Identification of a fluorometabolite from Streptomyces sp. MA37: (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid

Article abstract of DOI:10.1039/c4sc03540b

(2R3S4S)-5-Fluoro-2,3,4-trihydroxypentanoic acid (5-FHPA) has been discovered as a new fluorometabolite in the soil bacterium Streptomyces sp. MA37. Exogenous addition of 5-fluoro-5-deoxy-d-ribose (5-FDR) into the cell free extract of MA37 demonstrated that 5-FDR was an intermediate to a range of unidentified fluorometabolites, distinct from fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Bioinformatics analysis allowed identification of a gene cluster (fdr), encoding a pathway to the biosynthesis of 5-FHPA. Over-expression and in vitro assay of FdrC indicated that FdrC is a NAD⁺ dependent dehydrogenase responsible for oxidation of 5-FDR into 5-fluoro-5-deoxy-lactone, followed by hydrolysis to 5-FHPA. The identity of 5-FHPA in the fermentation broth was confirmed by synthesis of a reference compound and then co-correlation by ¹⁹F-NMR and GC-MS analysis. The occurrence of 5-FHPA proves the existence of a new fluorometabolite pathway.

Full text of DOI:10.1039/c4sc03540b

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RSCPublishing
ARTICLE
Identification of a fluorometabolite from Streptomyces
sp. MA37: (2R,3S,4S)-5-Fluoro-2,3,4-
trihydroxypentanoic acid
Cite this: DOI: 10.1039/x0xx00000x
Long Maaǂ, Axel Bartholomea, Ming Him Tongb, Zhiwei Qinb, Yi Yuc, Thomas
Shepherdd, Kwaku Kyeremehe, Hai Dengb* and David O’Hagana*
Received 00th January 2012,
Accepted 00th January 2012
(2
fluorometabolite in the soil bacterium Streptomyces sp. MA37. Exogenous addition of 5ꢀ
fluoroꢀ5ꢀdeoxyꢀ ꢀribose (5ꢀFDR) into the cell free extract of MA37 demonstrated that 5ꢀFDR
R, 3S, 4S)ꢀ5ꢀFluoroꢀ2,3,4ꢀtrihydroxypentanoic acid (5ꢀFHPA) has been discovered as a new
DOI: 10.1039/x0xx00000x
D
www.rsc.org/
was an intermediate to a range of unidentified fluorometabolites, distinct from fluoroacetate
(FAc) and 4ꢀfluorothreonine (4ꢀFT). Bioinformatics analysis allowed identification of a gene
cluster (fdr), encoding a pathway to the biosynthesis of 5ꢀFHPA. Overꢀexpression and in vitro
assay of FdrC indicated that FdrC is a NAD+ dependent dehydrogenase responsible for
oxidation of 5ꢀFDR into 5ꢀfluoroꢀ5ꢀdeoxyꢀlactone, followed by hydrolysis to 5ꢀFHPA. The
identity of 5ꢀFHPA in the fermentation broth was confirmed by synthesis of a reference
compound and then coꢀcorrelation by 19FꢀNMR and GCꢀMS analysis. The occurrence of 5ꢀ
FHPA proves the existence of a new fluorometabolite pathway.
xinghaiensis (NRRL Bꢀ24674) was also shown to have a
functional fluorinase.15 S. xinghaiensis was found to produce
Introduction
The introduction of fluorine into organic molecules can FAc
1 only (no 4ꢀFT 2) and FAc 1 production is seaꢀsalt
substantially modulate their physicochemical properties.1 About dependent.15 S. xinghaiensis is the first fluorometabolite
30% of current drugs, including many top sellers, contain at producer from a marine microorganism. All of these fluorinase
least one fluorine atom.2 In contrast to manꢀmade molecules, genes have greater than 80% sequence identity to the original
fluorinated natural products are extremely rare.3
fluorinase. In culture, N. brasiliensis was unable to produce a
Fluoroacetate (FAc)
1 is the most ubiquitous fluorometabolite trace of fluorometabolite under laboratory culture
found as a toxic selfꢀdefence agent in many tropical and subꢀ conditions.13,14 For the Actinoplanes sp. strain, a synthetic gene
tropical plants.4 In 1986, the Actinomycete soil bacterium was constructed and the product enzyme was an active
Streptomyces cattleya was found to secrete FAc
1 and 4ꢀ fluorinase, however the actual strain is not in the public domain
fluorothreonine (4ꢀFT)
2 as part of its metabolic profile when and it remains to be determined if it has the ability to produce
grown in the presence of inorganic fluoride.5 Over the last fluorometabolites in culture.
decade, intermediates on the fluorometabolite pathway in S.
