Substrate Specificity and Regioselectivity of Tryptophan 7-Halogenase from Pseudomonas fluorescens BL915

Article abstract of DOI:10.1002/1615-4169(200108)343:6/7<591::AID-ADSC591>3.0.CO;2-E

Tryptophan 7-halogenase which is involved in pyrrolnitrin biosynthesis is the first halogenating enzyme to be isolated that has substrate specificity and regioselectivity. This FADH₂-dependent halogenase catalyzes the chlorination of its natural substrate tryptophan exclusively at the 7-position, a position at which direct chemical chlorination is not possible. Other substrates such as N-Ω-methyltryptamine, 5-methyltryptamine, 5-methylindole, 3-methylindole, or indole-3-acetonitrile are also chlorinated by the enzyme, whereas compounds like 1-methyltryptophan, indole-3-carboxylic acid, indole-3-acetic acid, or indole are not accepted as substrates. In addition, phenylpyrrole derivatives are also chlorinated by the enzyme. However, in contrast to tryptophan, the tryptophan and indole derivatives are chlorinated at positions 2 or/and 3 of the indole ring system and not at the 7-position. Chlorination of the phenylpyrrole derivatives also proceeds without regioselectivity and a mixture of mono- and dichlorinated products is obtained.

Full text of DOI:10.1002/1615-4169(200108)343:6/7<591::AID-ADSC591>3.0.CO;2-E

COMMUNICATION
Substrate Specificity and Regioselectivity of Tryptophan
7-Halogenase from Pseudomonas fluorescens BL915
Manuela HoÈlzer,a Wassily Burd,b Hans-Ulrich Reiûig,c Karl-Heinz van PeÂed,*
a JEP GmbH, Rheingaustr. 190
-196, 65203 Wiesbaden, Germany
b Kupala Grodno State University, 22 Ozkeshko Street, Grodno, 230023 Belarus
c Institut fuÈ r Chemie ± Organische Chemie, Freie UniversitaÈt Berlin, Takustr. 3, 14195 Berlin, Germany
d Institut fuÈ r Biochemie, Technische UniversitaÈt Dresden, 01062 Dresden, Germany
Phone: (+49) 351
-
463-
4494; Fax: (+49) 351
-
463-
5506; e-mail: karl-heinz.vanpee@chemie.tu-dresden.de
Received April 27, 2001; Accepted June 26, 2001
chanisms have shown that they produce hypohalous
-
acids as the actual halogenating agent.[2 6] Thus, ha-
Abstract: Tryptophan 7-halogenase which is in-
volved in pyrrolnitrin biosynthesis is the first halo-
genating enzyme to be isolated that has substrate
specificity and regioselectivity. This FADH2-depen-
dent halogenase catalyzes the chlorination of its
natural substrate tryptophan exclusively at the 7-
position, a position at which direct chemical chlor-
ination is not possible. Other substrates such as
N-X-methyltryptamine, 5-methyltryptamine, 5-
methylindole, 3-methylindole, or indole-3-acetoni-
trile are also chlorinated by the enzyme, whereas
compounds like 1-methyltryptophan, indole-3-car-
boxylic acid, indole-3-acetic acid, or indole are not
accepted as substrates. In addition, phenylpyrrole
derivatives are also chlorinated by the enzyme.
However, in contrast to tryptophan, the tryptophan
and indole derivatives are chlorinated at positions
2 or/and 3 of the indole ring system and not at the
7-position. Chlorination of the phenylpyrrole deri-
vatives also proceeds without regioselectivity and
a mixture of mono- and dichlorinated products is
obtained.
logenation reactions catalyzed by haloperoxidases
and perhydrolases proceed without substrate specifi-
city and regioselectivity which is not in agreement
with results obtained by investigations of the bio-
synthesis of halogenated metabolites. These investi-
gations had shown that halogenating enzymes in-
volved in halometabolite biosynthesis must have
substrate specificity and regioselectivity.[6] The first
two halogenating enzymes with these properties
were detected in a pyrrolnitrin-producing Pseudomo-
nas fluorescens strain. These two halogenases were
originally believed to require NADH as a co-factor,[7]
but more detailed investigations revealed that it was
actually FADH2, produced by non-specific flavin re-
ductases from FAD and NADH, that was required by
these halogenases.[8] One of these halogenases cata-
lyzes the first step in pyrrolnitrin biosynthesis, the re-
gioselective chlorination of tryptophan (1) to 7-chloro
-
tryptophan (2).[9] In this paper, we describe the first
investigations of the substrate specificity and regio-
selectivity of tryptophan 7-halogenase (Trp 7-hal)
and their implication for pyrrolnitrin biosynthesis.
Since Trp 7-hal activity cannot be measured in cell-
free extracts of the wild-type strains producing pyr-
rolnitrin, a recombinant mutant of P. fluorescens
BL915, overexpressing Trp 7-hal from P. fluorescens
BL915 was used.[9] The enzyme was partially purified
which resulted in separation of most of the flavin re-
ductase activity from the Trp 7-hal. Therefore, par-
tially purified flavin reductase had to be added to the
enzyme assays.[8]
Keywords: biohalogenation; chlorination; enzyme
catalysis; enzymatic halogenation; oxidoreduc-
tases; regioselectivity
In contrast to other enzymes involved in secondary
metabolism, halogenating enzymes seemed to lack
substrate specificity and regioselectivity.[1] The halo-
genating enzymes that were known until 1997 were
all haloperoxidases or perhydrolases. Elucidation of
their three-dimensional structures and reaction me-
To investigate which other tryptophan and indole
derivatives in addition to the natural substrate 1 are
chlorinated by Trp 7-hal, we tested a number of com-
mercially available compounds. While 1-methyltryp-
tophan and N-acetyltryptophan were not accepted as
substrates, N-X-methyltryptamine (3) and 5-methyl-
tryptamine (5) were chlorinated by the enzyme. How-
ever, whereas 1 is exclusively chlorinated in the 7-po-
sition, the tryptamine derivatives 3 and 5 were
Supporting information for this article is available on the
WWW under http://www.wiley-vch.de/home/asc/ or from
the author.
Adv. Synth. Catal. 2001, 343, No. 6±7
Ó WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2001
1615-4150/01/34306±7-0±0 $ 17.50-.50/0
591

