Regio- and chemoselective enzymatic N-oxygenation in vivo, in vitro, and in flow

Article abstract of DOI:10.1002/anie.200603060

(Chemical Equation Presented) Action by the para: Evaluation of the nitro-group-forming N-oxygenase AurF in vivo, in vitro, and immobilized as a fusion protein with simply H₂O₂ as oxidant (peroxide shunt) reveals para-regioselective oxygenation of aromatic amines (see scheme). This effect includes the selective oxygenation of diamino compounds.

Full text of DOI:10.1002/anie.200603060

Communications
Biotransformations
DOI: 10.1002/anie.200603060
Regio- and Chemoselective Enzymatic
N-Oxygenation In Vivo, In Vitro, and in Flow**
Robert Winkler, Martin E. A. Richter, Uwe Knüpfer,
Dirk Merten, and Christian Hertweck*
Scheme 1. Proposed mechanism of N-oxygenation by AurF. PHABA=
para-(hydroxyamino)benzoic acid.
The use of oxygenases in biotransformations can expand
[
1]
vastly the repertoire of synthetic methods. A hallmark of
these enzymes is their exquisite specificity and their applica-
tion under environmentally friendly and technically safe
isomers, ortho- and meta-aminobenzoic acid, was oxygenated
(Scheme 2, entries B and C; Figure 1). This result clearly
[
2–4]
conditions.
However, the use of these biocatalysts may be
[
5]
restricted by the need for cofactor recycling, which can be
[
1]
circumvented by whole-cell biotransformations. The most
notable success of oxygenases has come with the oxygenation
of nonactivated carbon or the regioselective oxidation of
[
1]
polyhydroxy compounds.
Considering the breadth of biotransformations available,
it is remarkable that enzymes catalyzing the selective
oxidation of amino groups are only little explored. To date,
only two genuine nitro-group-forming N-oxygenases have
been identified. Aminopyrrolnitrin N-oxygenase (PrnD) is
involved in pyrrolnitrin biosynthesis in Pseudomonas fluo-
[
6]
rescens, and belongs to the Rieske-type oxygenases. In
contrast, para-aminobenzoate N-oxygenase (AurF) from
Streptomyces thioluteus represents an unparalleled enzyme
that does not show any sequence similarity to known oxy-
[
7]
genases. This unusual biocatalyst transforms para-amino-
benzoic acid (PABA) into para-nitrobenzoic acid (PNBA),
the rare aureothin polyketide synthase starter unit
[
7,8]
(
Scheme 1).
Recent in vivo studies coupled to an “at-
line” liquid chromatographic (LC) analysis revealed that the
oxygenation reaction occurs stepwise via hydroxylamine and,
[
9,10]
most probably, nitroso intermediates.
With the intention of exploring the synthetic potential of
AurF, we have addressed the scope of this unique oxygenase,
and its chemo- and regioselectivity, and have established an
in vitro N-oxygenation method that circumvents the need for
cumbersome cofactor regeneration. We first carried out a
comparative study in a validated whole-cell approach using
the geometrical isomers of aminobenzoate. While AurF
readily transforms PABA into PNBA, neither of the two
Scheme 2. Substrate specificity of AurF.
[*] R. Winkler, M. E. A. Richter, U. Knüpfer, Prof. Dr. C. Hertweck
Abteilung Biomolekulare Chemie
Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie,
HKI
Beutenbergstrasse 11a, 07745 Jena (Germany)
Fax: (+49)3641-656-705
indicates a high para regioselectivity of AurF. Most PABA
analogues substituted at the carboxy moiety with various
substituents, such as methoxy (para-anisidine), acetyl (para-
aminoacetophenone), alaninyl (para-amino-l-phenylala-
nine), and methyl (para-toluidine), are not transformed
E-mail: Christian.Hertweck@hki-jena.de
Dr. D. Merten, Prof. Dr. C. Hertweck
Friedrich-Schiller-Universität Jena
(Scheme 2, entry M). However, AurF also accepts para-
0
7745 Jena (Germany)
aminophenyl acetic acid and para-aminophenyl sulfonic acid
as substrates and converts them into para-nitrophenyl acetic
acid and para-nitrophenyl sulfonic acid, respectively
(Scheme 2, entries D and E). Hence, it can be concluded
[
**] This project has been generously funded by the DFG.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
8016
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Angewandte
Chemie
(HCDC) in minimal salt medium. Enrichment of
acetate was prevented by controlled feeding of glu-
[
12]
À1
cose, which yielded a final wet cell mass of 133 gL .
Protein expression was induced by addition of isopro-
pyl-b-d-thiogalactopyranoside (IPTG; 1 mm) and per-
formed at 268C to support correct protein folding by
[
13]
chaperones. One-step affinity purification of clarified
disintegrates from a 20-g cell pellet on a 17-mL amylose
column yielded over 100 mg of fusion protein with more
than 90% purity (SDS-PAGE) per batch.
