Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal

JBC Papers in Press. Published on December 17, 2018 as Manuscript RA118.005393

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.005393

Ann-Christin Moritzer^1†, Hannah Minges^2†, Tina Prior¹, Marcel Frese², Norbert Sewald²,

Hartmut H. Niemann^1*

From the Department of Chemistry, ¹Structural Biochemistry and ²Organic and Bioorganic Chemistry,

Bielefeld University^,Universitätsstraße 25, D-33615 Bielefeld, Germany

Running title: Regioselectivity switch of tryptophan 6-halogenase Thal

*To whom correspondence should be addressed: Hartmut H. Niemann, Department of Chemistry,

Bielefeld University, Universitätsstraße 25, D-33615 Bielefeld, Germany;

Email: hartmut.niemann@uni-bielefeld.de; Tel. +49 521 106 6154; Fax: +49 521 106 6146

^†Equal contribution

Keywords: biocatalysis; enzyme catalysis, crystal structure; enzyme structure, flavoprotein, FAD-

dependent halogenase; tryptophan, protein engineering, protein design, enzyme design

from C6 to C7 as in RebH. We conclude that

structure-based protein engineering with

targeted substitution of a few residues is an

efficient approach to tailoring flavin-dependent

halogenases.

ABSTRACT

Flavin-dependent halogenases increasingly

attract attention as biocatalysts in organic

synthesis, facilitating environmentally friendly

halogenation strategies that require only

FADH₂, oxygen and halide salts. Different

______________________________________

flavin-dependent

tryptophan

halogenases

While chemical halogenation of aromatic

compounds requires toxic elemental halogens

and Lewis acids as catalysts, flavin-dependent

halogenases (FDHs) can catalyze electrophilic

aromatic substitutions with innoxious halide

salts and O₂as substrates and FADH₂as co-

factor (1, 2) . FDHs are responsible for the

regioselective chlorination or bromination of a

vast variety of natural products (3) and have

attracted a lot of attention due to their potential

use as biocatalysts (2, 4, 5). Slow turnover and

low stability still limit their large scale

application, but various strategies have been

pursued to tackle these challenges (6–8).

Bacterial tryptophan halogenases were the first

FDHs to be described (9–11). In addition,

regioselectively chlorinate or brominate

tryptophan’s indole moiety at C5, C6, or C7.

Here, we present the first substrate-bound

structure of a tryptophan 6-halogenase, namely

Thal, also known as ThdH, from the bacterium

Streptomyces

albogriseolus

at

2.55 Å

resolution. The structure revealed that the C6

of tryptophan is positioned next to the ε-amino

group of a conserved lysine, confirming the

hypothesis that proximity to the catalytic

residue determines the site of electrophilic

aromatic substitution. Although Thal is more

similar in sequence and structure to the

tryptophan 7-halogenase RebH than to the

tryptophan 5-halogenase PyrH, the indole

binding pose in the Thal active site more

closely resembled that of PyrH than that of

RebH. The difference in indole orientation

between Thal and RebH appeared to be largely

governed by residues positioning the Trp

backbone atoms. The sequences of Thal and

RebH lining the substrate binding site differ in

only few residues. Therefore, we exchanged

five amino acids in the Thal active site with the

FDHs

dichlorinated

were

identified

that

produce

from

tryptophan

chlorotryptophan (12, 13) or chlorinate

tryptophan only when it is part of a substrate

oligopeptide (14). FDHs acting on free

substrates other than tryptophan were also

described (15–18), including fungal enzymes

that accept large substrates (19–21) or have

rather broad substrate specificity (22, 23)

raising the hope that diverse compounds can be

enzymatically halogenated in vitro. Moreover,

FDHs have been integrated into engineered

biosynthetic pathways in vivo (24, 25). The

first structure of an FDH showed that these

corresponding

counterparts

in

RebH,

generating the quintuple variant Thal-RebH5.

Overall conversion of Trp by the Thal-RebH5

variant resembled that of wildtype Thal, but its

regioselectivity

of

chlorination

and

bromination was almost completely switched

enzymes consist of two subdomains,

a

Regioselectivity switch of tryptophan 6-halogenase Thal

conserved box-shaped subdomain harboring

the FAD binding site and a variable subdomain

that is pyramidal in tryptophan halogenases

(26). Tryptophan is bound at the interface of

the box and the pyramid.

protein conformational changes that occur

upon substrate binding (28, 35, 36). One would

expect that in tryptophan 6-halogenases C6 of

the indole moiety occupies a position roughly

equivalent to the position of C7 or C5 in

tryptophan 7-halogenases or 5-halogenases,

respectively. This positioning of C6 could be

achieved by slightly rotating the indole moiety

starting either from the binding pose observed

in the tryptophan 7-halogenases PrnA and

RebH or from the flipped binding pose

observed in the tryptophan 5-halogenase PyrH

(37). Thus, it remains elusive how L-Trp binds

to the active site of tryptophan 6-halogenases.

With their application as biocatalysts in

mind, enzyme optimization has been

performed on FDHs aiming at increasing their

lifetime and changing their substrate scope or

regioselectivity (1, 2). A large amount of work

has gone both into structure-based engineering

and directed evolution (27). These efforts

resulted in enzymes with higher stability (7, 8),

higher activity (23, 38), different substrate

scope (24, 37, 39, 40) or altered

regioselectivity (34, 37, 40, 41). Despite of the

already achieved success, these tasks still pose

formidable challenges. Directed evolution

requires many rounds of high throughput

screening and frequently it remains unclear,

how beneficial mutations exert their positive

effect (37). Attempts to alter the

regioselectivity with tryptophan as substrate

have so far resulted in an incomplete change of

selectivity so that only one third of substrate

was converted to a new regioisomer, while the

majority was still halogenated on the initially

preferred position (34, 41).

In addition to using benign reactants under

mild conditions, the regioselectivity of FDHs

provides a further advantage of enzymatic

halogenation in contrast to chemical

approaches. Different FDHs are known that

halogenate tryptophan selectively on C5 or C6

or C7 of the indole moiety, while chemical

halogenation preferably occurs at C2 (27).

Thus, for the natural substrate L-Trp the site of

enzymatic halogenation by flavin-dependent

tryptophan halogenases is not governed by

electronic factors of the substrate but

determined sterically by the orientation of the

substrate in the active site. This model was

suggested based on the structure of the

tryptophan 7-halogenase PrnA (26) and

confirmed by the structure of the tryptophan 5-

halogenase PyrH (28), both in complex with

their native substrate tryptophan. These

structures showed that the carbon atom to be

halogenated is positioned most closely to the ε-

amino group of the catalytic lysine, which is

conserved among FDHs. To achieve this

positioning, tryptophan is bound with distinctly

different poses by tryptophan 7-halogenases

and by tryptophan 5-halogenases. The indole

moiety of L-Trp bound to PyrH is flipped by

about 180° relative to the orientation observed

in the tryptophan 7-halogenases PrnA and

RebH. Several FDHs have been described that

halogenate tryptophan on C6, namely Thal

(29) (also known as ThdH (30)), SttH (31) and

thermophilic Th-Hal (32) that are bona fide

tryptophan 6-halogenases and KtzR that

produces 6-chlorotryptophan from tryptophan

but has higher catalytic efficiency for

converting 7-Cl-L-Trp to 6,7-di-Cl-L-Trp (12).

BorH is another putative tryptophan 6-

halogenase, however its activity has not yet

been confirmed in vitro (33). Based on

sequence identity, SttH, Th-Hal and KtzR are

closely related (Table 1) to each other and to

the tryptophan 5-halogenase PyrH, whereas on

the other hand, Thal and BorH are closely

related to each other and to the tryptophan 7-

halogenases RebH and PrnA. Crystal structures

of SttH (34) and Th-Hal (32) have been

published, but only in the absence of substrate.

No structural data is available for Thal, BorH

or KtzR. Modeling the substrate into the active

site of the empty enzyme is hampered by

We embarked on a project to further

elucidate the structural basis of regioselectivity

of tryptophan halogenases. We aimed at

crystallizing the tryptophan 6-halogenase Thal

together with its native substrate L-Trp. Based

on its structure we designed a Thal variant with

few amino acid substitutions in the active site

with the goal to switch the regioselectivity

from the 6- to the 7-position.

Results

Overall structure

The crystal structure of Thal without FAD,

chloride and tryptophan (apo-Thal) and a

structure in complex with its substrate L-Trp

(Trp-Thal) obtained by soaking contained two

copies of Thal per asymmetric unit (Table S1).

2

Regioselectivity switch of tryptophan 6-halogenase Thal

Amino acids (aa) 2-529 could be modelled in

all chains except for chain B of the apo-Thal

structure, which lacked interpretable electron

density for residues 450-456. In the apo-Thal

structure, the active site of chain A contains

positive difference density, which we could not

faithfully model. This difference density

suggests that the substrate binding site of apo-

Thal chain A is not truly empty. Clear electron

density was observed for the substrate L-Trp in

both chains of Trp-Thal.

Accordingly, structural differences between

Thal and PyrH, Th-Hal or SttH are bigger than

between Thal and RebH. As a L-Trp-bound

structure is available only for PyrH, we will

focus on a structural comparison of Thal with

PyrH rather than SttH or Th-Hal. Thal (PDB

ID 6H44) and PyrH (PDB ID 2WEU)

protomers align with only 466 structurally

corresponding residues and an r.m.s.d. value of

1.22 Å (Fig. S2b). For the dimer, the r.m.s.d.

between Thal and PyrH is even higher with

1.73 Å for 928 structurally corresponding

residues instead of the total of 1056 possible

In solution Thal behaved as monomer

according to the elution volume from size

exclusion

chromatography

and

its

residues. Structural conservation is highest in

the box shaped FAD binding module. In the

pyramidal subdomain some secondary

structure elements are rearranged or connected

differently, which is mostly due to insertions or

deletions (Fig. 1).

hydrodynamic radius as determined by

dynamic light scattering (data not shown).

