

Thal-WT

D-Trp to D-6-Br-Trp by Thal-WT and variants.

Figure 5. Conversion of L-Trp (1 mM) catalyzed by Thal WT and different

Conversion of D-Trp / %

variants at 25 °C indicates accelerated conversion compared to WT enzyme.

Thal-GWV catalyzed reactions stop at a conversion of 75% whereas all other

halogenases reach quantitative substrate turnover. Among all variants Thal-

GLV turns out as most valuable biocatalyst whereas Thal-K374W exhibits

faster conversion of substrate.

>99

20

Thal-GWV

Thal-S359G

Thal-K374R

Thal-K374L

Thal-K374W

Thal-I393V

17

>99

In contrast, Thal-GWV, the most thermostable variant obtained

so far, suffers from incomplete conversion and stagnates at

75 % bromination of L-Trp. The reason of this deficiency remains

elusive until now. Notably, Thal-GLV is endowed with significant-

ly improved T₅₀and T_Mand reaches >98% conversion albeit

slightly retarded. Concluding from these results, two considera-

bly improved halogenase variants were obtained: Thal-GLV was

confirmed as a thermostable biocatalyst with significantly in-

creased halogenation activity also observed at 25 °C. Thal-

K374W shows higher activity but less thermostability than GLV.

Thal-D378N

Thal-A476T

Reaction conditions: 25 µM Thal variant, 1 mM

3 h at 25 °C.

D-Trp, cofactor regeneration,

Further characterization of evolved variant confirmed main-

tained chlorination ability but altered enantiomer selectivity

As recently disclosed by Neubauer et al., the FAD-dependent

halogenase BrvH, identified from a marine metagenome of

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To establish the selectivity of the S359G mutant, relative con-

evident from two signals ascribed to the

containing diastereomers. In contrast, Thal-S359G’s strong

preference for -Trp was evident from its quantitative conversion

to -6-Br-Trp, whereas the -configured substrate enantiomer

was not converted and accumulated in the reaction mixture

(Figure S9).

L- and D-6-Br-Trp-

version rates of separate bromination reactions of

were prepared. Applying a lower enzyme loading further re-

duced conversion of the -enantiomer that dropped to 3.5% for

Thal S359G (Figure 6). However, this mutant revealed steepest

conversion of the -enantiomer compared to Thal-WT. Not sur-

D- and L-Trp

L

D

L

D

L

prisingly, the native enzyme exhibited comparable conversion of

both enantiomers, whereas a slight preference for the native

The active-site Ser residue plays a crucial role in enantio-

mer selectivity

substrate

L-Trp was noted. In addition, a competition experiment

applying a mixture of

L

- and -tryptophan in a ratio of 1:1 was

D

carried out to provide unambiguous insight into altered enantio-

mer preference. After incubation for 24 h at 25 °C Marfey’s

derivatization of reaction mixtures combined with LC-MS analy-

sis permitted separation of resulting diastereomers comprising

As these previous findings underline the peculiar role of the

active site mutation, we questioned whether the advantages of

S359G mutation are also applicable to related halogenases.

Thus, the corresponding point mutants were generated in RebH

and PyrH, respectively (RebH-S358G / PyrH-S355G). In con-

trast to the observations made for Thal-S359G, the RebH and

PyrH mutants did not reveal a significant increase in perfor-

mance. Even though RebH-S358G exhibited 23% elevated

activity compared to the WT at 25 °C, this effect was not ob-

served at 40 °C and not as pronounced as for the Thal-S359G

variant. Introduction of the mutation in PyrH completely abol-

ished its activity (Figure 7A). Moreover, these active site variants

were even detrimental on the thermostability of both halogenas-

es (Table S3). To deepen the study of the catalytic role of S359

the corresponding Ala mutants were generated in accordance to

an analogous PrnA mutant previously reported by van Pée

et al.^[54]As site-saturation mutagenesis of Thal-GRV confirmed

Gly as the most beneficial amino acid, the corresponding mutant

either

signals formed upon coupling with N^α-(2,4-dinitro-5-

fluorophenyl)- -alaninamide (FDAA) were identified by LC-MS

D-/L-Trp or D-/L-6-Br-Trp. The expected diastereomer

L

and comparison with authentic standards. Thal-WT exhibited

complete conversion of both enantiomers as

Thal-S359A was therefore of minor interest. RebH-S358A and

PyrH-S355A exhibited lower performance than the WT with

regard to activity and thermostability (Figure 7A, Table S3). To

conclude, the beneficial effects on activity and thermostability

observed for Gly359 are specific for the 6-halogenase Thal

whereas they are not valid for other FAD-dependent halogenas-

es such as its close homologue RebH. However, similar to the

Thal-S359G variant, the RebH-S358G mutant displayed a signif-

Figure 6. Thal-WT and Thal-S359G catalyzed bromination of

S359G showed highest conversion of the -enantiomer whereas negligible

bromination is noted for the -enantiomer. Thal-WT exhibits comparable

conversion of both - and -Trp, with a slight preference for the native

-enantiomer. Reaction conditions: 7 µM Thal variant, 1 mM -Trp or -Trp,

cofactor regeneration, 25 °C.