Interestingly, UV/Vis and fluorescence spectroscopy
gave no hint of flavin, heme, or other typical oxygenase
cofactors. However, inductively coupled plasma optical
emission spectrometry (ICP-OES) investigations
showed significant contents of iron and manganese in
the purified protein. This finding was confirmed by
colorimetric assays for iron and manganese and com-
parison with the culture media, which also revealed that
manganese is enriched by a factor of about 20 in relation to
iron. Such a high selectivity of the protein for manganese
provides strong evidence that AurF employs manganese as an
oxygen-activating cofactor.
Figure 1. Relative reaction rates (with reference to PABA (substrate A)).
that the enzyme allows only some degree of variation at the
carboxy group. Comparing these probes, it is notable that all
converted substrates bear an acidic group adjacent to the
aromatic ring. This fact strongly suggests that the presence of
[
11]
an acidic residue is important for substrate binding.
To investigate the impact of additional ring substitutions,
substrates with extra methyl and hydroxy groups in either the
ortho or meta position to the carboxy group were adminis-
tered. All the tested monosubstituted PABA derivatives
resulted in the corresponding nitro product (Scheme 2,
entries F–I)). Acceptance of both methyl and hydroxy
substituents on either the ortho or meta position is amazing,
as the first accounts for a +I effect and the latter for a ÀI
effect. Apparently some variation of the electrochemical state
of the substrate seems to be tolerated. The substrate-binding
site allows for a certain range of flexibility, which was already
indicated by the successful transformation of para-amino-
phenyl acetic acid.
It is astonishing that the addition of H O to MalE–AurF
2
2
preparations restored the enzymatic activity in vitro without
the need for other supplements. This is in strong contrast to
the N-oxygenase PrnD, which requires proteolytic cleavage of
the fusion enzyme and additional coenzymes for in vitro
[
14]
activity. Presumably, the reaction mechanism employed by
AurF is similar to that of heme or iron-dependent oxygenases,
which allow bypassing of the native enzyme regeneration by
[
15]
H O via a so-called “peroxide shunt”.
2
2
The strict para selectivity of AurF can be rationalized by a
simplified model (Figure 2). The substrate is most likely
The absent oxygenation of 3-amino-4-methylbenzoic acid
and 3-amino-5-methylbenzoic acid (not shown) further sup-
ports the strict regioselectivity of AurF in regard to the amino
group, whilst the failure in oxidizing 4-amino-2,3,5,6-tetra-
fluorobenzoic acid (not shown) could be a result of exagger-
ated electron withdrawal from the ring system, or may be
caused by space constraints at the active site.
To exploit the limited substrate tolerance in conjunction
with the strict para specificity, the diamino surrogates 2,4-
diaminobenzoic acid, 3,4-diaminobenzoic acid, and 2,4-dia-
minobenzenesulfonic acid were probed. Strikingly, all trans-
formations resulted exclusively in products with a nitro group
at the para position and unaltered amino groups at the meta
and ortho positions (Scheme 2, entries J–L). Such strict site-
directed N-oxygenations are unprecedented and have not
been achieved by standard synthetic protocols.
To establish a convenient in vitro N-oxygenation method
for this unique biocatalyst, various attempts to yield pure
enzyme were undertaken. Initial trials to overexpress native
or 6 ” His-tagged protein in S. lividans or Escherichia coli
failed. However, soluble MalE–AurF fusion protein was
produced at high levels in E. coli, and in vivo tests proved the
catalytic activity of the chimeric enzyme. Large amounts of
the enzyme were produced by high-cell-density cultivation
Figure 2. Model for regioselective N-oxygenation by AurF; R’=H,
NH ; X=COO, CH COO, SO .
2
2
3
tethered by ion interactions to a positively charged protein
residue, which allows the para-substituted amino group to
enter the reactive hot spot, probably a Mn–oxo species. In
stark contrast to AurF, haloperoxidases, which are not
genuine N-oxygenases, catalyze nitro group formation in a
nonspecific way through the formation of reactive peroxo
[
16–18]
species in the absence of halide ions.
Apart from its remarkable selectivity, AurF has another
advantage over other systems. Most conveniently, the loaded
amylose column with immobilized enzyme can be used
directly as an N-oxygenator “in flow”. This is simply achieved
by addition of substrate and H O to the loading buffer. In a
2
2
pilot experiment, a single pass of PABA and H O immedi-
2
2
ately resulted in 27% conversion to PNBA. To challenge the
Angew. Chem. Int. Ed. 2006, 45, 8016 –8018
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Communications
method, a mixture of 2-, 3-, and 4-aminobenzoic acids was
Preparative amounts of an active MalE–AurF fusion
protein were manufactured and the enzyme was proven
functional in vitro by addition of H O (peroxide shunt). The
circulated about 140 times. LC analyses demonstrated that a
steady state is reached at 47% conversion to PNBA, and that
only 4-aminobenzoic acid is oxidized, while the regioisomers
remain unchanged (Figure 3).
2
2
chimeric enzyme could be immobilized and successfully
applied in a fixed-bed tube reactor for regioselective amine
transformation. To our knowledge, this is the first report of an
enzymatic process for the chemo- and regioselective oxygen-
ation of aromatic amines to nitro compounds, which could not
be emulated in a single step using current synthetic protocols.