Nevertheless, Thal appears to form a dimer in

the crystal (Fig. S1) like other tryptophan

halogenases (26, 28, 32, 34–36). The

arrangement of protomers in the dimer is very

similar to that of other Trp-halogenases with a

root mean square deviation (r.m.s.d.) between

the apo-Thal dimer and the apo-RebH (PDB

ID 2OAM) dimer of only 1.4426 Å for 1027

structurally corresponding residues.

The structural similarity is even higher for

the protomers with an r.m.s.d. of 0.9675 Å and

0.9013 Å for 521 and 519 structurally

corresponding residues, respectively. This is

not surprising given the sequence identity

between Thal and RebH of 64 % with almost

no insertions or deletions, except for His104

and the C-terminal Ser531 that are inserted in

Thal and Asp449 inserted in RebH (Fig. 1).

Each monomer consists of a single domain,

which folds into a larger rectangular box

containing the FAD binding site, and a smaller

triangular pyramid (Fig. 2), as described for

PrnA (26) and other tryptophan halogenases

(28, 32, 34–36). The substrate binding site is

located between the box and the pyramid and

occupies the same position as in RebH (Fig.

S2a). Thal residues Lys79 and Glu358 align

with the catalytic key residues Lys79 and

Glu357 of RebH. The difference to the

catalytic residues Lys83 and Glu362

previously reported for Thal (29) is due to a

difference in numbering with an offset of four.

At the level of the backbone, just few

structural differences between Thal and RebH

become apparent, mainly at the sites of

insertions (Fig. S2a).

Tryptophan binding site

The structure of apo-Thal, which we had

determined first, revealed a substrate binding

site very similar to that of RebH. All residues

of the substrate binding site that differ between

Thal and RebH are smaller in Thal, leaving

more space for the substrate. As we were not

able to determine the L-Trp binding pose or to

deduce

a

reason for the different

regioselectivity from the structure of apo-Thal,

we determined the structure of Thal in

complex with its native substrate L-Trp. As

expected, L-Trp is oriented in such a way that

its C6 position points toward the catalytic

residues Lys79 and Glu358 (Fig. 3). The

substrate binding site can be divided into three

regions, namely the catalytic, the π-stacking,

and the backbone-binding region (Fig. 4a). The

catalytic region harbors the Lys79 that is

universally conserved among flavin-dependent

halogenases and Glu358 that is conserved

among tryptophan halogenases (Fig. 4b). In

addition, we assign several small amino acids

including Val52, Val82 and Ser360 to this

region. The π-stacking region fixes the indole

ring (Fig. 4c) and may shield the non-

halogenated aromatic carbon atoms of the

indole from reactive HOCl. It consists of the

aromatic residues His110, Phe112, Phe465 and

Trp466. In addition, we assign Pro53 and

Pro111 to this middle region (Fig. 4a and c).

The backbone-binding region, which is in part

poorly ordered in the absence of substrate,

contains residues that coordinate the α-amino

and α-carboxyl group of L-Trp. It contains

Gly113 and Lys114 from a loop that flips into

Sequence conservation between Thal and

the tryptophan 5-halogenase PyrH or the

tryptophan 6-halogenases Th-Hal and SttH is

lower than between Thal and RebH (Table 1).

3

Regioselectivity switch of tryptophan 6-halogenase Thal

position upon substrate binding, Tyr454,

Tyr455 and Glu461 from the substrate-binding

loop and other polar residues like Thr467 and

Ser470 (Fig. 4a and d).

halogenated carbon atoms (C6 in Trp-Thal and

C5 in Trp-PyrH) again overlap closely with a

distance of only 0.2 Å. The indole nitrogen

forms a H-bond to structurally equivalent

residues in Thal (Pro111) and PyrH (Pro93),

but to a different residue in RebH (Glu357).

The rather low sequence and structural

conservation between Thal and PyrH makes a

meaningful comparison of substrate binding to

these proteins difficult. The catalytic and π-

stacking regions of the Trp-PyrH (PDB ID

2WEU) active site show only few substitutions

(V52F, V82I, S360T, W466F; numbering

according to Thal) and align fairly well with

the structure of Trp-Thal. In contrast, the

backbone-binding region is completely

different in structure and sequence (Fig. 1 and

Fig. S2b). The rearranged structure of the

active site lid is the most obvious difference. In

Thal, residues 445-465 form two mini helices

and loops covering the tryptophan binding site.

The corresponding PyrH loop aa 441-451 only

partly covers the area above the active site.

Therefore, in PyrH the active site is further

covered by an α-helix (aa 157-162), which

structurally corresponds to residues 451-459 of

Thal. In Thal, Tyr454, Tyr455 and Glu461 are

key residues of the substrate-binding loop and

form direct or water-mediated H-bonds and a

salt bridge with L-Trp. These residues are

replaced by Gln160, Gln163 and Tyr454

(which does not superpose with Thal Tyr454)

in PyrH. Accordingly the distance of the

backbone atoms of L-Trp are larger between

Thal and PyrH than between Thal and RebH.

Structural changes upon L-Trp binding

A comparison of apo-Thal and Trp-Thal

indicates structural changes upon binding of L-

Trp. The B factors of the loop covering the

FAD binding site (FAD loop; aa 38-45)

increase while the substrate-binding loop (aa

450-457) becomes more ordered. In chain B,

Phe112 undergoes a peptide flip, moving

Gly113 and Leu114 closer to the substrate by

2.4 Å and 1.4 Å, respectively. A similar

peptide flip involving the equivalent residue

Phe111 is observed for RebH and Trp-RebH

(36, 42). Phe112 in chain A of apo-Thal adopts

the same conformation as chain A and B of

Trp-Thal, presumably because the substrate

binding site is not truly empty as indicated by

positive difference density. Upon substrate

binding, residues of the backbone-binding

region that form polar contacts with L-Trp

become ordered and block the way out through

the substrate tunnel to the dimer interface

(Fig. S3). The α-amino group of L-Trp forms a

salt bridge with the carboxyl group of Glu461

and two H-bonds, one to the hydroxyl group of

Tyr454 and the other to the backbone carbonyl

of Tyr465 (Fig. 4d). The carboxyl group of L-

Trp forms H-bonds to the hydroxyl group of

Tyr455 and to two conserved water molecules

(see below). Upon substrate binding, several

residues move out of the active site (Fig. 5).

This makes room for L-Trp both in the

catalytic region (Thr51: 0.6 Å; Val52: 0.7 Å;

Glu358: 1.0 Å) and in the π-stacking region

(Pro53: 0.7 Å; Phe112: 1.0 Å; Trp466: 0.6 Å).

Due to the higher conservation,

a

comparison of Thal with RebH appears more

rewarding. Considering the direct surrounding

of L-Trp in Thal and RebH (i.e. residues with

atoms within 4 Å to L-Trp), there are only four

substitutions, namely V52I, V82I, S470N and

P111S110 (Thal  RebH). In a slightly

extended definition of the surroundings (i.e.

residues with atoms within 5 Å to L-Trp or one

Comparison of substrate binding in Thal,

RebH and PyrH

The indole moiety in Trp-Thal is almost

parallel to that in Trp-RebH but flipped by

180°, so that the five-membered rings do not

superpose at all (Fig. 3). In contrast, the

halogenated carbon atoms (C6 in Trp-Thal and

C7 in Trp-RebH) are only 0.5 Å apart. The

same flip of ~180° relative to the orientation in

RebH is present in PyrH. Hence, the binding

pose of the substrate indole moiety in Thal is

more like that in PyrH than that in RebH.

Interestingly, L-Trp adopts different rotameric

conformations in Thal and in RebH, while it is

of two conserved water molecules

(see

below)), there are another three substitutions,

namely S360T359, T467N and G469S

(Fig. S4). The side chains of Ser110 in RebH

and Pro111 in Thal point away from the

substrate. Thus, the larger Pro side chain in

Thal presumably has little influence on L-Trp

binding, as the backbone atoms that line the

substrate binding site superpose well. The

other six substitutions result in smaller side

chains in Thal than in RebH. Thus, the

substrate binding site of Thal should be more

bound to PyrH in

a

non-favorable

conformation. In Thal and PyrH, the

4

Regioselectivity switch of tryptophan 6-halogenase Thal

spacious than that of RebH. Three of the

substitutions (V52I, V82I and S360T359)

are in the catalytic region and, therefore, in

close contact to the indole moiety. The

remaining three substitutions are in the

backbone-binding region. Two substitutions

(T467N and G469S) will potentially impact the

water structure involved in coordinating the L-

Trp carboxyl group. Finally, the S470N

substitution impacts both the coordination of a

conserved water molecule (see below) and the

indole binding site. Serin in position 470

provides the necessary space for the five-

membered ring of L-Trp that is required upon

the 180° flip of the indole moiety.

B713, W3 in Fig. 6a, which is conserved

among FDHs ), which in turn is bound to Thal

via two H-bonds to the hydroxyl groups of

Thr467 and Ser470. W3 is conserved between

Trp-Thal monomers, but not among Trp-Thal

and Trp-RebH. This difference between Thal

and RebH is presumably due to the amino acid

substitutions in the backbone-binding region of

the active site.