D- or L-Trp. Thal-

L

D

L

D

L

D

L

icantly reduced conversion of

D-Trp, dropping from 97% (WT) to

merely 14% (Figure 7B). Obviously, this observation is con-

Figure 7. Impact of the active site Ser residue on activity and enantioselectivity of the halogenases Thal, RebH and PyrH. A) Determination of specific activity at

25 °C and 40 °C revealed elevated activity of the Thal mutant whereas a mutation to Gly in RebH or PyrH did not significantly improve activity. B) The conver-

sion assay of D-Trp to D-6-Br-Trp resulted in significantly reduced performance of mutants indicating higher enantiospecificity as a result of the mentioned amino

acid exchange.

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sistent among the halogenases tested herein. This consistency

provides evidence that this position is decisive for substrate

selectivity. The mutation to Ala did not alter enantiomer selectivi-

ty merely reducing the overall activity of RebH.

evolution campaign also opens up novel motifs having a strong

influence on fundamental enzyme properties. Thermostable and

more active Thal variants obtained herein will find their way into

chemoenzymatic synthesis for efficient production of haloarenes.

Based on these findings novel halogenases with altered sub-

strate spectrum and high robustness may be developed that

further expand the scope of enzymatic halogenation.

Obviously, Ser influences enantiomer selectivity which is rather

surprising as this residue is oriented proximally to the indole

moiety and undergoes no direct interactions to the amino acid

backbone (Figure 3). Noteworthy, the neighbouring residue

Glu346 supports positioning of the substrate tryptophan by a

hydrogen bond between the NH group of the indole ring and the

carbonyl group of peptide bond between Glu346 and

Ser347.^[45,54]Recently, desymmetrization of prochiral methylene

dianilines by a RebH mutant was demonstrated by Lewis et al.,

providing an example of engineering the enantioselectivity of a

flavin-dependent halogenase.^[56]In our study, alteration of one

residue within the active site allows for a tremendous influence

Experimental Section

Analytics

Reversed phase-high performance liquid chromatography (RP-

HPLC)

A Shimadzu CBM-20A chromatography system was employed for analyt-

ical measurements equipped with a diode array detector SPD-M20A

IVDD (Shimadzu), pump Nexera XR liquid chromatography LC-20AD_XR

(Shimadzu), column oven CTO-20A (Shimadzu) and an autosampler

Nexera XR SIL-20A_xr(Shimadzu). For separation a Luna 3 µm C₁₈col-

on L-/D-selectivity and we were able to alter specificity of the

substrate enantiomer. This points to an unexpected crucial role

of Ser in substrate recognition presumably attractive for engi-

neering of halogenase specificity.

umn 100 Å, 100 × 2 mm from Phenomenex with

a flow rate of

0.65 mL min^-1was used. Absorbance was measured simultaneously at

220, 254 and 280 nm. Eluents: A: water/trifluoroacetic acid (99.9:0.1),

B: acetonitrile/trifluoroacetic acid (99.9:0.1). Elution profile at 40 °C

column temperature: (A %): 0-5.5 min: linear gradient from 95 % to 5 %,

5.5-6 min: 5 %, 6-6.1 min: gradient from 5 % to 95 %, 6.1-9.0 min: 95 %.