Future studies of AurF will include elucidation of the
structure of the enzyme and protein engineering techniques,
with the goal of increasing reaction rates and altering the
scope of accepted substrates.
Received: July 28, 2006
Published online: November 2, 2006
Keywords: amines · biotransformations · enzymes ·
.
oxygenation · regioselectivity
[
1] K. Faber, Biotransformations in Organic Chemistry: ATextbook,
Springer, Heidelberg, 2004.
[
2] Z. Li, J. B. van Beilen, W. A. Duetz, A. Schmid, A. de Raadt, H.
Griengl, B. Withold, Curr. Opin. Chem. Biol. 2002, 6, 136.
3] R. D. Schmid, V. Urlacher, Curr. Opin. Biotechnol. 2002, 13, 557.
4] A. Straathof, S. Panke, A. Schmid, Curr. Opin. Biotechnol. 2002,
[
[
13, 548.
[
[
5] S. Fetzner, Appl. Microbiol. Biotechnol. 2002, 60, 243.
6] S. Kirner, P. E. Hammer, D. S. Hill, A. Altmann, I. Fischer, L. J.
Weislo, M. Lanahan, K.-H. van Pee, J. M. Ligon, J. Bacteriol.
1998, 180, 1939.
Figure 3. Top: Continuous regio- and chemoselective N-oxygenation
on a fixed bed (column with thermostat) with immobilized MalE–AurF
enzyme (spheres). Bottom: Chromatographic profiles of selective
biotransformation; the asterisk indicates an unknown side product
from PABA. The peak of 4 is significantly reduced in relation to that of
[7] J. He, C. Hertweck, Chem. Biol. 2003, 10, 1225.
[8] M. Ziehl, J. He, H.-M. Dahse, C. Hertweck, Angew. Chem. 2005,
117, 12 2 6 A; ngew. Chem. Int. Ed. 2005, 44, 1202.
[9] R. Winkler, C. Hertweck, Angew. Chem. 2005, 117, 4152; Angew.
Chem. Int. Ed. 2005, 44, 4083.
1
as a result of lower absorption of the nitro compound at 280 nm.
[
10] This finding has been confirmed for PrnD; D. J. Lee, H. Zhao,
Angew. Chem. 2006, 118, 638; Angew. Chem. Int. Ed. 2006, 45,
The possibility of using an immobilized enzyme, supplied
622.
with only H O , for the selective biotransformation of
[11] Previously reported results obtained with a S. thioluteus strain (S.
Kawai, K. Kobayashi, T. Oshima, F. Egami, Arch. Biochem.
Biophys. 1965, 112, 537) could not be reproduced.
2
2
aromatic amines is notable, because it allows for a number
of applications, such as the design of continuous flow
processes, which are increasingly relevant to the future of
chemoenzymatic syntheses. The urgent need for such novel
techniques is a contemporary challenge in organic synthe-
[
12] U. Horn, W. Strittmatter, A. Krebber, U. Knüpfer, M. Kujau, R.
Wenderoth, K. Müller, S. Matzku, A. Plückthun, D. Riesenberg,
Appl. Microbiol. Biotechnol. 1996, 46, 524.
[
13] M. Ferrer, T. Chernikova, M. Yakimov, P. Golyshin, K. Timmis,
Nat. Biotechnol. 2003, 21, 1266.
[
19]
ses. Possible uses of enzymatic N-oxygenation are “green”
regio- and chemoselective modification of valuable fine
[14] J. Lee, M. Simurdiak, H. Zhao, J. Biol. Chem. 2005, 280, 36719.
[
20]
[
[
[
15] M. Sono, M. P. Roach, E. D. Coulter, J. H. Dawson, Chem. Rev.
996, 96, 2841.
16] N. Itoh, N. Morinaga, T. Kouzai, Biochem. Mol. Biol. Int. 1993,
9, 785.
17] M. D. Corbett, B. R. Chipko, A. O. Batchelor, Biochem. J. 1980,
87, 893.
chemicals,
or the separation and extraction of aromatic
1
amines by previous oxidation, as well as in analytical
applications.
2
In summary, various substrates have been probed for N-
oxygenation by AurF, which revealed a strict regio- and
chemoselectivity for an aromatic amino group para to an
acidic group. A number of modifications on the carboxy
group (carboxymethyl, sulfonate) and additional ring sub-
stituents (CH , OH, NH ) are tolerated by AurF, although
1
[18] S. Kirner, K.-H. van Pee, Angew. Chem. 1994, 106, 346; Angew.
Chem. Int. Ed. Engl. 1994, 33, 352.
[
[
19] G. Jas, A. Kirschning, Chem. Eur. J. 2003, 9, 5708.
20] A. Bruggink, A. Straathof, L. van der Wielen, Adv. Biochem.
Eng. Biotechnol. 2003, 80, 69.
3
2
reaction rates are reduced compared to those of PABA
Figure 1). The most remarkable result is the selective
(
oxygenation of diamino compounds. The excellent para
specificity can be rationalized on the basis of an active-site
model.
8018
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Angew. Chem. Int. Ed. 2006, 45, 8016 –8018