Switching the regioselectivity by structure-

guided mutagenesis

To test whether a few targeted mutations are

sufficient to switch the regioselectivity, we

substituted five residues in the substrate

binding site of Thal by the corresponding

amino acids of RebH. The resulting variant

Thal-RebH5 harboured the substitutions V52I,

V82I, S360T, G469S and S470N already

described above (Fig. S4). In order to assess

the overall conversion of tryptophan, assay

mixtures using purified protein and

concomitant cofactor regeneration were

analyzed via HPLC-MS (Fig. 7). After a

reaction time of 86 h 70 % conversion of

5 mM L-Trp to L-chlorotryptophan (L-Cl-Trp)

was reached. This proved that these

substitutions in the active site do not affect the

overall conversion. In addition to the main

signal observed in the chromatogram, a less

intense signal indicated the formation of a

second product (Fig. 7c). As both signals were

identified as monochlorinated tryptophan with

the same m/z pattern from their corresponding

mass spectra, we assumed that another

regioisomer might have formed. HPLC-MS

analysis showed that both isomers were formed

in a ratio of 95:5, while comparison with

authentic standards revealed that 7-

chlorotryptophan was formed as the main

product. The minor signal corresponds to 6-

chlorotryptophan.

Water within the Trp binding site

Along with the substrate L-Trp some water

molecules get trapped in the active site. The

position of these waters is conserved between

the two crystallographically independent Thal

monomers and sometimes even among Thal

and RebH. There are two prominent water

molecules forming H-bonds with the L-Trp

carboxyl group. Their positions are conserved

between Thal and RebH and here we call them

W1 and W2 for simplicity (Fig. 6). W1

corresponds to A710 and B721 in Trp-Thal

and to water A1031 and B1125 in Trp-RebH

(PDB ID 2E4G). W1 forms a H-bond with the

carboxyl group of the substrate and two H-

bonds with the protein via the side chain

carboxyl group of the Glu461 and the

backbone NH of Asn54 (Fig. 6a and c). The H-

bond pattern between W1 and the protein is

identical in Thal and RebH and W1 is also

present in chain A of apo-Thal (water A718)

(Fig. 6b) and in apo-RebH (waters A777 and

B646; PDB ID 2OAM) (Fig. 6d). W2

corresponds to A752 and B712 in Trp-Thal

and to waters A1080 and B1091 in Trp-RebH

(PDB ID 2E4G)). In contrast to W1, W2 is

missing in the apo-structures of Thal and RebH

(Fig. 6b and d). Like W1, W2 forms a H-bond

with the carboxyl group of the substrate L-Trp.

In addition, W2 forms a H-bond with the

carbonyl oxygen of Gly113 (Gly112 in RebH).

This H-bond in combination with the absence

of W2 in the apo-structures may be one reason

for the peptide flip of Phe112 upon substrate

binding. Further H-bonds between W2 and the

protein differ between Thal and RebH. W2

forms two direct H-bonds with RebH via the

side chain amides of Asn467 and Asn470 (Fig.

6c). In contrast, W2 in Thal forms a H-bond

with a secondary water molecule (A717 and

In addition, chlorination of L-Trp was

conducted on a preparative scale using E. coli

lysate containing overexpressed halogenase.

After 26 h a maximum conversion of 30 % into

the expected monochlorinated products (95:5

ratio) was observed via HPLC-MS. Separation

and purification of the main product by

reversed-phase HPLC finally allowed to

identify L-7-chlorotryptophan by one- and two-

dimensional NMR spectroscopy (Supporting

Information). This proves the successful and

nearly

quantitative

change

of

the

regioselectivity from position 6 to 7 by

5

Regioselectivity switch of tryptophan 6-halogenase Thal

substituting five residues of Thal by the

corresponding RebH residues.

the plane of the indole moiety was well

defined, each conformer could be rotated or

translated to a certain degree in this plane.

Positional refinement of L-Trp did not

converge to a uniquely defined solution,

presumably due to the partial occupancy and

the limited resolution. Therefore, we decided

not to deposit this structure in the PDB but

provide coordinates and structure factors as

Supporting Information. The Trp-Thal-RebH5

structure strongly suggests that Thal-RebH5 is

permissive to binding L-Trp with its C7

positioned most closely to the ε-amino group

of the catalytic Lys79.

Enzymatic bromination reactions were

performed on a preparative scale analogously

to the chlorination described above. After a

reaction time of 48 h, HPLC-MS data revealed

15 % conversion to monobrominated L-Trp.

Like the chlorination, two product signals in a

ratio of 95:5 were observed. As the overall

conversion was lower, the minor isomer was

hardly visible in the UV trace, but its quantity

was sufficient for MS detection. After

purification C7-brominated L-Trp was isolated

as the major bromination product of Thal-

RebH5, which was also confirmed by NMR

analysis. Corresponding to the previous

observations made for Thal-RebH5-catalyzed

chlorination, 6-bromotryptophan had been

formed in negligible quantity as identified via

an authentic HPLC standard (Supporting

Information).

Discussion

Here we present the first experimental

structure of a tryptophan 6-halogenase in

complex with its native substrate L-Trp. The

structure

confirms

the

model

of

regioselectivity put forward by the Naismith

and van Pée groups (26, 28). As in the

tryptophan 7-halogenases PrnA and RebH and

the tryptophan 5-halogenase PyrH, the

halogenated aromatic carbon atom is

positioned most closely to the ε-amino group

of the catalytic lysine in Thal. It clearly

appears to be the binding pose of the substrate

that determines the regioselectivity.

Our apo- and L-Trp-bound structures of

Thal suggest that the empty active site of the

enzyme may not be well suited to reliably

model the substrate binding pose for three

reasons: First, in the absence of L-Trp several

conserved residues in the catalytic region and

the π-stacking region move toward the center

of the substrate binding site. If one places L-

Trp from the Trp-Thal structure into

structurally aligned apo-Trp, there would be

severe clashes (Fig. S7). Second, major

conformational changes between the apo-Thal

and Trp-Thal structure affect key residues for

L-Trp binding in the backbone-binding region

(Fig. 4d and Fig. S3). Finally, two water

molecules that are conserved between Thal and

RebH mediate interactions between the L-Trp

carboxyl group and the protein, but only one of

these waters is present in the substrate-free

structures of Thal and RebH (Fig. 6b).

Structure of Thal-RebH5

Thal-RebH5 crystallized like the wildtype

protein and the 2.12 Å structure (Table S1)

showed clear electron density for the five

mutated residues. The rotamers of substituted

residues in Thal-RebH5 correspond to those of

RebH except for Ser469. An overlay of apo-

Thal, apo-Thal-RebH5 and apo-RebH showed

that in Thal-RebH5 most active site residues,

including many conserved residues, adopt an

intermediate conformation between Thal and

RebH (Fig. S5). In some cases like Thr360 or

Phe465, Thal-RebH5 more closely resembles

RebH than wildtype Thal. We also soaked L-

Trp into Thal-RebH5 crystals and obtained a

structure at 2.55 Å resolution. There was clear

positive difference density in the active site of

chain

A

(Fig. S6a) and some positive

difference density in the active site of chain B.

The difference density in chain A is akin to the

shape of L-Trp but cannot be modelled

faithfully. The best interpretation that we

achieved is a superposition of L-Trp as it is

bound to Thal (conformer A) and to RebH

(conformer

B)

(Fig. S6b).

Occupancy

refinement converged to values of about 0.5

for conformer A and about 0.36 for conformer

B. If only one conformer was modeled, the

occupancy refined to values of about 0.8-0.9.

Placing a single conformer, however, resulted

in positive difference density in the region

occupied by the five-membered ring of the

other conformer (Fig. S6c and d), supporting

the presence of the second conformer. While

Given the high degree of sequence and

structural similarity between Thal and RebH,

also in the active site, it may have come as

some surprise to find in Thal an indole binding

pose that more closely resembles that of PyrH

than that of RebH. Altogether, this work

6

Regioselectivity switch of tryptophan 6-halogenase Thal

underscores the importance of experimental

structure determination, even in cases where

structures of highly homologous proteins are

available.

To gain more insight into which residues

may be key to determine the regioselectivity in

RebH and Thal, we performed a structural

overlay of Trp-Thal and Trp-RebH (PDB ID

2E4G) and copied the L-Trp from Trp-RebH to

Trp-Thal and vice versa. We then checked for

clashes indicating which residues might

prevent binding of L-Trp to Thal in the same

binding pose as in RebH and vice versa.

observed in Trp-Thal. Interestingly, a serine is

also present at this position in BorH (Ser469),

SttH (Ser464), Th-Hal (Ser460) and PyrH

(Ser455). In PyrH, however, mutating this Ser

to Asn would not result in clashes with the L-

Trp indole moiety. Still, the N470S

substitution may be an enabling mutation in

RebH that allows flipping the indole moiety

relative to the orientation found in Trp-RebH.

Due to the higher activity of the N470S variant

on tryptamine, Andorfer et al. used RebH

N470S as starting point for their directed

evolution of RebH towards C5 or C6

selectivity (37). Interestingly, RebH N470S

retained regioselectivity for the 7-position. It

produced >99% 7-chlorotryptamine and only

When placing L-Trp from RebH in the Trp-

Thal structure (Fig. 8a), clashes mainly occur

with conserved residues. This may not be

surprising given the fact that all sequence

differences in the active sites of Thal and

RebH have smaller side chains in Thal. Several

conserved residues of the catalytic and the π-

stacking region move towards the substrate in

Trp-Thal compared to Trp-RebH. Accordingly,

there would be close contacts between L-Trp

from RebH and Thal residues Pro53, Phe112

and Trp466. Reasons why these residues move

into the active site in Thal are not easily

apparent, as even the shell of residues

surrounding Pro53, Phe112 and Trp466 is

highly conserved between Thal and RebH. We

suspect that the indole binding pose in Thal

may not be determined primarily by steric

hindrance from residues surrounding the indole

moiety but by the different positioning of L-

Trp Cα due to the different H-bond pattern

between the L-Trp carboxylate and the

backbone-binding region of the protein.

In contrast, moving L-Trp from Trp-Thal to

Trp-RebH results in distinct clashes between

RebH and L-Trp (Fig. 8b). Two RebH residues

would form particularly short contacts with L-

Trp. First, the side chain of Asn470 would

clash with C2 and C3 of the indole moiety.