cattleya have been uncovered and are shown in Scheme 1.6 The
formation of the CꢀF bond is catalysed by the fluorinase, which
mediates the biotransformation of
(SAM) and inorganic fluoride into 5’ꢀfluoroꢀ5ꢀdeoxyꢀ
adenosine (5’ꢀFDA) 4 7
5’ꢀFDA is then phosphorylated
generating 5ꢀfluoroꢀ5ꢀdeoxyꢀribose phosphate (5ꢀFDRP) 5 8
followed by ring opening to generate 5ꢀfluoroꢀ5ꢀdeoxyꢀ
ribuloseꢀphosphate (5ꢀFDRulP) 6 9
An aldolase catalyses a
retroꢀaldol reaction to generate fluoroacetaldehyde , the last
common intermediate on the pathway.10 Fluoroacetaldehyde
SꢀadenesylꢀLꢀmethionine
3
.
,
.
7
7
2
is either oxidised to FAc 1 11
or biotransformed into 4ꢀFT
,
catalysed by a PLPꢀdependent transaldolase.12 The fluorinase
gene remained a sole representative in the genome databases
for a decade, however between 2012ꢀ2014, three fluorinase
genes appeared in the genomes of sequenced microorganisms
Scheme 1 Biosynthetic pathway to FAc 1 and 4ꢀFT 2 in actinomycete
bacteria.
(
Streptomyces sp
.
MA37, Actinoplanes sp
). PCR amplification of these genes
.
N902ꢀ109,13
Norcardia brasiliensis 13,14
,
from genomic DNA, or the expression of synthetic genes in E.
coli demonstrated that these were all functional fluorinases.
Most recently the marine actinomycete Streptomyces
Streptomyces sp. MA37, an isolate from a Ghanaian soil
sample, produces FAc
1 and 4ꢀFT 2
in culture.13 Unlike S.
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cattleya and S. xinghaiensis
,
Streptomyces sp. MA37 also In this context it appeared appropriate to explore a role for the
in Streptomyces sp. MA37 metabolism to establish if
, as determined by 19FꢀNMR of a any of the additional unknown metabolites are derived from the
supernatant extract.13 The identity of these novel free sugar. To this end, 5ꢀFDR was prepared by synthesis8
fluorometabolites is of interest given that this class of natural and was incubated with cellꢀfree extract (CFE) of
Streptomyces sp. MA37. Products were monitored by 19Fꢀ
is a branchpoint metabolite NMR. The usual dominance of FAc and 4ꢀFT , found in full
produced a range of unidentified fluorometabolites in addition 5ꢀFDR
to FAc and 4ꢀFT
8
1
2
8
a
products is so rare.
Here we report that 5ꢀFDRP
5
1
2
between two pathways. Exogenous addition of 5ꢀfluoroꢀ5ꢀ whole cell incubations with added fluoride ion was no longer
deoxyribose (5ꢀFDR) to cell free extracts was found to the observed profile. Instead these signals were substantially
support biosynthesis predominantly of the unidentified diminished and some of the minor unknowns now dominated
fluorometabolites and could not significantly support FAc
and the 19FꢀNMR spectrum. (Figure 1). These unknown signals
4ꢀFT biosynthesis. Genomicꢀdriven analysis allowed were not observed in control experiments using boiled CFE
identification of the fdr gene cluster, encoding elements of the incubated with 5ꢀFDR or the CFE alone. Therefore 5ꢀFDR
new pathway. In vitro assay of overꢀexpressed FdrC appears to be an intermediate to some of the fluorometabolites
demonstrated that the protein is
NAD+ dependent in Streptomyces sp. MA37, and does not appear to support FAc
dehydrogenase that oxidizes 5ꢀFDR to 5ꢀfluoroꢀ5ꢀdeoxyꢀDꢀ or 4ꢀFT biosynthesis in a primary manner.
ribonoꢀ ꢀlactone (5ꢀFRL) , followed by lactone hydrolysis to
(2 , 3 , 4 )ꢀ5ꢀfluoroꢀ2,3,4ꢀtrihydroxypentanoic acid (5ꢀFHPA)
8
1
2
8
8
a
8
1
2
γ
S
9
R
S
10. This sequence from 5ꢀFDRP forms the first steps of a new
fluorometabolite pathway.
Results and discussion
5ꢀFDRP
FAc and 4ꢀFT
established that the deꢀphosphorylated free ribose 5ꢀFDR
5
is known to be an intermediate in the biosynthesis of
in Streptomyces cattleya.8 In that study it was
was
1
2
8
unable to recover fluorometabolite biosynthesis in cell free
extracts. It was unable to support fluorometabolite biosynthesis,
and it was also unable to become phosphorylated to reꢀgenerate
5
and channel back into the fluorometabolite pathway to
8,16
generate FAc
1
or 4ꢀFT
2
.