asc.wiley-vch.de
chlorinated in the 2-position (Table 1). Indole deriva- position 7, but at the 2-position (Table 1). All the
tives with only a carboxy group in the side chain such above-mentioned substrates 3, 5, 7, and 9 were mono-
as indole-3-carboxylic acid and indole-3-acetic acid chlorinated. A substrate from which a dichlorinated
were not accepted as substrates. This was also the product is obtained is indole-3-acetonitrile (11). The
case for indole and some indole derivatives substi- main product was 2-chloroindole-3-acetonitrile (12)
tuted in position 3 such as indole-3-carbonitrile, in- and as minor products 2,3-dichloroindole-3-acetoni-
dole-3-carboxaldehyde, and gramine. Surprisingly, trile (13) and 3-chloroindole-3-acetonitrile (14) were
5-methylindole (7) and 3-methylindole (9) were obtained (Table 1).
chlorinated by Trp 7-hal. As in the case of the trypta-
mine derivatives 3 and 5, chlorination did not occur at monodechloroaminopyrrolnitrin
Chlorination of 3-(2'-aminophenyl)pyrrole (19) and
[4-(3'-chloro-2'-
Table 1. Chlorination of tryptophan and several tryptophan and indole derivatives by tryptophan 7-halogenase in the pre-
sence of an FADH2-producing flavin reductase, chloride, and oxygen.
592
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COMMUNICATION
Table 2. Chlorination of 3-(2'-aminophenyl)pyrrole (19) and monodechloroaminopyrrolnitrin (20) by tryptophan 7-halogen-
ase in the presence of an FADH2-producing flavin reductase, chloride and oxygen.
aminophenyl)pyrrole] (20) resulted in a mixture of formed tryptophan 7-halogenase-bound flavin hydro-
products, chlorinated at different positions of the peroxide activates the organic substrate by a mono-
phenyl and the pyrrole rings. The main products oxygenase-like reaction leading to the formation of
obtained with 19 as the substrate are 20 and 3-(5'- an epoxide which would not leave the enzyme. The
chloro-2'-aminophenyl)pyrrole (22); the other prod- epoxide would be opened by the nucleophilic attack
ucts are formed by further chlorination of these two of a chloride ion resulting in formation of a chlorohy-
compounds (Table 2). Chlorination of the corre- drin. Specific removal of water would result in the
sponding nitro compound [3-(2'-nitrophenyl)pyrrole] formation of the chlorinated product.[6] However, so
could not be observed. Although Trp 7-hal can chlori- far none of the postulated intermediates has been iso-
nate 19 to 20, further chlorination of 20 to amino- lated.
pyrrolnitrin (21) hardly occurs. This could explain
Trp 7-hal can accept a number of different trypto-
why chlorination of 20 to 21 by tryptophan 7-halogen- phan, indole, and phenylpyrrole derivatives, but only
ase obviously does not occur in vivo, since a mutant 1 is regioselectively chlorinated in the 7-position of
with an inactive monodechloroaminopyrrolnitrin 3- the indole ring. With all the other compounds, the re-
halogenase (Mcap 3-hal) cannot produce pyrrolni- action proceeds with a relaxed regioselectivity. Ob-
trin, showing that Trp 7-hal cannot substitute the viously, only 1 is positioned at the active site of the en-
Mcap 3-hal.[9]
zyme in a way that allows the chlorination to take
The formation of the obtained products is consis- place at a position at which direct chemical halogena-
tent with the proposed mechanism for FADH2-depen- tion is not possible. This shows that halogenating en-
dent halogenases (Figure 1). In this hypothetical me- zymes can have substrate specificity and that they can
chanism, FADH2 is formed by the reduction of FAD catalyze regioselective halogenation reactions, the
with NADH catalyzed by a non-specific flavin reduc- latter being strongly dependent on the chemical
tase. FADH2 is suggested to be bound by the trypto- structure of the substrate, and that in some cases they
phan 7-halogenase where it reacts with oxygen. The are even superior to chemical halogenation.
Adv. Synth. Catal. 2001, 343, 591±595