Second, the aromatic ring of Tyr455 would

clash with the L-Trp carboxyl group. Our

conclusions are consistent with the previous

notion of the van Pée and Naismith groups that

in PrnA the equivalent residues Tyr444 and

Asn459 prevent L-Trp from adopting the same

orientation as that seen in PyrH (41).

Intriguingly, even a random mutagenesis

approach to select RebH variants that can

halogenate larger substrates picked up the

N470S substitution (39).

minute

amounts

of

5-

and/or

6-

chlorotryptamine. However, all the later

variants that halogenated up to 95% of

tryptamine on C5 or C6 had this founding

N470S substitution.

The second RebH residue that would clash

with L-Trp from Thal, namely Tyr455, is

conserved between RebH and Thal. Despite

sequence conservation, its spatial arrangement

is markedly different between RebH and Thal.

In RebH, Tyr455 is pushed toward the

substrate relative to Thal. This displacement

may be caused by the only insertion in RebH

relative to Thal. A structure-based sequence

alignment identifies Asp449 as the inserted

residue (Fig. 1). This different location of

Tyr455 decreases the space available to the L-

Trp backbone atoms in RebH. Again, the

extensive mutagenesis work on RebH by

Andorfer et al. provides circumstantial support

for this hypothesis, as their RebH substitution

variants with good halogenation activity on C5

or C6 of tryptamine had no activity towards the

larger L-Trp (37).

demonstrated

that

halogenase regioselectivity can be altered

using different mutagenesis strategies. In a

rational approach

a

partial change of

regioselectivity yielding a 2:1 ratio of 7- and 5-

bromotryptophan was achieved for PrnA (41).

In an approach more similar to ours

Micklefield and co-workers tried to switch the

regioselectivity of the tryptophan 6-halogenase

SttH to position 5 (34). Their work is based on

structural differences between SttH and the

closely related tryptophan 5-halogenase PyrH.

A triple variant of SttH showed activity like

wild-type SttH with relaxed regioselectivity

leading to 32 % 5-Cl-L-Trp. A larger switch in

regioselectivity was observed for the smaller

These observations suggest that the N470S

substitution (RebHThal) may be of central

importance to facilitate the indole binding pose

7

Regioselectivity switch of tryptophan 6-halogenase Thal

substrate 3-indolepropionic acid, yielding 75 %

5-chlorinated product compared to just 10 % 5-

chlorination for wild-type SttH. In a directed

evolution approach, Lewis and co-workers

used random mutagenesis in combination with

structure-guided mutation of active site

residues to modify the regioselectivity of RebH

(37). Their efforts to evolve the tryptophan 7-

that of the roughly 200 sequence differences

between RebH and Thal, only a handful is

required to switch regioselectivity. All of these

substitutions affect active site residues.

Substitution of residues distant to the substrate

binding site that are frequently picked up in

random mutagenesis but almost impossible to

predict in rational structure-based approaches

halogenase RebH into

a

C5- or C6-

were not required.

regioselective biocatalyst resulted in the

generation of two new variants capable of

chlorinating tryptamine with up to 95 % and

90 % regioselectivity for position 5 and 6,

respectively. For 2-methyltryptamine and N-

methyltryptamine the selectivity was even

higher. Both the C5- and the C6-selective

variant were inactive on the native substrate L-

Trp.

In summary, previous studies achieved

nearly complete selectivity switches for non-

native substrates like 3-indolepropionic acid

and tryptamine both by rational, structure-

based engineering (34) and by (random)

mutagenesis coupled to high-throughput

screening (37). For L-Trp, however, structure-

based mutagenesis of both PrnA (41) and SttH

(34) shifted regioselectivity to only about 1/3

of 5-Cl-L-Trp from 100% each of 7-Cl-L-Trp

and 6-Cl-L-Trp, respectively. Our Thal-RebH5

Experimental procedures

Plasmids and molecular cloning

The pET-28a plasmid containing a codon-

optimized gene for E. coli encoding Thal

(Uniprot ID: A1E280) was reported earlier

(43). The genes encoding Thal and Thal-

RebH5 were separately subcloned into the

vector pETM-11 (Gunter Stier, Heidelberg)

using the NcoI and NotI restriction sites with

primers listed in Table S2. The plasmid vector

pET-21-adh encoding alcohol dehydrogenase

from Rhodococcus ruber was kindly provided

by Prof. Dr. Werner Hummel, Bielefeld

University, and pCIBhis-prnF encoding the

flavin reductase PrnF from Pseudomonas

fluorescens 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. Generation of the Thal-RebH5

quintuple variant was performed according to

the QuikChange II Site-Directed Mutagenesis

Kit with primers listed in Table S2. Mutations

were introduced consecutively starting with

pET-28a-thal wildtype as DNA template.

Mutations were confirmed by DNA

sequencing (GATC Biotech).

variant exhibited

a

95 % change in

regioselectivity towards C7 halogenation for

both chlorination and bromination of L-Trp,

constituting a nearly quantitative switch that

has not been reported for any tryptophan

halogenase until now. Both 3-indolepropionic

acid and tryptamine are smaller than L-Trp and

should have more flexibility to accommodate

in the active site, offering a potential

explanation why switching regioselectivity for

smaller substrates was more successful. This

may be particularly relevant for RebH,

because, as pointed out before, the insertion of

D449 in RebH pushes the conserved Y455 into

the active site in comparison to Thal. Thus,

Thal, which provides more space for the L-Trp

backbone atoms, may represent a better

starting point than RebH to evolve or engineer

regioselectivity on the larger substrate L-Trp.

The success of our protein engineering is

based on a careful structural analysis of

substrate binding in closely related tryptophan

halogenases with different regioselectivity. As

such, our approach most closely resembles that

of Shepherd et al. who also based their

mutations on structures of two closely related

tryptophan halogenases (34). Our results show

Expression and purification of recombinant

Thal for crystallization

N-terminal His₆-tagged Thal was over-

expressed in E. coli BL21(DE3) using a

pETM-11 vector. The culture was grown in LB

medium with kanamycin (30 mg·L^-1) at 37 °C

until an optical density OD₆₀₀of 0.6 was

achieved. After that, the culture was induced

with

0.1 mM

isopropyl-β-D-

thiogalactopyranoside (IPTG) and shaken for

an additional 20 h at 25 °C. The cells were

harvested by centrifugation (30 min, 4 °C,

5180 × g), resuspended in lysis buffer (50 mM

sodium phosphate pH 7.4, 300 mM NaCl) with

additional DNase I and a cOmplete^TMEDTA-

free protease inhibitor tablet (Roche), and

disrupted using

a

SPCH high-pressure

8

Regioselectivity switch of tryptophan 6-halogenase Thal

homogenizer (Stansted Fluid Power Ltd UK).

The lysate was clarified by centrifugation

(4 °C, 1 h, 30966 × g). Soluble Thal was

purified by nickel-nitrilotriacetic acid (Ni-

NTA) agarose chromatography (pure Cube Ni-

NTA agarose, Cube Biotech; buffer: 50 mM

sodium phosphate pH 7.4, 300 mM NaCl,

1 mM β-ME, 10-300 mM imidazole), followed

by ion exchange (Source 15Q, GE Healthcare;

buffer: 50 mM Tris pH 7.4, 50-500 mM NaCl,

resin, flow rate: 0.5 mL·min^-1), followed by

washing steps (10 mL, flow rate 1 mL·min^-1)

with 50 mM NaH₂PO₄pH 7.4, 300 mM NaCl

and with 50 mM NaH₂PO₄pH 7.4, 300 mM

NaCl, 10 mM imidazole. The fusion protein

was eluted by addition of 300 mM NaCl,

300 mM imidazole, 50 mM Na₂HPO₄pH 7.4

(flow rate: 1 mL·min^-1). Fractions of 0.5 mL

were collected and protein concentration was

determined by Nano-Drop UV spectroscopy.

1 mM

DTT)

and

size

exclusion

Crystallization and data collection

chromatography (HiLoad 16/60 Superdex 200

Prep grade, GE Healthcare; buffer: 10 mM Tris

pH 7.4, 50 mM NaCl, 1 mM DTT). After the

Ni-NTA and during buffer exchange through

dialysis the N-terminal His₆-tag was cleaved

via tobacco etch virus (TEV) protease at 20 °C

overnight using a mass ratio (w/w) of 1:50.

Purified Thal was concentrated up to

25 mg·mL^-1using Vivaspin concentrators

(Sartorius AG, MWCO: 10.000) and stored in

crystallization buffer (10 mM Tris pH 7.4,

50 mM NaCl and 1 mM TCEP) at -80 °C until

further usage. The variantThal-RebH5 was

expressed and purified as described for Thal,

except using a Co-NTA (pure Cube Co-NTA

agarose, Cube Biotech) instead of Ni-NTA.

Purified Thal was crystallized using the sitting

drop vapor-diffusion method at 20 °C with a

drop ratio of 2:1 of protein solution

(~15 mg·mL^-1) and reservoir solution (0.1 M

Tris pH 8.5, 1.5 M K₂HPO₄/KH₂PO₄). In order

to obtain an L-Trp-bound structure, Thal

crystals were soaked in reservoir solution

additionally containing 5 mM L-tryptophan.

The Thal crystals for soaking experiments

were obtained from a different crystallization

condition (reservoir solution: 0.1 M bicine

pH 9.0, 1.6 M K₂HPO₄/NaH₂PO₄; drop ratio:

1:1). Thal-RebH5 was crystallized as the

wildtype but using another reservoir solution

(0.1 M

bicine

pH 8.4,

1.3 M

K₂HPO₄/KH₂PO₄). Trp-Thal-RebH5 crystals

were obtained by soaking Thal-RebH5 crystals

in reservoir solution saturated with L-

tryptophan for 75 minutes. Crystals appeared

as hexagonal prisms within seven days. For

cryoprotection the crystals were transferred to

reservoir solution supplemented with 25 %

glycerol, before being flash cooled and stored

in liquid nitrogen. All data were collected at a

temperature of 100 K at beamline P13 operated

by EMBL Hamburg at the PETRA III storage

ring at DESY, Hamburg, Germany (44).