Interestingly in the marine
microorganism Salinospora tropica, a closely related pathway
operates as illustrated in Scheme 2.17 This bacterium produces
salinosporamide A 11 a chlorinated metabolite which has
received attention as an antiꢀcancer therapeutic.18 Investigations
into salinosporamideꢀA 11 biosynthesis have shown that the
chlorine is introduced by a SAMꢀdependent chlorinase to
Figure 1. 19F NMR spectroscopic analyses of fluorometabolites: (A)
fluoroacetate, 4ꢀfluorothreonine and seven unidentified
fluorometabolitesin the supernatant of cultures from Streptomyces sp.
MA37; (B) fluoroacetate, 4ꢀfluorothreonine, and four unidentified
fluorometabolites in the cell free extract of MA37 strain incubated with
5ꢀFDR 8. The signals of excess exogenously added 5ꢀFDR 8 are
highlighted.
generate 5’ꢀClDA 12, an enzyme closely analogous to the
19
fluorinase of S. cattleya
.
The two pathways also share the
second step. In S. cattleya, depurination of 5’ꢀFDA generates 5ꢀ
A homolog search of the Streptomyces sp. MA37 genome
FDRP
5 and analogously in S. tropica 5’ꢀClRP 13 is generated.
revealed an open reading frame (orf) fdrA that is predicted to
The two pathways appear to diverge at this point.17 For
salinosporamideꢀA 11, SalN catalyses dephosphorylation of 5ꢀ
ClRP 13 to generate the free sugar 5ꢀchloroꢀ5ꢀdeoxyꢀDꢀribose
(5ꢀClR) 14 as a key intermediate on the biosynthetic pathway to
salinosporamide A 11. The free sugar does not appear to be
encode metalꢀdependent phosphoesterase, sharing high
a
sequence identity (56% identity) with SalN of the
salinosporamide gene cluster. Notably, immediately
downstream of fdrA are two orfs, fdrB and fdrC, that are
divergently transcribed. These are FdrB and FdrC which also
share high sequence identities with SalH (68% identity) and
SalM (69% identity), respectively (Fig. 2 and Fig. S1) and are
predicted to encode a dihydroxyꢀacid dehydratase and a short
chain dehydrogenase (SDRs). SalM was found to catalyse the
NAD+ dependant oxidation of 5ꢀClR 14 to 5ꢀchlororibolactone
15, which is then hydrolysed to 5ꢀchlororibonate 16 during
relevant in S. cattleya. Interestingly, 5ꢀFDR
8 was also found to
support this biotransformation to form the analogous
fluorinated derivative of salinosporamide A, demonstrating that
the salinosporamideꢀA pathway can channel fluoromethyl and
well as chloromethyl intermediates.20
NH2
NH2
N
N
studies exploring the biosynthesis of salinisporamide A 11 21
.
N
N
NH3
N
Cl
O
P
O
N
O
Cl
O
O
SalN
O
S
Cl
O
OH
SalL
Cl
SalT
N
N
O
O
O
HO
OH
HO
OH
HO
OH
HO
OH
5-ClRP 13
5-ClR 14
12
5'-ClDA
SCoA
SAM
NH
3
SalM
H
COO-
OH
COO-
O
HO
HO
O
O
Cl
SalQ
H2O
SalH
O
O
OH
HO
HO
O
O
HO
OH
Cl
Cl
Cl
Cl
22
11
16
15
17
salinosporamide A
Scheme2 Early steps in salinosporamide A 11 biosynthesis.17
2 | J. Name., 2012, 00, 1-3
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Figure 2. Organisation and proposed functions of the encoded proteins
of the identified fluorometabolite gene cluster (8.55 kb; accession no
LN612605) predicted to direct the biosynthesis of 5ꢀFHPA 10 in
Streptomyces sp. MA37.
It was an objective in this study to explore the function of
fdrC. Overꢀexpression of a codonꢀoptimized synthetic gene for
fdrC in E. coli, with a His6 tag and a TEV protease cleavage
site added, allowed isolation and purification of the coded
protein. The resultant FdrC appeared on SDSꢀpage with an
estimated mol wt ~31 kDa (Fig. S2). Incubations of the
Figure 3. 19F NMR spectroscopic analyses of (A) 19F{1H}ꢀNMR of FdrC
o
after overnight incubation with 5ꢀFDR 8 at 37 C; (B) 19F{1H}ꢀNMR of
the FdrC enzymatic reaction after overnight incubation with NADP+ and
5ꢀFDR 8 at 37 oC; (C) 19F{1H}ꢀNMR of the FdrC enzymatic reaction
after overnight incubation with NAD+ and 5ꢀFDR 8 at 37 oC; (D)
19F{1H}ꢀNMR of FdrC enzymatic reaction after overnight incubation
with NAD+ and 5ꢀFRL 9; (E) 19F{1H}ꢀNMR of aqueous solution of 9
after overnight incubation at 37oC; (F) 19FꢀNMR of synthetic 5ꢀFRL 9;
(G) 19FꢀNMR of synthetic 5ꢀFDR 8; (H) 19F{1H}ꢀNMR of synthetic 5ꢀ
FDR 8. Left insert: the enzymatic reaction; right insert: the GCꢀMS
fragmentation pattern of 5ꢀFHPA 10.