High-performance liquid chromatography-mass spectrometry (LC-

MS)

Conclusions

Halogenase stability often cannot be increased without affecting

conformational flexibility as a crucial factor lowering enzyme

activity. This observation is ascribed to a stability-activity trade-

off typically occurring for engineered enzymes. Therefore, a

balance between stabilization and maintenance of activity is

Separation was performed with a 1200 HPLC system consisting of an

autosampler, degasser, binary pump, column oven and diode array

detector (Agilent Technologies, Santa Clara, CA, USA) using a C₁₈

Hypersil Gold column, 3 µm, 150 × 2.1 mm (Thermo Scientific) and

solvent flow was directly introduced to ESI-oa-ToF (orthogonal accelera-

tion time-of-flight) mass spectrometer. ESI mass spectra were acquired

employing an Agilent 6220 time-of-flight mass spectrometer from Agilent

Technologies (Santa Clara, CA, USA) in extended dynamic range mode

essential to encounter the requirements for biocatalysis.^[57,58]

A

combination of different evolution strategies including directed

evolution, rational design as well as site-saturation mutagenesis

was employed aiming to the development of a significantly more

thermostable halogenase with elevated activity. Seven different

residues were identified in the tryptophan 6-halogenase Thal

and their impact on thermostability and activity was analyzed in

detail. Thal-GLV displays significantly increased thermostability

compared to Thal WT (∆T₅₀= 16.0 K; ∆T_M= 23.5 K) and strongly

elevated enzyme activity at 25°C. Moreover, the single mutation

K374W turned out to strongly enhance overall halogenation

efficiency at 25 °C. We were able to identify beneficial residues

within the protein scaffold (amino acid position 359, 374 and

393) that contribute to thermostability as well as elevated activity.

Interestingly, hydrophobic interactions located at the interface

between the Thal monomers were favourable for improved

thermostability. MS analysis of selected Thal mutants indicates

an enhanced tendency for dimer formation compared to the WT.

These results suggest that dimerization occurs as a beneficial

factor for halogenase stabilization essential to an enhanced

synthetic utility. The mutation S359G tremendously increases

thermostability and activity despite its location within the active

site. Similar effects of this mutation could not be observed for

related tryptophan halogenases. Noteworthy, this mutation also

increases enantiomer selectivity giving significantly reduced

containing

a Dual-ESI source, operating with a nitrogen generator

NGM 11. The mass axis was externally calibrated with ESI-L Tuning Mix

(Agilent Technologies, Santa Clara, CA, USA) as calibration standard.

HPLC solvent A: water/acetonitrile/formic acid (94.9:5:0.1); HPLC solvent

B: water/acetonitrile/formic acid (5:94.9:0.1). Gradient elution: (A %):

0-10 min gradient from 98 % to 2 %, 10-11 min: 2 % isocratic,

11-11.5 min: gradient from 2 % to 100 %, 11.5-15 min: 100 %) with a flow

rate of 0.3 mL min^-1and column temperature of 40 °C.

Protein analysis using nano-electrospray ionization-mass spec-

trometry (nano-ESI-MS)

His₆-tagged halogenase was purified via HisTalon affinity chromatog-

raphy, dialyzed against 100 mM ammonium acetate buffer, pH 7.0 and

applied for nano-ESI-MS measurements under native conditions (final

concentration: 10 µM). Under denaturing conditions, his₆-tagged halo-

genase (final concentration 10 µM) was dissolved in 50% MeCN, 0.1%

formic acid. Nano-ESI measurements were performed using a Q-IMS-oa-

TOF mass spectrometer Synapt G2Si (Waters GmbH, Manchester, UK)

in resolution mode, interfaced to a nano-ESI ion source. Nitrogen served

both as the nebulizer gas and the dry gas for nano-ESI. Nitrogen was

generated by a nitrogen generator NGM 11. Samples were dissolved in

ammonium acetate buffer (100 mM, pH 7.0) and introduced by static

nano-ESI using in-house pulled glass emitters. The mass axis was

externally calibrated with ESI-L Tuning Mix (Agilent Technologies) as

calibration standard. Scan accumulation and data processing was per-

formed with MassLynx 4.1 (Waters GmbH, Manchester, UK) on a PC

Workstation. The spectra shown here were generated by the accumula-

tion and averaging of several single spectra. Determination of protein

conversion of

D-Trp. The results shown herein underscore that

halogenases, promising tools for enzymatic C–H activation, can

be improved by random or rational evolution strategies. This

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masses was performed using centroided data using the ESIprot soft-

ware.^[59]

15 mM Na₂HPO₄, 30 mM NaBr, pH 7.4 (5 L reservoir) overnight to re-

move contaminating chloride ions.

General procedures

Directed evolution of tryptophan 6-halogenase Thal

Standard chemicals and solvents were obtained from commercial suppli-

ers in highest purity suitable for analytical applications (p. a.). Restriction

enzymes were purchased from New England Biolabs, DNA polymerase

was supplied from Roboklon and Random Mutagenesis GeneMorph II Kit

obtained from Agilent.