Protein expression and purification for

enzymatic assays

Expression of PrnF and alcohol dehydrogenase

was performed according to published

procedures (5). For Thal and Thal-RebH5,

1.5 L LB medium containing kanamycin

(60 mg·L^-1) and chloramphenicol (50 mg·L^-1)

were inoculated with 20 mL·L^-1overnight

culture of E. coli BL21 (DE3) pGro7 pET28a-

thal wt or –thal-rebh5. 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. His₆-tagged

fusion protein was extracted from E. coli cells

by resuspending the 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 0.2 µm Whatman filter. The

cleared lysate was loaded on HisTALON

agarose affinity resin (bed volume of 1.5 mL

Data processing, structure determination and

refinement

The data sets were processed with XDS and

scaled with XSCALE (45). All crystals

belonged to the space group P6₄and showed

no indication of twinning using the program

aimless (46) from the CCP4 package (47). A

summary of the data processing is shown in

Table 2. The structure of apo-Thal was

determined by molecular replacement using

the program Phaser (48). The search model

consisted of an ensemble of structures of the

Trp-halogenases RebH (PDB ID 2OAM),

PrnA (PDB ID 2AQJ), PyrH (PDB ID 2WEU)

and SttH (PDB ID 5HY5). Phaser placed two

molecules per asymmetric unit with no clashes

9

Regioselectivity switch of tryptophan 6-halogenase Thal

and rotation function Z-scores of 21.0 and 9.3,

and translation function Z-scores of 42.2 and

84.8. The Trp-Thal structure was determined

using the apo-Thal structure as a search model

and was used as search model for the apo-

Thal-RebH5 and Trp-Thal-RebH5 structures.

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, 30 mM NaCl, in a

final volume of 30 mL. The reaction mixture

was incubated at 25 °C in a shaking incubator

(150 rpm) and the reaction progress monitored

by HPLC-MS as described above. Bromination

assays were performed analogously, except

that an aqueous buffer consisting of 10 mM

K₂HPO₄pH 7.4 and 30 mM NaBr was used for

resuspending the cells and subsequent

biocatalysis.

The first layout of the apo-Thal structure was

generated with AutoBuild (49) from the

PHENIX suite (50) and improved by

modelling in COOT (51) and restrained

refinement in Refmac5 (52) using non-

crystallographic symmetry (NCS) restraints or,

in case of Thal-RebH5, improved by

phenix.refine using TLS refinement and NCS

restraints. All structures were optimized until

R values converged (Table S1). The occupancy

of L-Trp in Trp-Thal refined to a value of one.

Figures of the final structures were generated

using PyMOL. All r.m.s.d values were

calculated with the CCP4 program TOPP with

the setting “superpose using secondary

structure matching“.

Analytical reversed phase-high performance

liquid chromatography

A

Shimadzu CBM-20A chromatography

system was employed for analytical

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

Hypersil Gold C18 column, 3 µm,

Enzymatic chlorination of L-tryptophan using

purified halogenase

150 × 2.1 mm from Thermo Scientific with a

flow rate: 0.5 mL·min^-1was used. Absorbance

was measured simultaneously at 220 nm,

Enzymatic chlorination was assayed in a total

volume of 500 μL while shaking gently at

25 °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^-1alcohol

dehydrogenase from Rhodococcus ruber (RR-

ADH), 2.5 U·mL^-1flavin redcutase PrnF,

5 % (v/v) iso-propanol, 10 mM Na₂HPO₄

pH 7.4 and 30 mM NaCl. Purified halogenase

was added to a final concentration of 89 µM.

Reactions were performed as triplicates and

reaction progress was monitored by HPLC-MS

or analytical HPLC. Aliquots of 30 µL were

taken from the reaction mixture at different

time points and quenched by adding an equal

volume of methanol. The mixture was

centrifuged (12000 × g, 10 min) and the

supernatant analyzed via HPLC-MS or

analytical HPLC.

254 nm

A: water/trifluoroacetic

and

280 nm.

acid

Eluents:

(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 %.

Preparative reversed-phase high performance

liquid chromatography

A Merck-Hitachi LaChrom HPLC was used

for purification consisting of interface D-7000,

pump L-7150 and detector L-7420 equipped

with Hypersil Gold C18 8 µm column,

250 × 21.2 mm, Thermo Scientific. Flow rate:

10 mL·min^-1, injection volume: 2.00 mL,

detection

at

λ = 280 nm,

solvents:

A: water/acetonitrile/trifluoroacetic acid

(94.9:5:0.1);

B: water/acetonitrile/

Enzymatic bromination/chlorination of L-

tryptophan using E. coli crude lysate

trifluoroacetic acid (5:94.9:0.1). Gradient

elution: (A %): 0-5 min: 100 %, 5-105 min:

linear gradient from 100 % to 0 %, 105-

110 min: gradient from 0 % to 100 %, 110-

115 min: 100 %.

E. coli BL21(DE3) pGro7 cells overexpressing

Thal-RebH5 from 1.5 L culture were

resuspended in 20 mL chlorination buffer

(10 mM Na₂HPO₄pH 7.4, 30 mM NaCl). The

cells were lysed by French press (3x) and cell

debris was removed by centrifugation

(10000 × g, 30 min, 4 °C). Subsequently,

lysate was added to 5 mM L-tryptophan, 1 mM

High performance liquid chromatography

mass spectrometry (LC-MS)

Electrospray ionization (ESI) mass spectra

were acquired employing an Agilent 6220

10

Regioselectivity switch of tryptophan 6-halogenase Thal

time-of-flight mass spectrometer from Agilent

Technologies (Santa Clara, CA, USA) in

extended dynamic range mode 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. Samples were

introduced from an HPLC outlet and

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.

HPLC solvent A: water/acetonitrile/formic

NMR spectroscopy

Nuclear magnetic resonance (NMR) spectra

were recorded on a Bruker Avance 500

(¹H: 500 MHz, ¹³C: 126 MHz), Avance 500

HD (¹H: 500 MHz, ¹³C: 126 MHz) or Avance

600 (¹H: 600.13 MHz, ¹³C: 150.92 MHz)

spectrometer. Chemical shifts (δ) were

reported in ppm relative to tetramethylsilan

(TMS) (δ_TMS= 0 ppm) and referenced to the

residual non-deuterated solvent signal as

internal standard. (DMSO-d₆: ¹H: 2.50 ppm;

¹³C: 39.5 ppm). Coupling constants (J) were

reported in Hertz (Hz) with 0.05 Hz resolution.

Multiplicities were described as singlet (s),

doublet (d), triplet (t) quartet (q) or multiplet

(m). The assignment of ¹³C- and H-NMR

1

acid

(94.9:5:0.1);

B:

(5:94.9:0.1).

signals was supported by 2D-NMR techniques

(COSY, ROESY, HSQC).

water/acetonitrile/formic acid

Gradient elution: (A %): 0-10 min gradient

from 100 % to 60 %, 10-11 min: gradient from

60 % to 2 %, 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.

Accession numbers

The atomic coordinates and structure factors of

apo-Thal, Trp-Thal and apo-Thal-RebH5 have

been deposited in the PDB as entries 6H43,

6H44 and 6IB5, respectively.

11

Regioselectivity switch of tryptophan 6-halogenase Thal

Acknowledgements: The synchrotron MX data was collected at beamline P13 operated by EMBL

Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). We would like to thank Dr.

Guillaume Pompidor, Dr. Isabel Bento and Johanna Hakanpää for the assistance in using the beamline.

We thank Christiane Widmann and Dominic Gilzer for careful reading of the manuscript.

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of

this article.

12

Regioselectivity switch of tryptophan 6-halogenase Thal

References

1.

2.

3.

4.

5.

Schnepel, C., and Sewald, N. (2017) Enzymatic Halogenation: A Timely Strategy for

Regioselective C−H Activation. Chem. - A Eur. J. 23, 12064–12086

Latham, J., Brandenburger, E., Shepherd, S. A., Menon, B. R. K., and Micklefield, J. (2018)

Development of Halogenase Enzymes for Use in Synthesis. Chem. Rev. 118, 232–269

van Pée, K. H. (2001) Microbial biosynthesis of halometabolites. Arch. Microbiol. 175, 250–

258

Payne, J. T., Andorfer, M. C., and Lewis, J. C. (2013) Regioselective Arene Halogenation

using the FAD-Dependent Halogenase RebH. Angew. Chemie - Int. Ed. 52, 5271–5274

Frese, M., Guzowska, P. H., Voß, H., and Sewald, N. (2014) Regioselective Enzymatic

Halogenation of Substituted Tryptophan Derivatives using the FAD-Dependent Halogenase

RebH. ChemCatChem. 6, 1270–1276

6.

7.

Frese, M., and Sewald, N. (2015) Enzymatic Halogenation of Tryptophan on a Gram Scale.

Angew. Chemie - Int. Ed. 54, 298–301

Schnepel, C., Minges, H., Frese, M., and Sewald, N. (2016) A High-Throughput Fluorescence

Assay to Determine the Activity of Tryptophan Halogenases. Angew. Chemie - Int. Ed. 55,

14159–14163

8.

9.

Poor, C. B., Andorfer, M. C., and Lewis, J. C. (2014) Improving the Stability and Catalyst

Lifetime of the Halogenase RebH by Directed Evolution. ChemBioChem. 15, 1286–1289

Hammer, P. E., Hill, D. S., Lam, S. T., van Pée, K. H., and Ligon, J. M. (1997) Four Genes

from Pseudomonas fluorescens That Encode the Biosynthesis of Pyrrolnitrin. Appl. Environ.