The 19FꢀNMR signals became coincident when a sample of
the enzyme reaction mixture was spiked into the supernatant of
the Streptomyces sp. MA37 fermentation (Fig 4C). The
synthetic reference of 5ꢀFHPA 10 was also used to confirm this
recombinant enzyme with 5ꢀFDR
8
and NAD+ or NADP+, were
followed by 19F NMR spectroscopy. When the assay was
conducted in the absence of NAD+ or in the presence of
NADP+, there was no turnover (Fig 3A and B, respectively).
Reactions with NAD+ however resulted in an efficient
conversion of 5ꢀFDR
8 to a new organofluorine compound
3
(19FꢀNMR; ꢀ233.15 ppm, dt
,
2JHF =25 Hz, JHF=47 Hz)
demonstrating that FdrC is a NAD+ dependent enzyme (Fig
3C). Given the similarity of this pathway to that in
salinosporamideꢀA 11, it was anticipated that the enzymatic
product might be carboxylic acid 10. This would arise by
oxidation of 5ꢀFDR
8 to lactone 5ꢀFRL 9 and then hydrolysis to
the ring opened carboxylic acid 5ꢀFHPA 10. The identity of 5ꢀ
FHPA 10 in the FribC reaction mixture was confirmed by
comparison with a synthetic sample. A synthesis of 5ꢀFHPA 10
was carried out following the protocol22 illustrated in Scheme 3.
species as
Streptomyces sp. MA37. This was achieved by global
persilylation of the components of the CFE using ꢀmethylꢀ
a component of the product mixture from
N
Nꢀ
(trimethylsilyl)ꢀtrifluoroacetamide (MSTFA). The extract was
then analysed by GCꢀMS (Fig. 3 and Fig. S3). Comparison with
the persilylated derivative of synthetic 5ꢀFHPA 10 revealed a
constituent of the CFE with an identical fragmentation pattern
and retention time (Fig. S3). Therefore these studies are
consistent with 5ꢀFHPA 10 as the product of FdrC enzyme, and
as an intermediate in fluorometabolite biosynthesis in
Streptomyces sp. MA37.
HO
HO
HO
OH
O
O
a
b
O
O
OH
O
O
O
O
OH OH
18
19
20
c
F
F
OH OH
O
d
e
O
O
F
O
COOH
O
O
OH
OH OH
10
9
21
Scheme 3. Reagents and conditions: a) Acetone, conc. H2SO4 (cat.), 2 h,
24%; b) I2, K2CO3, DCM, 2 h, 97%; c) Deoxoꢀfluor ®, DCM, 40 ˚C, 30
min, 52%; d) TFA, water, 5 h, 96%; e) LiOH, water, 3 d, quant.
The 2’ꢀ and 3’ꢀ hydroxyl groups of
by preparation of acetonide 19 22
D
ꢀribose 18 were protected
.
Oxidation of the anomeric
alcohol to give lactone 20 was efficiently accomplished using
iodine in DCM in the presence of potassium carbonate,23 and
then fluorination with Deoxofluor® afforded fluorolactone 21
in good yield.24 Acetonide hydrolysis using TFA in water gave
5ꢀFRL
9 and finally hydrolysis was accomplished with aqueous
LiOH to give 10. This synthetic 5ꢀFHPA 10 was found to be
identical by 19FꢀNMR to the enzyme (FdrC) reaction product.
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O
NH2
N
NH2
N
H3N
O
N
N
N
N
purine nucleotide
phosphorylase
(PNP)
+
N
fluorinase
N
S
Me
O
F
O
P
O
FAc 1
F
F
O
O
+
2
4-FT
O
O
OH OH
OH OH
OH OH
5-FDRP 5
3
SAM
5'-FDA 4
dephosphorylation
FdrA
FdrC+
F
OH OH
COOH
F
NAD+
O
F
O
O
OH
OH
10
OH OH
9
OH OH
8
NADH
Scheme 4. The proposed fluorometabolite pathway toward the synthesis
of 5ꢀFHPA 10 from SAM 3 in Streptomyces sp MA37. Bracket: 9,
although synthesized, is an unstable intermediate in the aqueous solution
and was not observed in the supernatant of the MA37 fermentation
during the 19F NMR analysis.