Generation of megaprimers (epPCR)

Construction of mutant libraries was performed based on the already

described procedure by Weiß et al.^[61]For error-prone PCR libraries, the

genes encoding the respective variants of Thal were amplified by apply-

ing the GeneMorph II Random Mutagenesis Kit (Agilent Technologies)

using the following primers: pET28a-NdeI-FW and pET28a-BamHI-Stop-

RV (Table S1). 50 ng of parental plasmid template (1^stround of evolution:

pET28-Thal-GR, 2^ndround of evolution: pET28-Thal-GRV ) was em-

ployed in a total volume of 50 µL and amplified over 30 cycles of muta-

genesis as follows: (a) 95 °C, 2 min; (b) 30 cycles: 95 °C, 30 s; 55 °C, 30

s; 72 °C, 90 s; (c) 72 °C, 10 min. Three randomly selected colonies were

sent for sequencing and revealed an average mutation frequency of

three nucleotide exchanges per gene. PCR products were purified ac-

cording to the manual instructions using the NucleoSpin Gel and PCR

Clean-up Kit (Macherey & Nagel). The sample was eluted in 25 µL for

use as megaprimers in the following MEGAWHOP-PCR.^[62]

Molecular cloning

Molecular biology was performed according to standard procedures as

described.^[60]Codon-optimized gene for E. coli encoding Thal (Uniprot ID:

A1E280), RebH (Uniprot ID: Q8KHZ8) or PyrH (Uniprot ID: A4D0H5) was

subcloned into pET28a as reported earlier and employed for heterolo-

gous expression in E. coli BL21 (DE3) pGro7.^[18]The plasmid vector

pET21-ADH encoding alcohol dehydrogenase was kindly supplied from

Prof. Dr. Werner Hummel, Bielefeld University, and pCIBhis-PrnF encod-

ing the flavin reductase was donated by Prof. Dr. Karl-Heinz van Pée, TU

Dresden. Plasmid pGro7 was purchased from TaKaRa. E. coli DH5α and

BL21(DE3) were purchased from Novagen.

Performance of MEGAWHOP (whole-plasmid PCR)

Heterologous expression in E. coli

Mutated halogenase genes were inserted into plasmid vector by making

use of MEGAWHOP according to the following procedure in a total

volume of 50 µL: Pfu-buffer, 0.8 mM dNTPs, 75 ng parental plasmid

(pET28-Thal-GR), 6 µL purified megaprimers as described above and

1.0 µL of Pfu Plus! DNA polymerase. The amplification was performed as

follows: (a) 94 °C, 2 min; (b) 25 cycles: 94 °C, 25 s; 58.5 °C, 30 s; 72 °C,

7.0 min; (c) 7 °C, 14 min. The PCR product was digested with DpnI (20 U,

2 h at 37 °C) and subsequently the restriction enzyme was inactivated by

incubation at 80 °C for 20 min. 5 µL sample volume was directly trans-

formed into a 75 µL aliquot of electro-competent E. coli DH5α (2 kV,

5 ms). After transformation, 1 mL SOC medium was added and after

55 min incubation (37 °C, 500 rpm) a sample of 50 µL was plated out on

LB agar supplemented with Kan (60 mg L^–1kanamycin) and the remain-

ing sample was employed for inoculation of a 50 mL LB-Kan (60 mg L^–1

kanamycin) overnight culture (37 °C, 150 rpm). After overnight incubation

colonies were counted to calculate the number of transformants

(5000 transformants were usually obtained). The overnight culture was

employed for plasmid isolation and chemo-competent E. coli BL21(DE3)

pGro7 cells were transformed via heat-shock with 40-80 ng of the library

plasmid DNA. After incubation at 37 °C 0.5-1.0 mL was plated out on

Kan/Cam plates (60 mg L^-1kanamycin, 50 mg L^–1chloramphenicol) for

Thal library screening as described below.

Flavin reductase PrnF and alcohol dehydrogenase RR-ADH

Expression of PrnF and alcohol dehydrogenase was conducted accord-

ing to our already published procedure.^[11]

Halogenase Thal, RebH, PyrH and respective variants

1.5 L LB medium containing kanamycin (60 mg L^–1) and chloramphenicol

(50 mg L^–1) was inoculated with 20 mL L^-1overnight culture of E. coli

BL21 (DE3) pGro7 pET28a-Thal, pET28a-RebH, pET28a-PyrH or its

respective variants. The expression culture was incubated at 37 °C until

an OD₆₀₀= 0.6 was reached. Temperature was decreased to 25 °C for

30 min and overexpression subsequently induced by addition of 0.1 mM

IPTG and 2 g∙ L^-1l-arabinose. Cells were shaken at 150 rpm for 20 h,

harvested by centrifugation (3220 × g, 30 min, 4 °C), washed with 40 mL

Na₂HPO₄buffer (0.1 M, pH 7.4) and stored at –20 °C.