Microbiol. 63, 2147–2154

10.

11.

12.

13.

14.

Kirner, S., Hammer, P. E., Hill, D. S., Altmann, A., Fischer, I., Weislo, L. J., Lanahan, M., van

Pée, K. H., and Ligon, J. M. (1998) Functions Encoded by Pyrrolnitrin Biosynthetic Genes

from Pseudomonas fluorescens. 180, 1939–1943

Keller, S., Wage, T., Hohaus, K., Hölzer, M., Eichhorn, E., and van Pée, K. H. (2000)

Purification and Partial Characterization of Tryptophan 7-Halogenase (PrnA) from

Pseudomonas fluorescens. Angew. Chem. Int. Ed. Engl. 39, 2300–2302

Heemstra Jr, J. R., and Walsh, C. T. (2008) Tandem Action of the O2- and FADH2-Dependent

Halogenases KtzQ and KtzR Produce 6,7-Dichlorotryptophan for Kutzneride Assembly.

Society. 10.1021/ja806467a

Smith, D. R. M., Uria, A. R., Helfrich, E. J. N., Milbredt, D., van Pée, K. H., Piel, J., and Goss,

R. J. M. (2017) An Unusual Flavin-Dependent Halogenase from the Metagenome of the

Marine Sponge Theonella swinhoei WA. ACS Chem. Biol. 12, 1281–1287

Ortega, M. A., Cogan, D. P., Mukherjee, S., Garg, N., Li, B., Thibodeaux, G. N., Maffioli, S.

I., Donadio, S., Sosio, M., Escano, J., Smith, L., Nair, S. K., and van Der Donk, W. A. (2017)

Two Flavoenzymes Catalyze the Post-Translational Generation of 5-Chlorotryptophan and 2-

Aminovinyl-Cysteine during NAI-107 Biosynthesis. ACS Chem. Biol. 12, 548–557

15.

16.

17.

Agarwal, V., El Gamal, A. A., Yamanaka, K., Poth, D., Kersten, R. D., Schorn, M., Allen, E.

E., and Moore, B. S. (2014) Biosynthesis of polybrominated aromatic organic compounds by

marine bacteria. Nat. Chem. Biol. 10, 640–647

Neubauer, P. R., Widmann, C., Wibberg, D., Schröder, L., Frese, M., Kottke, T., Kalinowski,

J., Niemann, H. H., and Sewald, N. (2018) A flavin-dependent halogenase from metagenomic

analysis prefers bromination over chlorination. PLoS One. 10.1371/journal.pone.0196797

Zeng, J., and Zhan, J. (2010) A novel Fungal Flavin-Dependent Halogenase for Natural

13

Regioselectivity switch of tryptophan 6-halogenase Thal

Product Biosynthesis. ChemBioChem. 11, 2119–2123

18.

19.

Zhu, T., Cheng, X., Liu, Y., Deng, Z., and You, D. (2013) Deciphering and engineering of the

final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces

aureofaciens. Metab. Eng. 19, 69–78

Fraley, A. E., Garcia-Borràs, M., Tripathi, A., Khare, D., Mercado-Marin, E. V., Tran, H., Dan,

Q., Webb, G. P., Watts, K. R., Crews, P., Sarpong, R., Williams, R. M., Smith, J. L., Houk, K.

N., and Sherman, D. H. (2017) Function and Structure of MalA/MalA′, Iterative Halogenases

for Late-Stage C-H Functionalization of Indole Alkaloids. J. Am. Chem. Soc. 139, 12060–

12068

20.

21.

Chankhamjon, P., Tsunematsu, Y., Ishida-Ito, M., Sasa, Y., Meyer, F., Boettger-Schmidt, D.,

Urbansky, B., Menzel, K. D., Scherlach, K., Watanabe, K., and Hertweck, C. (2016)

Regioselective Dichlorination of a Non-Activated Aliphatic Carbon Atom and Phenolic

Bismethylation by a Multifunctional Fungal Flavoenzyme. Angew. Chemie - Int. Ed. 55,

11955–11959

Almeida, C., Pérez-Victoria, I., González-Menéndez, V., de Pedro, N., Martín, J., Crespo, G.,

Mackenzie, T., Cautain, B., Reyes, F., Vicente, F., and Genilloud, O. (2018) Non-geminal

Aliphatic Dihalogenation Pattern in Dichlorinated Diaporthins from Hamigera fusca NRRL

35721. J. Nat. Prod. 81, 1488–1492

22.

23.

Zeng, J., Lytle, A. K., Gage, D., Johnson, S. J., and Zhan, J. (2013) Specific chlorination of

isoquinolines by a fungal flavin-dependent halogenase. Bioorganic Med. Chem. Lett. 23, 1001–

1003

Menon, B. R. K., Brandenburger, E., Sharif, H. H., Klemstein, U., Shepherd, S. A., Greaney,

M. F., and Micklefield, J. (2017) RadH: A Versatile Halogenase for Integration into Synthetic

Pathways. Angew. Chemie - Int. Ed. 56, 11841–11845

24.

25.

26.

Glenn, W. S., Nims, E., and O’Connor, S. E. (2011) Reengineering a Tryptophan Halogenase

to Preferentially Chlorinate a Direct Alkaloid Precursor. J. Am. Chem. Soc. 133, 19346–19349

Runguphan, W., Qu, X., and O’Connor, S. E. (2010) Integrating carbon-halogen bond

formation into medicinal plant metabolism. Nature. 468, 461–467

Dong, C., Flecks, S., Unversucht, S., Haupt, C., van Pée, K. H., and Naismith, J. H. (2005)

Tryptophan 7-Halogenase (PrnA) Structure Suggests a Mechanism for Regioselective

Chlorination. Science (80-. ). 309, 2216–2219

27.

28.

29.

Andorfer, M. C., and Lewis, J. C. (2018) Understanding and Improving the Activity of Flavin-

Dependent Halogenases via Random and Targeted Mutagenesis. Annu. Rev. Biochem.

10.1146/annurev-biochem-062917-012042

Zhu, X., De Laurentis, W., Leang, K., Herrmann, J., Ihlefeld, K., van Pée, K. H., and Naismith,

J. H. (2009) Structural Insights into Regioselectivity in the Enzymatic Chlorination of

Tryptophan. J. Mol. Biol. 391, 74–85

Seibold, C., Schnerr, H., Rumpf, J., Kunzendorf, A., Hatscher, C., Wage, T., Ernyei, A. J.,

Dong, C., Naismith, J. H., and van Pée, K. H. (2006) A flavin-dependent tryptophan 6-

halogenase and its use in modification of pyrrolnitrin biosynthesis. Biocatal.

Biotransformation. 24, 401–408

30.

31.

32.

Milbredt, D., Patallo, E. P., and van Pée, K. H. (2014) A Tryptophan 6-Halogenase and an

Amidotransferase Are Involved in Thienodolin Biosynthesis. ChemBioChem. 15, 1011–1020

Zeng, J., and Zhan, J. (2011) Characterization of a tryptophan 6-halogenase from Streptomyces

toxytricini. Biotechnol. Lett. 33, 1607–1613

Menon, B. R. K., Latham, J., Dunstan, M. S., Brandenburger, E., Klemstein, U., Leys, D.,

14

Regioselectivity switch of tryptophan 6-halogenase Thal

Karthikeyan, C., Greaney, M. F., Shepherd, S. A., and Micklefield, J. (2016) Structure and

biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

Org. Biomol. Chem. 14, 9354–9361

33.

34.

Chang, F.-Y., and Brady, S. F. (2013) Discovery of indolotryptoline antiproliferative agents by

homology-guided metagenomic screening. Proc. Natl. Acad. Sci. U. S. A. 110, 2478–2483

Shepherd, S. A., Menon, B. R. K., Fisk, H., Struck, A. W., Levy, C., Leys, D., and Micklefield,

J. (2016) A Structure-Guided Switch in the Regioselectivity of a Tryptophan Halogenase.

ChemBioChem. 17, 821–824

35.

36.

Yeh, E., Blasiak, L. C., Koglin, A., Drennan, C. L., and Walsh, C. T. (2007) Chlorination by a

Long-Lived Intermediate in the Mechanism of Flavin-Dependent Halogenases. Biochemistry.

46, 1284–1292

Bitto, E., Huang, Y., Bingman, C. A., Singh, S., Thorson, J. S., and Phillips, G. N. (2008) The

structure of flavin-dependent tryptophan 7-halogenase RebH. Proteins Struct. Funct. Genet. 70,

289–293

37.

38.

Andorfer, M. C., Park, H. J., Vergara-Coll, J., and Lewis, J. C. (2016) Directed evolution of

RebH for catalyst-controlled halogenation of indole C–H bonds. Chem. Sci. 7, 3720–3729

Belsare, K. D., Andorfer, M. C., Cardenas, F. S., Chael, J. R., Park, H. J., and Lewis, J. C.

(2017) A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

ACS Synth. Biol. 6, 416–420

39.

40.

41.

42.

43.

44.

Payne, J. T., Poor, C. B., and Lewis, J. C. (2015) Directed Evolution of Rebh for Site-Selective

Halogenation of Large Biologically Active Molecules. Angew. Chemie - Int. Ed. 54, 4226–

4230

Shepherd, S. A., Karthikeyan, C., Latham, J., Struck, A.-W., Thompson, M. L., Menon, B. R.

K., Styles, M. Q., Levy, C., Leys, D., and Micklefield, J. (2015) Extending the biocatalytic

scope of regiocomplementary flavin-dependent halogenase enzymes. Chem. Sci. 6, 3454–3460

Lang, A., Polnick, S., Nicke, T., William, P., Patallo, E. P., Naismith, J. H., and van Pée, K. H.