We finish with a commentary on the genes that surround
fdrAꢀC in the gene custer (Fig. 2). The fdr cluster was found
Figure 4. 19F{1H}ꢀNMR spectroscopic analyses of (insert)
fluorometabolites in the supernatant of the culture medium from
Streptomyces sp. MA37; (A) the 19F NMR expansion showing two 19F
NMR signals of 4ꢀFT 2 and 5ꢀFHPA 10; (B) the FribC enzymatic
reaction after overnight incubation with NAD+ and 5ꢀFDR 8; (C) Coꢀ
addition of the A and B samples showing the identical chemical shifts;
(D) Coꢀaddition of synthetic 5ꢀFHPA 10 and sample A with {1H}
couplings; (E) Coꢀaddition of synthetic 5ꢀFHPA 10 and sample A with
{1H} decoupling.
located in a different scaffold of the draft genome assembly of
the MA37 strain, indicating that it is physically remote to the fl
gene cluster in the previous report.13 The orf fdrD is located
immediately downstream of fdrC, encoding a putative cyclase
that shares high sequence identity (61% identity) with SalO.
Knockout of salO had no obvious effect on salinosporamide A
11 production,25 suggesting that salO is not involved directly in
salinosporamide A synthesis, so a role for FdrD is not obvious.
The gene fdrE is a LuxR family regulator gene and it shares
moderate sequence identity (38% identity) with SalRII, a
Incubation of synthetic 5ꢀFRL
9 with FdrC (Fig 3D) resulted in
its complete conversion to 10, suggesting lactone hydrolysis is
also catalysed by the enzyme. Two step enzymatic catalysis is
probably also the case for SalM (conversion of 15 to 16) on the
salinosporamide A 11 pathway.25
pathwayꢀspecific
salinisporamide A. Overꢀexpression of SalRII led to
regulator
in
the
biosynthesis
of
a
significant increase in the production of salinosporamide A 11
thus there are perhaps prospects for upꢀregulation of
fluorometabolism by overꢀexpression of this gene.25
A study of the reaction kinetics for FdrC mediated NAD+
oxidation indicates that FdrC has a higher affinity for Dꢀribose
over 5ꢀFDR
8 as measured by Km, but overall both riboses
Immediately upstream of fdrA lie two orfs, fdrF and fdrG. FdrG
oxidise with a similar efficiency (kcat/Km) of less than 0.4 ꢁmꢀ
1minꢀ1 (Table 1 and Fig. S4).
belongs to the ABC transporter family and an obvious role is
not clear. The gene fdrF is predicted to encode a ribuloseꢀ5ꢀ
phosphateꢀ4ꢀepimerase, which may be relevant to the
Specificity
Vmax
(µM/min)
Km
(µM)
kcat
constant
(kcat/Km)(µMꢀ
1.minꢀ1)
downstream metabolism of 5ꢀFDR 8. Interestingly, the main
(minꢀ1)
difference between the newlyꢀidentified fdr cluster in the MA37
and the sal cluster in S. tropica is that there is no homolog of
salQ in the proximity of the fdr cluster or anywhere in the draft
5ꢀFDR 8
4.96 ± 0.25
2.73 ± 0.05
2.73 ± 0.73
0.84 ± 0.14
0.58
0.32
0.21
genome of MA37. SalQ, a putative
αꢀoxoacid ferredoxin
Dꢀribose
0.38
18
oxidoreductase, was proposed to be the key enzyme catalysing
oxidative decarboxylation from a 5ꢀcarbon intermediate 5ꢀ
chloroꢀhydroxyꢀ2ꢀoxopentanoate 17 to a 4ꢀcarbon intermediate
4ꢀchloroꢀ3ꢀhydroxybutyrylꢀCoA 21. This observation suggested
that 5ꢀFHPA 10 cannot be metabolised further and accumulated
extracellularly, consistent with our chemical identification of 5ꢀ
FHPA 10 as one of the most abundant fluorometabolite in the
supernatant of the MA37 culture. We are currently focussing on
establishing the biochemical steps and structure of the other
fluorometabolites beyond 5ꢀFHPA 10 on this branch pathway.
Table 1. Kinetic data of FdrC mediated oxidation using 5ꢀFDR 8 and Dꢀ
ribose 18 as substrates, respectively.
This study identifies 10 as a novel fluorometabolite and
extends the very small collection of this rare class of natural
products to six. Organic chemists have only identified five
unique fluorine containing natural products so far.3a The
pathway to 10 branches from that already established for FAc
and 4ꢀFT biosynthesis. The branch point occurs at 5ꢀFDRP
whereby phosphorolysis generates 5ꢀFDR . This sugar is then
oxidised by FdrC to 5ꢀFRL , which undergoes hydrolysis to
1
5,
2
8
9
generate 5ꢀFHPA 10 (Scheme 4).