Determination of flavin reductase activity at 25 °C and 40 °C

Determination of PrnF activity was performed according to our already

published procedure including the following modifications.^[11]The volu-

metric activity of the elution fractions of the flavin reductase PrnF from

Pseudomonas fluorescens was determined as triplicates by monitoring

the decrease of absorption at λ = 340 nm due to the oxidation of NADH +

H⁺to NAD⁺(ε = 6.3 mL µmol^-1cm^-1) in a final volume of 1 mL containing

20 µL of diluted flavin reductase (dilution 1:100), 10 mM Na₂HPO₄pH 7.4,

50 µM FAD and 160 µM NADH. All components were pre-equilibrated at

25 °C / 40 °C for 30 min. Afterwards pre-equilibrated PrnF was added to

residual components and the conversion rate of the substrate was de-

termined using a UV-3100 PC spectrometer (VWR) by regression of the

linear range (15 s after addition of the enzyme).

Library Expression

Library expression, halogenase characterization and screening for in-

creased thermostability based on high-throughput Suzuki-Miyaura cross-

coupling was performed with some modifications following our already

published procedure.^[38]Colonies were picked and arrayed by inoculation

of 150 µL LB medium (Kan: 60 mg L^–1, Cam: 50 mg L^-1) in 96-well plates.

Plates were sealed with AeraSeal and grown for 18 h at 30 °C in an

orbital shaker (800 rpm, Heidolph Titramax 1000). Deep-well plates

containing 1.0 mL TB medium per well supplemented with the appropri-

ate antibiotics was inoculated with 50 µL precultures and grown at 37 °C,

800 rpm. After 3 h temperature was decreased to 25 °C and plates were

incubated for additional 30 min. Protein expression was induced by

addition of 0.1 mM IPTG and 2 mg mL^-1l-arabinose and shaken for 20 h

(1000 rpm) at 25 °C. Cells were harvested by centrifugation (3220 × g,

45 min, 4 °C), supernatant was discarded and expression plates were

stored at -20 °C.

Determination of alcohol dehydrogenase activity

Activity determination of RR-ADH from Rhodococcus spp. was performed

as previously published.^[11]

Protein purification

His₆-tagged fusion protein was extracted from E. coli cells after expres-

sion by resuspending bacteria in 40 mL 50 mM Na₂HPO₄pH 7.4,

300 mM NaCl for subsequent lysis by French Press (3x). Cell debris was

removed by centrifugation (10000 × g, 30 min, 4 °C) and the supernatant

filtered through 0.45 µm Whatman filter. The cleared lysate was loaded

on HisTALON agarose affinity resin (bed volume of 1.5 mL resin), incu-

bated for 1 h at 4 °C to enable protein binding, followed by two washing

steps adding 10 mL 50 mM Na₂HPO₄pH 7.4, 300 mM NaCl and after-

wards 50 mM Na₂HPO₄pH 7.4, 300 mM NaCl, 10 mM imidazole. The

fusion protein was isolated by addition of 300 mM NaCl, 300 mM imidaz-

ole, 50 mM Na₂HPO₄pH 7.4. Fractions of 0.5 mL were collected and

protein concentration was determined by Nano-Drop UV spectroscopy.

For following enzyme characterization the protein was dialyzed against

Cell lysis and screening for thermostability

Expression plates were thawed for 20 min on ice and 150 µL lysozyme

(2 mg mL^-1) as well as 10 µL DNAseI (1 mg mL^-1), diluted in bromination

buffer (10 mM K₂HPO₄pH 7.4, 30 mM NaBr) were added to each well.

Cell pellets were resuspended and shaken vigorously at 25 °C for 3 h in

an orbital shaker. To screen for increased thermostability the microtiter

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plate was incubated in a water bath at 59 °C for 20 min (1^stround) and at

64 °C (2^ndround). Afterwards cell debris was removed by centrifugation

(3220 × g, 30 min, 4 °C). 50 µL thermally treated lysate were used for

halogenation at 25 °C by addition of 50 µL bromination buffer containing

was performed using NT Melting Control software (NanoTemper Tech-

nologies GmbH). The T_Mwas determined by fitting the tryptophan fluo-

rescence emission ratio of 350 nm to 330 nm using a polynomial function,

in which the maximum slope is indicated by the peak of its first derivative.

