Angewandte
Communications
Chemie
[
9]
by the enzyme from Coprococcus sp. PGRcl-his, PGReu-
his, and PGRde-his were individually applied to 26 different
compounds, including bicyclic compounds such as flaviolin
and 2-tetralone, and several aromatic and nonaromatic
monocyclic compounds, to check for possible reductase
activity. Surprisingly, none of the compounds were converted
by any of the three enzymes (Figure S1 in the Supporting
Information). Menadione and 1,4-naphthoquinone showed
false-positive results because they were converted nonenzy-
matically in the presence of the reduced cofactor NADPH.
Regarding the compounds tested so far, we propose that
a 1,3,5-arrangement of the hydroxy groups is essential for
being accepted by PGRs. Therefore, the phloroglucinol
range (1, 7–9) and allowed menadione to be excluded as
a substrate. Nevertheless, PGRs possess an unusually narrow
substrate range in comparison to other SDRs, such as the tri-
and tetrahydroxynaphthalene reductases (T HNR, T HNR).
3
4
The latter are known for the reduction of a variety of
compounds (Figure S1 in the Supporting Information); how-
ever, they do not share any common substrates with the
[
8,15]
PGRs.
Although PGRs and T HNR/T HNR share the
3 4
common catalytic function of participating in dearomatiza-
tion reactions, they have crucially different biological func-
tions. T HNR and T HNR are involved in the secondary
3
4
metabolism of fungi, and therefore display high substrate
[
28,29]
promiscuity.
In contrast, PGRs are involved in the
[26]
derivatives 7–9 were tested and were found to be converted
in the same fashion as 1 (Table 1). Nevertheless, the
primary metabolism of bacteria, where they participate in
the anaerobic degradation of 1 as a carbon source. Moreover,
and in regard to structural comparison within the SDR family,
substrate promiscuity and substrate specificity are probably
determined by variations in the flexible C-terminal seg-
Table 1: Enzymatic reduction of phloroglucinol derivatives 7–9 by PGRcl-
his, PGReu-his, and PGRde-his.
[30,31]
ment.
[
a]
Substrate
Product
Enzyme
Conversion [%]
Despite the low pairwise sequence identities between
SDRs (about 10–30%) in general, they still share a highly
similar three-dimensional structure within the a/b-folding
pattern known as the Rossmann fold for cofactor bind-
PGRcl-his 7a 29
8
9
a
a
8
11
[
32–34]
ing.
To check for differences in the active site between
PGReu-his 7a 64
PGRs (monocyclic substrates) and T HNR/T HNR (bicyclic
3
4
8
9
a
a
n.c.
14
substrates), especially in the C-terminal segment, the three-
dimensional structures of PGRcl in the apo form and with
bound NADPH were determined to 1.7 ꢀ and 1.8 ꢀ reso-
lution, respectively. The enzyme crystallizes as a homote-
tramer (125 kDa), with two monomers in the asymmetric unit.
Its NADPH binding motif (G -X-X-G -X-X-G ) differs
PGRde-his 7a 82
8
9
a
a
11
40
1
[
a] Conversions were determined by H NMR analysis of the crude
2
4
27
30
product; n.c.=no conversion.
slightly from that of T HNR/T HNR (G-X-X-X-G-X-G), but
3
4
shares the Y167-X-X-X-K171 motif typical for SDRs, which is
complemented by S154 and D to form the catalytic tetrad
125
[
33]
conversion of acetophenone 8 into the corresponding dihydro
product 8a was significantly diminished. PGReu-his did not
convert 8 under the tested conditions. Product 8a, a rare
natural product from the fungal endophyte Nodulisporium
that mediates catalysis. The C-terminal substrate binding
loop spans 33 amino acids (T199–E231) and is thus considerably
shorter than that of T HNR/T HNR (43 amino acids). In the
3
4
apo form, the loop attains a highly flexible open-state
conformation, leaving a wide cleft open for the substrate.
Upon cofactor binding, it adopts a semi-closed state, fixing
the nucleotide in place but still leaving the substrate binding
site open, as seen in the NADPH-bound structure, where
multiple loop conformations could be modelled. Owing to
a lack of suitable inhibitors, PGRcl could not be crystallized in
the closed state. Nevertheless, sequence and structure align-
[
27]
sp., induces chlorosis in Japanese barnyard millet.
The above-noted deuterium transfer onto 1 was repeated
with substrate 7 using PGRde-his, and the deuterium was
incorporated accordingly at C-4 of 7a (see the Supporting
Information). The formation of enantiomerically enriched
7
a–9a might be expected owing to a hydrogen bond between
the carbonyl and the enolic hydroxy group, which probably
1
hinders tautomerization (five H NMR signals for the ali-
ments of T HNR and PGRcl suggest the amino acids W208–
3
phatic hydrogens of 7a–9a relative to three signals for 2). The
transformation of 7 with PGRde-his was performed on
a preparative scale (71% conversion), with subsequent
purification and circular dichroism (CD) measurement of
product 7a. The resulting spectrum shows no CD effect, thus
the enzymatic products 7a–9a are probably racemic (see the
Supporting Information). Formation of the racemic products
E213 in the C-terminal loop and Y164 on the opposite site as
prime candidates for examination in further studies.
In summary, we have identified and characterized three
different PGRs from anaerobic fermenting bacteria that
exhibit a narrow substrate range comprising 1 and its
derivatives 7–9. Accordingly, substrates for these PGRs
need to consist of a monoaromatic compound with a 1,3,5-
arrangement of unsubstituted hydroxy groups. In a patent
application, Frost et al. have reported the sequence of a PGR
7
a–9a might be explained by variable orientation of the
substrates in the active site, or by a nonselective tautomeri-
zation step in the case of a Michael-type addition.
The identification and heterologous production of the
three phloroglucinol reductases enabled substrate character-
ization with purified enzymes, which broadened the substrate
[35]
from E. oxidoreducens. Our rational approach is validated
by the protein sequence identified by Frost et al., which shows
51–76% amino acid sequence identity to PGRcl, PGReu, and
PGRde.
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3
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