Conclusions
In conclusion, 5ꢀFHPA 10 is identified as
fluorometabolite branching from the established fluorinase
pathway to FAc and 4ꢀFT In silico analysis enabled
a
new
1
2.
identification of a biosynthetically relevant gene cluster. In
vitro assay of overꢀexpressed FdrC demonstrated that it can
oxidise (NAD+ dependant) 5ꢀFDR
8
to its corresponding 5ꢀFRL
9
followed by hydrolysis to generate 5ꢀFHPA 10. FdrC was
similarly active with Dꢀribose. GCꢀMS analysis and correlation
of synthetic or enzymatically prepared 5ꢀFHPA 10 with the
product of the supernatant of Streptomyces sp. MA37 indicated
4 | J. Name., 2012, 00, 1-3
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identical products, demonstrating that 5ꢀFHPA 10 is a new extinction coefficient was determined using the ExPAsy
natural product of Streptomyces sp. MA37. This is the first ProtParam tool. The identity of the protein was confirmed by
secure identification of a new fluorinated natural product since both polyacrylamide gel electrophoresis and ESIꢀMS analysis
1998.26ꢀ29
(Figure S2).
ESIMS analysis of FdrC enzyme reaction
ESIꢀFTꢀMS was used to determine the identity of enzymatic
products in the FdrCꢀmediated reactions. Reaction mixtures
consisted of FdrC (0.625 mg/mL), NAD+ (5 mM) and MgCl2
(10 mM) in the presence of either 5ꢀFDR (10 mM) or Dꢀribose
(10mM) in the TrisꢀHCl buffer (1 mL, 25 mM, pH 7.8). The
reactions were conducted overnight at 37 oC. Protein was
removed by heating the vial to 90 °C for 3 min, followed by
centrifugation (13,3000 rpm, 3 min). The supernatant was
subjected for ESIꢀFTꢀMS analysis.
Experimental Section
19F-NMR analysis
The samples from enzyme reaction and the supernatants of
Streptomyces sp. MAꢀ37 fermentation were subjected to 19Fꢀ
NMR analysis. The 19FꢀNMR spectra were recorded with and
without proton decoupling on
a
Bruker AVꢀ500MHz
instrument (19F at 470.3 MHz). The chemical shifts of 19Fꢀ
NMR were calculated with respect to CFCl3.
Cell-free extraction (CFE) reaction
Enzyme kinetics measurements for FdrC
The cells of Streptomyces sp. MA 37 was harvested by
centrifugation (13000 rpm × 20 mins) after 8ꢀday fermentation.
The cell pellets were washed three times using TrisꢀHCl buffer
(20 mM, pH 7.5) supplemented with 10 mM MgCl2 to remove
remnant culture media. The cells were reꢀsuspended in the same
buffer (0.1 g wetꢀcell weight per mL). The cells were disrupted
by ultraꢀsonication (60% duty cycle for 30ꢀ60s). Cell debris
was removed by centrifugation (13000 rpm, 30 mins) and the
resultant clear supernatant was used as the cell free extract for
incubation experiments. The cell free extracts (1 mL) were
supplemented with or without 5ꢀFDR at 37 °C for 6 hours. At
the end of the incubation period, protein was precipitated by
heating the vial to 90 °C for 3 min and the protein was then
removed by centrifugation. The supernatant was collected for
19FꢀNMR analysis.
A UVꢀVis spectroscopyꢀbased assay was employed to
monitor the enzyme activity of FdrC. The enzyme reactions
were initiated by mixing FdrC (final concentration was 0.26
mg/mL) with either 5ꢀFDR or
Dꢀribose, supplemented with 1
mM magnesium chloride and 2.5 mM nicotinamide adenine
dinucleotide (NAD+) in the TrisꢀHCl buffer (25 mM, pH 7.8).
The concentrations of the substrates used in the assay ranged
from 0.5 ꢁM to 200 ꢁM. After mixing with the substrate, the
UV spectra were recorded immediately, scanning from 300 nm
to 450 nm every 0.2 minutes for 3 min at ambient temperature.
An increase in absorbance at 340 nm was attributed to the
production of NADH. Initial FdrC reaction velocities were
plotted against substrate concentration and a MichaelisꢀMenten
plot was established (See Figure S4). Enzyme kinetics
parameters Vmax, Km, kcat, and specificity constant were all
calculated accordingly and are listed in Table 1.