Temperature-conversion gradient for T₅₀determination of Thal

variants

5 mM L

-tryptophan, 2.5 U mL^–1PrnF, 1 U mL^–1alcohol dehydrogenase,

10 µM FAD, 1 mM NAD⁺, 5% (v/v) iso-propanol, 10 mM K₂HPO₄(pH 7.4),

30 mM NaBr, 0.1 mg mL^–1ampicillin and 5 µM PMSF. Plates were

sealed with AeraSeal and bromination was conducted at 150 rpm, 25 °C

for 20 h. Endpoint conversion was determined by high-throughput Suzu-

ki-Miyaura cross-coupling as described below.

A stock solution of Thal or corresponding variants (145 µM) in 15 mM

Na₂HPO₄pH 7.4 and 30 mM NaBr) was pipetted as triplicate into a 96-

well PCR plate (60 µL per well) and incubated in a thermocycler (Peqlab,

Primus 96 advanced) using a gradient from 42.0-75.0 °C for 20 min. After

centrifugation (4000 × g, 10 min, 4 °C) 40 µL of each sample (40 µM

final halogenase concentration) was applied to determine residual halo-

genase activity and employed in a 150 µL reaction mixture containing

High-throughput Suzuki-Miyaura cross-coupling

Subsequent to enzymatic bromination catalyzed by thermally treated

Thal variants the assay plates were centrifuged (3220 × g, 10 min, 4 °C)

and 10 μL supernatant from each well was transferred into a 96-well PCR

plate. Coupling mastermix containing 3-aminophenylboronic acid

(6.7 mM) and K₃PO₄(10 mM) in aqueous solution was prepared. 75 μL

mastermix were added to each well with regard to a final concentration of

5 mM boronic acid and 7.5 mM base in a total volume of 100 μL in the

coupling assay. Catalyst mastermix consisting of 1.7 mM Na₂PdCl₄as

well as 5 mM sSPhos (sodium 2′-dicyclohexylphosphino-2,6-dimethoxy-

1,1′-biphenyl-3-sulfonate hydrate) was pre-activated at 40 °C for 10 min

under vigorous shaking. 15 μL Pd catalyst solution was added to each

well giving 0.25 mM Na₂PdCl₄and 0.75 mM sSPhos as end concentra-

tion. Plates were sealed with adhesive film and incubated for 2 h at 95 °C

in a drying oven. Reaction was quenched by addition of 90 μL 2% TFA

added to 15 μL sample per well and fluorescence emission was recorded

at 430 nm (excitation: 300 nm) using a microplate reader (Tecan, Infinite

M200).

5 mM L

-tryptophan, 2.5 U mL^-1flavin reductase PrnF, 1 U mL^-1alcohol

dehydrogenase, 10 µM FAD, 1 mM NAD⁺, 10 mM K₂HPO₄pH 7.4,

30 mM NaBr and 5% iso-propanol in a 96-well plate. The plate was

sealed with AeraSeal and shaken at 150 rpm and 25 °C. After 20 h, final

conversion of Trp was determined via RP-HPLC, plotted against temper-

ature and half-maximal conversion (T₅₀) was calculated from sigmoidal

regression.

Determination of specific activity for Thal, RebH, PyrH and variants

The calculation of the specific activity referred to the conversion of 1 mM

L

-tryptophan at 25 °C or 40 °C in presence of concomitant cofactor

regeneration. In a total volume of 750 µL, 1 mM substrate -tryptophan,

L

2.5 U mL^–1PrnF, 1 U mL^–1ADH, 10 µM FAD, 1 mM NAD⁺, 15 mM

Na₂HPO₄pH 7.4, 30 mM NaBr and 5 % iso-propanol were combined in

96-deep well plates. The mixture was pre-equilibrated at 25 °C or 40 °C

for 10 min and finally the reaction was initiated by addition of tryptophan

halogenase dissolved in 15 mM Na₂HPO₄, pH 7.4, 30 mM NaBr in a final

concentration of 7 µM. During incubation at 25 °C / 40 °C, 600 rpm, in

intervals of 5-10 min a 50 µL aliquot was taken and inactivated by addi-

tion of an equal volume of methanol. The resulting samples were centri-

fuged (5 min, 4000 × g) and 80 µL supernatant transferred to a new

96-well plate and diluted with an equal volume of H₂O. To determine the

specific activity the formation of Br-Trp was analyzed via analytical RP-

HPLC and plotted against reaction progress. The linear phase of the

enzyme reaction, usually from 0-30 min, was analyzed by linear regres-

sion and the specific activity was calculated accordingly based on the

amount of tryptophan halogenase added to the reaction (see Supporting

Information, Figure S1, Table S2).