(2011) Changing the Regioselectivity of the Tryptophan 7-Halogenase PrnA by Site-Directed

Mutagenesis. Angew. Chemie - Int. Ed. 50, 2951–2953

Yeh, E., Cole, L. J., Barr, E. W., Bollinger, J. M., Ballou, D. P., and Walsh, C. T. (2006) Flavin

Redox Chemistry Precedes Substrate Chlorination during the Reaction of the Flavin-Dependent

Halogenase RebH. Biochemistry. 45, 7904–7912

Frese, M., Schnepel, C., Minges, H., Voß, H., Feiner, R., and Sewald, N. (2016) Modular

Combination of Enzymatic Halogenation of Tryptophan with Suzuki-Miyaura Cross-Coupling

Reactions. ChemCatChem. 10.1002/cctc.201600317

Cianci, M., Bourenkov, G., Pompidor, G., Karpics, I., Kallio, J., Bento, I., Roessle, M.,

Cipriani, F., Fiedler, S., and Schneider, T. R. (2017) P13, the EMBL macromolecular

crystallography beamline at the low-emittance PETRA III ring for high- and low-energy

phasing with variable beam focusing. J. Synchrotron Radiat. 24, 323–332

45.

46.

Kabsch, W. (2010) XDS. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 125–132

Evans, P. R., and Murshudov, G. N. (2013) How good are my data and what is the resolution?

Acta Crystallogr. Sect. D Biol. Crystallogr. 69, 1204–1214

47.

Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan,

R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N.,

Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011)

Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D Biol.

Crystallogr. 67, 235–242

15

Regioselectivity switch of tryptophan 6-halogenase Thal

48.

49.

McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read,

R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674

Terwilliger, T. C., Grosse-Kunstleve, R. W., Afonine, P. V., Moriarty, N. W., Zwart, P. H.,

Hung, L. W., Read, R. J., and Adams, P. D. (2007) Iterative model building, structure

refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr.

Sect. D Biol. Crystallogr. 64, 61–69

50.

Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J.,

Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner,

R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010)

PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta

Crystallogr. Sect. D Biol. Crystallogr. 66, 213–221

51.

52.

Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of

Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486–501

Kovalevskiy, O., Nicholls, R. A., Long, F., Carlon, A., and Murshudov, G. N. (2018) Overview

of refinement procedures within REFMAC 5: utilizing data from different sources. Acta

Crystallogr. Sect. D Struct. Biol. 74, 215–227

53.

54.

Holm, L., and Laakso, L. M. (2016) Dali server update. Nucleic Acids Res.

10.1093/nar/gkw357

Gouet, P., Robert, X., and Courcelle, E. (2003) ESPript/ENDscript: Extracting and rendering

sequence and 3D information from atomic structures of proteins. Nucleic Acids Res.

10.1093/nar/gkg556

FOOTNOTES

The abbreviations used are: aa, amino acid; ESI, electrospray ionization; FDH, flavin-dependent

halogenase; HPLC-MS, high performance liquid chromatography-mass spectrometry; HSQC,

heteronuclear single quantum coherence; IPTG, isopropyl-β-D-thiogalactopyranoside; L-Cl-Trp, L-

chlorotryptophan; r.m.s.d., root mean square deviation; ROESY, rotating frame nuclear Overhauser

effect spectroscopy;

16

Regioselectivity switch of tryptophan 6-halogenase Thal

Tables

Table 1. Pairwise sequence identity of various tryptophan halogenases shows that the

tryptophan 6-halogenases. Thal and BorH are closely related to each other and to the tryptophan 7-

halogenase RebH. The tryptophan 6-halogenases SttH and Th-Hal are closely related to each other and

to the tryptophan 5-halogenase PyrH. The sequence identity is lower between Thal or BorH on the one

hand and SttH or Th-Hal on the other hand. The sequence identity was derived from pairwise

structure-based sequence alignments with the DALI server (53) except for BorH, for which no

structure is available.

ID%

Thal BorH* RebH PrnA PyrH SttH Th-Hal

Thal

100

70*

100

64

57

42

40

41

BorH

RebH

PrnA

PyrH

SttH

63*

100

56*

55

40*

40

39*

41

39*

41

100

42

40

41

100

58

56

100

74

Th-Hal

100

17

β1

α1

20

β2

β3

α2

α3

70

Thal

1

10

30

40

50

60

Thal .MDNRIKTVVILGGGTAGWMTAAYLGKALQNTVKIVVLEAP.TIPRIGVGEATVPNLQRAFFDYLGIPEEEWM

RebH .MSGKIDKILIVGGGTAGWMAASYLGKALQGTADITLLQAP.DIPTLGVGEATIPNLQTAFFDFLGIPEDEWM

PrnA .MNKPIKNIVIVGGGTAGWMAASYLVRALQQQANITLIESAAIPRI.GVGEATIPSLQKVFFDFLGIPEREWM

PyrH ....MIRSVVIVGGGTAGWMTASYLKAAFDDRIDVTLVESGNVRRIG.VGEATFSTVR.HFFDYLGLDEREWL

SttH MNTRNPDKVVIVGGGTAGWMTASYLKKAFGERVSVTLVESGTIGTVG.VGEATFSDIR.HFFEFLDLREEEWM

SttH

.

β1

α1

α2

η1

β2

α3

α4

α5

β4

80

β5

β6

110

β7

TT

120

β8

α4

130

Thal

TT

90

100

140

Thal RECNASYKMAVKFINWRTPGEGSPDPRTLDDGHTDTFHHPFGLLPSADQIPLSHYWAAKRLQGETDENFDEAC

RebH RECNASYKVAIKFINWRTAGEGTSEAREL.DGGPDHFYHSFGLLKYHEQIPLSHYWFDRSYRGKTVEPFDYAC

PrnA PQVNGAFKAAIKFVNWRKSP.....DPSR....DDHFYHLFGNVPNCDGVPLTHYWLRKREQG.FQQPMEYAC

PyrH PRCAGGYKLGIRFENWSEP..............GEYFYHPFERLRVVDGFNMAEWWLAVG...DRRTSFSEAC

SttH PACNATYKLAVRFQDWQRP..............GHHFYHPFEQMRSVDGFPLTDWWLQNG...PT.DRFDRDC

SttH

T..............T

TT

η2

β3

β4

β5

β6

β7

α5

α6

β9

170

α7

Thal

TT

160

150

180

190

Thal FADTAIMNAKKAPRFLDMRRA.........................TNYAWHFDASKVAAFLRNFAVTKQAVE

RebH YKEPVILDANRSPRRLDGSKV.........................TNYAWHFDAHLVADFLRRFATEKLGVR

PrnA YPQPGALDGKLAPCLSDGTRQ.........................MSHAWHFDAHLVADFLKRWAVE.RGVN

PyrH YLTHRLCEAKRAPRMLDGSLFASQVDE....SLGRSTLAEQ..RAQFPYAYHFDADEVARYLSEYAIA.RGVR

SttH FVMASLCDAGRSPRYLNGSLLQQEFDERAEEPAGL.TMSEHQGKTQFPYAYHFEAALLAEFLSGYSKD.RGVK

SttH

TT

α7

α8

β8

β15

α9

β10

β11

200

β12

β13

β14

η1

π1

β16

Thal

TT

210

220

230

240

250

260

Thal HVEDEMTEVLTDERGFITALRTKSGRILQGDLFVDCSGFRGLLINKAMEEPFIDMSDHLLCNSAVATAVPHDD

RebH HVEDRVEHVQRDANGNIESVRTATGRVFDADLFVDCSGFRGLLINKAMEEPFLDMSDHLLNDSAVATQVPHDD

PrnA RVVDEVVDVRLNNRGYISNLLTKEGRTLEADLFIDCSGMRGLLINQALKEPFIDMSDYLLCDSAVASAVPNDD

PyrH HVVDDVQHVGQDERGWISGVHTKQHGEISGDLFVDCTGFRGLLINQTLGGRFQSFSDVLPNNRAVALRVPREN

SttH HVVDEVLEVKLDDRGWISHVVTKEHGDIGGDLFVDCTGFRGVLLNQALGVPFVSYQDTLPNDSAVALQVPLDM

SttH

TT

β9

α8

β10

β11

β18

β12

β13

η3

π1

β14

β15

β17

280

β19

α9

β20

330

β21

Thal

TT

270

290

300

310

320

Thal EKNGVEPYTSSIAMEAGWTWKIPMLGRFGSGHVYSDHFATQDEATLAFSKLWGL.DPDNTEFNHVRFRVGRNR

RebH DANGVEPFTSAIAMKSGWTWKIPMLGRFGTGYVYSSRFATEDEAVREFCEMWHL.DPETQPLNRIRFRVGRNR

PrnA ARDGVEPYTSSIAMNSGWTWKIPMLGRFGSGYVFSSHFTSRDQATADFLKLWGL.S.DNQPLNQIKFRVGRNK

PyrH D.EDMRPYTTATAMSAGWMWTIPLFKRDGNGYVYSDEFISPEEAERELRSTVAPGR.DDLEANHIQMRIGRNE

SttH EARGIPPYTRATAKEAGWIWTIPLIGRIGTGYVYAKDYCSPEEAERTLREFVGPEA.ADVEANHIRMRIGRSE

SttH

β16

β17

β18

α10

η4

β19

β20

β22 β23

TT

η2 β24

350

α10

α11

Thal

TT

360

TT

380

340

370

390

400

410

Thal RAWVRNCVSVGLASCFVEPLESSGIYFIYAAIHMLAKHFPDKTFDKVLVDRFNREIEEMFDDTRDFLQAHYYF

RebH RAWVGNCVSIGTSSCFVEPLESTGIYFVYAALYQLVKHFPDKSLNPVLTARFNREIETMFDDTRDFIQAHFYF

PrnA RAWVNNCVSIGLSSCFLEPLESTGIYFIYAALYQLVKHFPDTSFDPRLSDAFNAEIVHMFDDCRDFVQAHYFT

PyrH RTWINNCVAVGLSAAFVEPLESTGIFFIQHAIEQLVKHFPGERWDPVLISAYNERMAHMVDGVKEFLVLHYKG

SttH QSWKNNCVAIGLSSGFVEPLESTGIFFIHHAIEQLVKHFPAGDWHPQLRAGYNSAVANVMDGVREFLVLHYLG

SttH

TT

β21 β22

TT

β23

α11

α12

420

α13

α14

α15

α16

Thal

430

440

450

460

470

480

Thal SPRVDTPFWRANK.ELKLADSIKDKVETYRAGLPVNLPVT.DEGTYYGNFEAEFRNFWTNGSYYCIFAGLG.L

RebH SPRTDTPFWRANK.ELRLADGMQEKIDMYRAGMAINAPASDDAQLYYGNFEEEFRNFWNNSNYYCVLAGLG.L

PrnA TSRDDTPFWLANRHDLRLSDAIKEKVQRYKAGLPLTTT.SFDDSTYYETFDYEFKNFWLNGNYYCIFAGLG.M

PyrH AQREDTPYWKAAKT.RAMPDGLARKLELSASHLLDEQ............TIYPYYHGFETYSWITMNLGLG.I

SttH AARNDTRYWKDTKT.RAVPDALAERIERWKVQLPDSE............NVFPYYHGLPPYSYMAILLGTGAI

SttH

TT

α13 η5

α14

α17

α15

η3

α18

Thal

.