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asc.wiley-vch.de
Figure 1. Hypothetical reaction mechanism for chlorination catalyzed by the FADH2-dependent tryptophan 7-halogenase
with N-X-methyltryptamine (3) as the substrate.
mixture except tryptophan 7-halogenase was incubated in
parallel and analyzed in the same way as the assay mixture.
After incubation at 30 °C for 16 h the reactions were stopped
by extraction with tert-butyl methyl ether. After extraction
the samples were evaporated to dryness under vacuum and
redissolved in 1 ± 2 mL of methanol. The conditions used for
the purification of the products for 1H NMR analysis are pre-
sented in Table 1 of the supporting information. Chlorina-
tion of the products was analyzed by HPLC-electrospray MS
The genes of similar halogenases have recently
been detected in a number of biosynthetic gene clus-
ters for halogenated antibiotics such as the vancomy-
cin-group antibiotics chloroeremomycin,[10] balhimy-
cin,[11] and the antifungal compound pyoluteorin.[12]
However, nothing is known about the corresponding
enzymes and their properties, but according to what
is known about the biosynthetic pathways for these
halometabolites it has to be expected that these halo-
genases also have substrate specificity and regio-
selectivity.
[substrate 5: LichroCRAT 250-4 column, CH3OH/H2O, 60:40
+ 1% HCO2H, 0.6 mL min±1] and GC-MS (substrates 3, 7, 9,
11, 19, and 20: capillary column DB-5, 25 m ´ 0.32 mm; tem-
perature program 100 ± 240 °C at 10 °C min±1, injection port
and detector 240 °C). The structures of the purified products
were elucidated by 1H NMR analysis and comparison of
these data with data from the literature.[13] The MS and
1H NMR data are presented as supporting information.
Experimental Section
General Procedure
Trp 7-hal and flavin reductase from a recombinant P. fluores-
cens BL915 mutant were partially purified by ion exchange
and hydrophobic interaction chromatography as described
previously.[8] The phenylpyrrole derivatives 19 ± 21 were
synthesized as described in the literature.[13] The enzyme
assays contained 4.45 mU Trp 7-hal, 50 mM NADH, 100 mM
NaCl, 550 lU flavin reductase, 10 lM FAD, 0.6 mM 1 or in-
dole derivative or 0.1 mM phenylpyrrole derivative in
10 mM potassium phosphate buffer, pH 7.2, in a total volume
of 64 mL in the case of the tryptophan and indole derivatives
and a total volume of 200 mL in the case of the phenylpyrrole
derivatives. A blank containing all components of the assay
Acknowledgements
We thank Dr. Gruner and Dr. Scheller, Institut fuÈ r Organische
Chemie, TU Dresden for measuring the H NMR spectra and
1
for help with their interpretation. We also wish to thank Ms
KoÈ lling-Speer, Institut fuÈ r Lebensmittelchemie, TU Dresden,
for measuring the GC-MS, and Dr. Hempel, AWD, Dresden
for HPLC-MS analysis. This work was supported by the SaÈ ch-
sische Landesamt fuÈ r Umwelt und Geologie, the Deutsche
Forschungsgemeinschaft (DFG) through the Graduiertenkol-
leg ``Struktur-Eigenschafts-Beziehungen bei Heterocyclen'',
594
Adv. Synth. Catal. 2001, 343, 591±595

COMMUNICATION
the Environment and Climate Research and Technology De-
velopment Programme of the European Union, and the Fonds
der Chemischen Industrie.
[8] S. Keller, T. Wage, K. Hohaus, M. HoÈlzer, E. Eichhorn,
K.-H. van PeÂe, Angew. Chem. 2000, 112, 2380±2382; An-
gew. Chem. Int. Ed. 2000, 39, 2300-2302
[9] S. Kirner, P. E. Hammer, D. S. Hill, A. Altmann, I.
Fischer, L. J. Weislo, M. Lanahan, K.-H. van PeÂe, J. M.
Ligon, J. Bacteriol. 1998, 180, 1939±1943.
[10] A. M. A. van Wageningen, P. M. Kirkpatrick, D. H. Wil-
liams, B. R. Harris, J. K. Kershaw, N. J. Lennard, M.
Jones, S. J. M. Jones, P. J. Solenberg, Chem. Biol. 1998,
5, 155±162.
[11] S. Pelzer, R. SuÈ ûmuth, D. Heckmann, J. Recktenwald,
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C. J. Chang, H. G. Floss, D. J. Hook, J. A. Mabe, P. E.
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