Site-directed and site-saturation mutagenesis

All Thal, RebH and PyrH variants were prepared following the Quik-

Change II Site-Directed Mutagenesis Kit with modifications. Primers were

designed with the desired mismatches to incorporate the corresponding

mutations. For site-saturation mutagenesis, primers with NNK degener-

ated codon in the position were applied (see Supporting Information,

Table S1). The PCR conditions were as follows: 100 ng plasmid DNA as

template, 5 µL 10x Pfu-reaction buffer, 200 µM dNTPs, 0.5 µM forward

primer, 0.5 µM reverse primer, 2.5 U/µl Pfu-Plus! polymerase. PCR was

performed in a volume of 50 µL by applying the following procedure:

95 °C 120 s, (95 °C 30 s, 58 °C 30 s, 68 °C 7.5 min) for 25 cycles, 68 °C

7 min. Amplification was verified by agarose gel electrophoresis and the

resulting amplificate was directly digested with DpnI by adding 20 U to

each amplification reaction. The digestion was conducted at 37 °C for 2 h

and the enzyme subsequently inactivated at 80 °C for 20 min. The mix-

ture was purified using QIAquick PCR Purification Kit (Qiagen), 10 µL of

DNA solution was transformed via heat shock into chemocompetent

E. coli DH5α, plated on LB agar supplemented with appropriate antibiot-

ics (kanamycin) and grown overnight at 37 °C. For plasmid isolation

10 mL LB medium containing the appropriate antibiotic (kanamycin,

60 mg L^-1) was inoculated with an individual colony and cells were grown

at 37 °C overnight. Plasmids were purified using QIAprep Spin Miniprep

Kit (Qiagen) according to the manufacturer’s instructions. After confirma-

tion of codon distribution in the case of a site-saturation mutagenesis or

sequence verification of point mutants (sequencing performed via GATC

Biotech), the plasmids were transformed into chemo-competent E. coli

BL21(DE3) pGro7 cells for halogenase expression.

Halogenation of

respective variants

Enzymatic halogenation of

200 μl under gentle shaking at 25 °C. Substrate

D-tryptophan using purified Thal, RebH, PyrH and

D-tryptophan was assayed in a total volume of

D-tryptophan was added

to a final concentration of 1 mM together with 1 mM NAD⁺, 0.01 mM FAD,

1 U mL^-1RR-ADH, 2.5 U mL^-1PrnF, 5 % (v/v) iso-propanol, 10 mM

Na₂HPO₄pH 7.4 and 30 mM NaBr or NaCl. Purified Thal-WT, RebH-WT,

PyrH-WT or variants were added to a final concentration of 25 µM. Reac-

tions were performed as duplicate and reaction progress was monitored

by analytical RP-HPLC. For this, at different time points aliquots of 50 µL

were taken from the reaction mixture and quenched by adding an equal

volume of methanol. The mixture was centrifuged (12000 × g, 10 min)

and the supernatant analyzed via analytical RP-HPLC.

Chlorination of

Enzymatic chlorination was assayed in a total volume of 500 μl under

gentle shaking at 25 °C. The substrate -tryptophan was added to a final

L -tryptophan employing purified Thal-GWV

L

concentration of 1 mM together with 1 mM NAD⁺, 0.01 mM FAD,

1 U mL^-1RR-ADH, 2.5 U mL^-1PrnF, 5 % (v/v) iso-propanol, 10 mM

Na₂HPO₄pH 7.4 and 30 mM NaCl. Purified Thal-GWV was added to a

final concentration of 80 µM. Reactions were performed as duplicate and

after 24 h final conversion was monitored by LC-MS. For this, aliquots of

50 µL were taken from the reaction mixture and quenched by adding an

equal volume of methanol. The mixture was centrifuged (12000 × g,

10 min) and the supernatant analyzed via LC-MS (see Supporting Infor-

mation, Figure S8).