490

500

510

520

530

Thal MPRNPLPALAYKPQSIAEAELLFADVKRKGDTLVESLPSTYDLLR.QLHGAS.................

RebH VPDAPSPRLAHMPQATESVDEVFGAVKDRQRNLLETLPSLHEFLR.QQHGR..................

PrnA LPDRSLPLLQHRPESIEKAEAMFASIRREAERLRTSLPTNYDYLRSLRDGDAGLSRGQRGPKLAAQESL

PyrH VPERPRPALLHMD..PAPALAEFERLRREGDELIAALPSCYEYLA.SIQ....................

SttH GLR.PSPALALAD..PAAAEKEFTAIRDRARFLVDTLPSQYEYFA.AMGQRV.................

SttH

η6

α16

α17

Figure 1. Structure-based sequence alignment. The tryptophan 6-halogenase Thal (PDB ID 6H44), the two tryptophan 7-halogenases

RebH (2E4G) and PrnA (2AQJ), the tryptophan 5-halogenase PyrH (2WEU) and the tryptophan 6-halogenase SttH (5HY5) are ordered by

decreasing sequence identity to Thal. Secondary structure elements of Thal and SttH are shown above and below the sequence,

respectively. The catalytic lysine and glutamate are highlighted in green. Fingerprint sequences conserved in FDHs (GxGxxG and

WxWxIP) are highlighted in orange. The Aps449 insertion in RebH relative to Thal is highlighted in yellow. The five residues mutated in

Thal-RebH5 are highlighted in cyan. The figure was generated with ESPpript (54). Pairwise structure-based sequence alignments were

performed with the DALI server (53).

18

Figure 2. Monomer of Trp‐Thal. The box and pyramid subdomains are shown in dark blue and

light blue, respectively. The substrate‐binding loop that undergoes a conformational change upon

L‐Trp binding and closes the substrate binding site is highlighted in green. The FAD loop is shown in

red. The substrate L‐Trp is shown as stick‐model with yellow carbon atoms.

19

(b)

(a)

tryptophan

Thal (C6)

RebH (C7)

PyrH (C5)

E358 Thal

E357 RebH

E354 PryH

K79 Thal

K79 RebH

K75 PyrH

Figure 3. Orientation of the substrate L‐Trp in the Trp‐Thal structure. (a) Fo‐Fc electron density

before building L‐Trp contoured at +3 σ (green) and the final 2Fo‐Fc electron density contoured at

1 σ (blue). (b) Superposition of the bound L‐Trp from the halogenases Thal (C6), RebH (C7) and

PyrH (C5). The halogenated carbon atoms of the indole moiety superpose well and are located

closest to the ε‐amino group of the catalytic lysine.

20

(a)

(b)

E461

Y454

F465

V52

S360

P53

Y455

T467

W466

V52

E358

F112

E358

K79

S360

V82

S470

H110

G469

V82

P111

E461

π-stacking

backbone binding

catalytic

F465

(d)

Y454

(c)

P53

F465

Y455

W466

F112

P111

H110

Figure 4. Depiction of the tryptophan binding site. (a) The binding site is divided into the three

regions catalytic (light blue), π‐stacking (light pink) and backbone‐binding (light green). The

substrate L‐Trp is shown with yellow carbon atoms and the halogenation position C6 is highlighted

with a star. Direct hydrogen bonds or salt bridges between Thal and L‐Trp are shown as dotted

yellow lines. (b) Catalytic region. (c) π‐stacking region. (d) Backbone‐binding region.

21

F465

P53

W466

F112

P111

apo-Thal

Trp-Thal

Trp-RebH

H110

Figure 5. Structural flexibility of conserved residues in the π‐stacking region. Overlay of the π‐

stacking region of chain A of apo‐Thal (grey), Trp‐Thal (dark blue) and Trp‐RebH (PDB ID 2E4G;

violet). The substrate tryptophan is shown in yellow for Thal and in violet for RebH. The

numbering of residues corresponds to that of Thal. RebH contains Ser110 instead of Pro111. In

apo‐Thal, Pro53, Phe112 and Trp466 have moved into the substrate binding site relative to Trp‐

Thal. In chain B the movement toward the empty substrate binding site of apo‐Thal is even larger

(see Fig. S6).

22

(a)

(b)

N54

W1

F112

G113

W2

W3

E461

S470

T467

(c)

(d)

N54

W1

F111

G112

F111

G112

E461

W2

N470

N467

Figure 6. Conserved water molecules that form H‐bonds to the L‐Trp carboxylate in the substrate

binding sites of Thal and RebH. Conserved water molecules W1 and W2 and their H‐bond

partners are shown. (a) In Trp‐Thal, W2 forms a H‐bond to Gly113. Further contacts to Thal are

mediated by water W3. (b) In chain A of apo‐Thal, W1 is also present. (c) In Trp‐RebH (PDB ID

2E4G), W2 forms three direct H‐bonds with the protein. The contact to Gly112 is conserved in

Thal but the contacts to Asn476 and Asn470 are not. (d) In apo‐RebH (PDB ID 2OAM), W1 is

present and Phe111 has undergone a peptide flip relative to Trp‐RebH so that the carbonyl oxygen

of Gly112 could no longer form a H‐bond to W2.

23

(a)

(b)

t_r= 6.6 min

L-7-halotryptophan

major regioisomer

(95.4 %)

(c)

(d)

Thal-RebH5

cofactor regeneration

NaX, O₂, aqueous buffer

pH 7.4, 25 °C

L-tryptophan

H

t_r= 6.9 min

Cl

N

NH₂

OH

O

L-6-halotryptophan

minor regioisomer

(4.6 %)

retention time / min

Figure 7. Thal‐RebH5‐catalyzed halogenation yielded L‐7‐halotryptophan. (a) Halogenation

scheme and final ratio of chlorotryptophan isomers resulting from Thal‐RebH5‐catalyzed

halogenation assays indicate the dominant formation of C7 isomer. (b‐d) LC‐MS analysis confirms

the formation of two monochlorinated L‐Trp isomers with same m/z pattern. m/z [M^35Cl+H]⁺

found 239.06, calc. 239.06, [M^37Cl+H]⁺found. 241.06, calc. 241.06. HPLC‐MS‐analysis of crude

reaction mixture was compared with authentic standards. (b) 7‐Cl‐Trp standard, t_r=6.6 min; The

trace of the reaction mixture revealed the formation of C7‐chlorinated L‐Trp as the major

regioisomer (95.4 %), whereas 6‐Cl‐Trp appeared as minor side product (4.6 %). Reaction

conditions: 89 µM Thal‐RebH5, 5 mM L‐Trp, 1 mM NAD⁺, 0.01 mM FAD, 1 U mL^‐1RR‐ADH, 2.5 U

mL^‐1PrnF, 5 % (v/v) 2‐propanol, 10 mM Na₂HPO₄pH 7.4, 30 mM NaX, X = Cl, Br. (d) 6‐Cl‐Trp

standard, t_r=6.9 min.

24

(a)

(b)

Y454

E461

Y455

E461

Y455

F465

N467

W466

T467

W466

P53

S469

V52

N470

S470

I52

F111

F112

T359

S360

S110

P111

H109

H110

E357

K79

E358

I82

V82

Figure 8. Steric conflicts upon placing L‐Trp from Trp‐RebH in Trp‐Thal and vice versa. Minor

clashes are indicated as small green hexagons, more severe clashes are shown as bigger red

hexagons. (a) L‐Trp from Trp‐RebH (PDB ID 2E4G) was placed in Trp‐Thal. Clashes mainly occur

between the L‐Trp indole and the side chains of conserved residues Phe112 and Trp466. (b) L‐Trp

from Trp‐Thal was placed in Trp‐RebH (PDB ID 2E4G). Clashes mainly occur between the L‐Trp

indole and the side chain of Asn470 and between the L‐Trp carboxylate and the side chain of

Tyr455.

25

Structure-based switch of regioselectivity in the flavin-dependent tryptophan

6-halogenase Thal

Ann-Christin Moritzer, Hannah Minges, Tina Prior, Marcel Frese, Norbert Sewald and

Hartmut H. Niemann

J. Biol. Chem. published online December 17, 2018

Access the most updated version of this article at doi: 10.1074/jbc.RA118.005393

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Article Doi

Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal

DOI: 10.1074/jbc.RA118.005393

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Article abstract of DOI:10.1074/jbc.RA118.005393

Full text of DOI:10.1074/jbc.RA118.005393

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