Plasmid construction, over-expression and purification of
Streptomyces sp. MA 37 FdrC enzyme
The plasmid encoding the chemically synthesised and
codonꢀoptimised fdrC gene was provided by the commercial
supplier DNA 2.0 (CA, USA). AGGAGGTAAAACAT was
incorporated as the ribosome binding site (RBS). T7 promoter
and kanamycinꢀresistance gene were incorporated. A Hisꢀtag
containing peptide (MetꢀSerꢀTyr2ꢀHis6ꢀAspꢀTyrꢀAspꢀIIeꢀProꢀ
Acknowledgements
DOH thanks EPSRC and the ERC for financial support. He also
acknowledges the Royal Society for a Wolfson Merit Award.
KK and HD thank the Leverhulme Trust; Royal Society Africa
Award (AA090088).
Thr2) was fused to the
Nꢀterminus of the enzyme. A TEV
proteinase cleavage site (ProꢀValꢀPheꢀSerꢀGly) was engineered
into the synthetic gene to enable cleavage of the Hisꢀtag. Two
restriction sites of NcoI and EcoRI were designed to locate
upstream and downstream of the fdrC gene, respectively. The
plasmid was transformed using the heat shock method. A
mixture of chemically competent bacteria and plasmid DNA
was placed at 42 oC for 90 s and then placed back on ice. E. coli
BL21(DE3) Gold cells, transformed with the synthetic fdrC
plasmids were grown in Lysogeny broth containing 50 mg/mL
Notes and references
EaStChem School of Chemistry, University of St Andrews, North
a
Haugh, St Andrews KY169ST, United Kingdom.
b
Marine Biodiscovery Centre, Department of Chemistry, Meston Walk,
University of Aberdeen, Aberdeen AB24 3UE, United Kingdom.
c
Key Laboratory of Combinatory Biosynthesis and Drug Discovery
(Ministry of Education), School of Pharmaceutical Sciences, Wuhan
University, 185 East Lake Road, Wuhan 430071 P. R. China.
o
kanamycin at 37 C until cell density reached an absorbance at
~0.6 at 600 nm. The culture was then cooled on ice for 30 min.
A final concentration of 0.3 mM isopropylthiogalactoside
(IPTG) was added as induction procedure for FdrC overꢀ
d
The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, United
Kingdom.
o
e
expression. The incubation was continued at 25 C for ~24 h.
Department of Chemistry, FGO Torto Building, University of Ghana,
The resultant E.coli cells were collected and lysed. After
centrifugation, the cell lysate was applied onto a benchꢀtop
column packed with Ni2+ꢀcharged HisꢀBind resin (Qiagen) for
protein purification. Recombinant protein bound on the resin
was firstly washed with a solution of TrisꢀHCl (20 mM, pH
8.0), imidazole (20 mM) and NaCl (0.5 M) buffer, followed by
a further wash with a solution of TrisꢀHCl (20 mM, pH 8.0),
imidazole (50 mM) and NaCl (0.5 M). The protein was
eventually eluted with a solution of TrisꢀHCl (20 mM, pH 8.0),
imidazole (400 mM) and NaCl (0.5 M). The protein
concentration was measured by OD280nm (Nanodrop). The
Legon, Ghana.
ǂCurrent address: School of Biotechnology, Tianjin University of Science
& Technology,Tianjin 300457, P. R. China.
†
The fdr gene cluster was deposited in EMBL Nucleotide
Sequence Database under the accession no: LN612605.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

Chemical Science
Page 6 of 6
ARTICLE
VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C4SC03540B
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
24. P. Nasomjai, D. O’Hagan, A. M. Z. Slawin, Beilstein J. Org. Chem.
,
2009, 5, A37.
25. A. Lechner, A. S. Eustáquio, T. A. M. Gulder, M. Hafner, B. S.
Moore. Chem. Biol., 2011, 18, 1527ꢀ1536.
1. D. O’Hagan, J. Fluorine Chem., 2010, 131, 1071ꢀ1081.
26. Recently a fluoroaromatic was claimed as a new metabolite from a
Streptomyces isolate,27 however it was subsequently shown to be an
insecure identification.28 Also some derivatives of 5ꢀfluorouracil
were claimed29 as isolates from a sponge in the South China Sea,
however these most likely arise from industrial contamination and
then bioꢀaccumulation. The last bone fide fluorometabolites to be
2. V. A. Brunet and D. O’Hagan, Angew. Chem. Int. Ed., 2008, 47
,
1179ꢀ1182.
3. (a) H. Deng and D. O’Hagan, Chem. Rev., 2014, in press; (b) H.
Deng, D. O’Hagan and C. Schaffrath, Nat. Prod. Rep., 2004, 21
773ꢀ784. (c) C. D. Murphy. Nat. Prod. Rep., 2006, 23, 147ꢀ152.