Biocatalytic halogenation assays

Determination of melting temperature (T_M) for Thal, RebH, PyrH and

respective variants

The melting temperature (T_M) of Thal-WT, RebH-WT, PyrH-WT and

evolved variants (8 μM) was evaluated employing a nanoDSF device

(Prometheus NT.48, NanoTemper Technologies GmbH). Capillaries

were filled from respective solutions (10 μL). Samples were measured in

the Prometheus NT.48 in a temperature range of 20-95 °C. Data analysis

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Determination of optimal reaction temperature (T_opt) for Thal-WT and

thermostable variants

Enzymatic bromination was assayed in a total volume of 200 μl under

comprising either

D

-/

L

- tryptophan or

D

-/

L

-6-bromotryptophan to provide

insight into altered enantiomer preference. In the resulting LC-MS chro-

matograms the expected diastereomer signals formed upon coupling with

FDAA were identified from the MS spectrum and comparison with au-

thentic standards. Therefore, after a reaction time of 24 h 100 µL reaction

sample was mixed with 50 µL 0.1 M aq. NaHCO₃solution and 50 µL

FDAA solution (10 mM in acetone) was added. The reaction sample was

incubated for 60 min at 40 °C in a thermoshaker to enable diastereomer

formation. The reaction was stopped by addition of 50 µL 0.1 M aq. HCl,

centrifuged (12000 × g, 10 min) and separated via LC-MS (see Support-

ing Information, Figure S9).

gentle shaking at 25 °C, 30 °C, 35 °C and 40 °C. Substrate L-tryptophan

was added to a final concentration of 5 mM together with 1 mM NAD⁺,

0.01 mM FAD, 1 U mL^-1RR-ADH, 2.5 U mL^-1PrnF, 5 % (v/v) iso-propanol,

10 mM Na₂HPO₄pH 7.4 and 30 mM NaBr. Purified Thal-WT, Thal-GR or

Thal-GLV was added to a final concentration of 20 µM. Reactions were

performed as duplicate at the temperature indicated and after 22 h final

conversion was analyzed by analytical RP-HPLC. Conversion-

temperature profiles were obtained by plotting conversion of substrate

against reaction temperature (see Supporting Information, Figure S2).

Long-term stability assay of Thal-WT and Thal-GLV

Purified tryptophan halogenase (50 µM in 15 mM Na₂HPO₄, pH 7.4 and

30 mM NaBr) was stored at 40 °C in a thermoshaker for several hours. At

different time points an aliquot of 10 µL was taken and applied for enzy-

matic bromination at 25 °C. For this, the sample was mixed with 2 mM

Acknowledgements

We thank Prof. Dr. Karl-Heinz van Pée for providing the plasmid

encoding for the flavin reductase PrnF, as well as Prof. Dr. Wer-

ner Hummel for providing the plasmid encoding for the alcohol

dehydrogenase. In addition, we are grateful to Pia Ferle and

Henrik Terholsen (Bielefeld University) for supporting this project.

L

-tryptophan, 2.5 U mL^-1PrnF, 1 U mL^-1RR-ADH, 0.01 mM FAD, 1 mM

NAD⁺, 5 % (v/v) iso-propanol, 10 mM Na₂HPO₄pH 7.4 and 30 mM NaBr

in a final volume of 100 µL. Reaction mixtures were incubated for 20 h at

25 °C in a thermoshaker (500 rpm) and afterwards aliquots of 50 µL were

taken and quenched by adding an equal volume of methanol. After

centrifugation (12000 × g, 10 min) the supernatant was analyzed by

analytical RP-HPLC. Measurements were performed as duplicate (see

Supporting Information, Table S4).

Keywords: directed evolution • enzyme catalysis • enzyme

stability • rational mutagenesis • tryptophan halogenase

Analysis of substrate panel of Thal-WT, Thal-S359G and Thal-GLV

The substrate scope of Thal-WT, Thal-S359G and Thal-GLV was tested

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10.1002/cctc.201901827

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Entry for the Table of Contents

FULL PAPER

Hannah Minges^[a], Christian Schne-

pel^[a], Dominique Böttcher^[b], Martin S.

Weiß^[b], Jens Sproß^[c],

A

comprehensive directed evolution cam-

paign led to a more thermostable tryptophan

6-halogenase. Successive rounds of random

evolution were combined with semi-rational

mutagenesis that generated a more robust

biocatalyst. Potential hotspots influencing

thermostability, activity and substrate selec-

tivity were combined to deduce a rationale

for halogenase stabilization. An enhanced

tendency of dimerization probably contrib-

uting to higher stability was observed for the

thermostable mutants by MS studies.

Uwe T. Bornscheuer^[b], and Norbert

Sewald*^[a]

Page No. – Page No.

Targeted Enzyme Engineering Un-

veiled Unexpected Patterns of Halo-

genase Stabilization

Article Doi

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

DOI: 10.1002/cctc.201901827

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Article abstract of DOI:10.1002/cctc.201901827

Full text of DOI:10.1002/cctc.201901827

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