,
4. D. O’Hagan, R. Perry, J. M. Lock, J. J. M. Meyer, L. Dasaradhi, J. T.
G. Hamilton and D. B. Harper, Phytochemistry, 1993, 33, 1043ꢀ
1046.
identified are derivatives of ωꢀfluoroꢀoleate from seeds of the West
African plant Dichapetalum toxicarium.30
27. N Jaivel, C. Uvarani, R. Rajesh, D. Velmurugan, P. Marimuthu. J.
Nat. Prod. 2014, 77, 2ꢀ8
5. M. Sanada, T. Miyano, S. Iwadare, J. M. Williamson, B. H. J. Arison,
L. Smith, A. W. Douglas, J. M. Liesch, E. Inamine, J. Antibiot., 28. M. S. Ayoup, D. B. Cordes, A. M. Z. Slawin, & D. O’Hagan. J. Nat.
1986, 39, 259ꢀ265.
Prod., 2014. 77, 1249–1251.
6. H. Deng, D. O’Hagan, Curr. Opin. Chem. Biol. 2008, 12, 582ꢀ592.
7. (a)D. O’Hagan, C. Schaffrath, SL. Cobb, J.T.G. Hamilton, C.D.
29. X. H. Xu, G. M. Yao; Y. M. Li; C. J. Lin; X. Wang,; C. H. Kong. J.
Nat. Prod., 2003, 66, 285ꢀ288.
Murphy. Nature. 2002, 416, 279; (b) C. Schaffrath, H. Deng, D. 30. W. W. Christie, J. T. G. Hamilton, D. B. Harper. Chem. Phys. Lipids
,
O’Hagan. FEBS Lett. 2003, 547, 111ꢀ114.
1998, 97, 41ꢀ47.
8. S. L. Cobb, H. Deng, J. T. G. Hamilton, R. P. McGlinchey, D.
O’Hagan, Chem. Comm., 2004, 592ꢀ593.
9. M. Onega, R. P. McGlinchey, H. Deng, J. T. G. Hamilton, D.
O’Hagan. Bioorg. Chem., 2007, 35, 375ꢀ385.
10. S. J. Moss, C. D. Murphy, D. O’Hagan, C. Schaffrath, J. T. G.
Hamilton, W. C. McRoberts, D. B. Harper. Chem. Commun. 2000
,
2281–2282.
11. C. D. Murphy, S. J. Moss, D. O’Hagan. Appl. Environ. Microbiol.
2001 67, 4919–4921.
,
12. (a) C. Murphy, D. O’Hagan, C. Schaffrath. Angew. Chem. Int. Ed.,
2001, 4479–4481. (b) H. Deng, S. M. Cross, R. P. McGlinchey, J. T.
G. Hamilton, D. O’Hagan. Chem. Biol. 2008, 15, 1268–1276.
13. H. Deng, L. Ma, N. Bandaranayaka, Z. Qin, G. Mann, K. Kyeremeh,
Y. Yu, T. Shepherd, J. H. Naismith, D. O’Hagan. ChemBioChem.
2014, 15, 364ꢀ368.
14. Y. Wang, Z. Deng, X. Qu. F1000Research, 2014, 61, 1ꢀ14.
15. S. Huang, L. Ma, M. H. Tong, Y. Yu, D. O’Hagan, H. Deng. Org.
Biomol. Chem. 2014, 12, 4828–4831.
16. S. L. Cobb, H. Deng, J. T. G. Hamilton, R. P. McGlinchey, D.
O’Hagan, C. Schaffrath. Bioorg. Chem. 2005, 33, 393–401.
17. A. S. Eustáquio, R. P. McGlinchey, Y. Liu, C. Hazzard, L. L. Beer,
G. Florova,; M. M. Alhamadsheh, A. Lechner, A. J. Kale, Y.
Kobayashi,; K. Reynolds, B. S. Moore. Proc. Natl. Acad. Sci. U. S.
A. 2009, 106, 12295–12300.
18. W. Fenical, P. R. Jensen, M. Palladino, K. S. Lam, G. K. Lloyd, B. C.
Potts. Bioorg. Med. Chem. 2009, 17, 2175–2180.
19. A. S. Eustáquio, F. Pojer, J. P. Noel, B. S. Moore. Nat. Chem. Biol.
,
2008, , 69ꢀ74.
4
20. A. S. Eustáquio, D. O’Hagan, B. S. Moore. J. Nat. Prod. 2010, 73
,
378–382.
21. A. J. Kale, R. P. McGlinchey, B. S. Moore. J. Biol. Chem. 2010, 285
,
33710–33717.
22. Y. H. Jin, C. K. Chu, Tetrahedron Lett., 2002, 43, 4141ꢀ4143.
23. M. B. Fusaro, V. Chagnault, S. Josse, D. Postel, Tetrahedron, 2013,
69, 5880ꢀ5883.
6 | J. Name., 2012, 00, 1-3
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