Characterization of the TDP-d-ravidosamine biosynthetic pathway: One-pot enzymatic synthesis of TDP-d-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate

Article abstract of DOI:10.1039/c0ob00854k

Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (d-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-d-ravidosamine biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-d-glucose synthase, TDP-4-keto-6-deoxy-d-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-d- glucose-3,4-ketoisomerase, TDP-3-keto-6-deoxy-d-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-d-galactose-N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of TDP-d-ravidosamine has been developed. The results presented here now set the stage to produce TDP-d-ravidosamine routinely for glycosylation studies.

Full text of DOI:10.1039/c0ob00854k

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 1799
www.rsc.org/obc
PAPER
Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot
enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and
glucose-1-phosphate†
Madan K. Kharel, Hui Lian and Ju¨rgen Rohr*
Received 8th October 2010, Accepted 29th November 2010
DOI: 10.1039/c0ob00854k
Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against
various cancer cell lines in the presence of visible light. The amino sugar moieties (D-ravidosamine and
its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and
analogues when compared to closely related compounds with neutral or branched sugars. Within the
ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-D-ravidosamine
biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is
demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-D-glucose synthase,
TDP-4-keto-6-deoxy-D-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase,
TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-D-galactose-
N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of
TDP-D-ravidosamine has been developed. The results presented here now set the stage to produce
TDP-D-ravidosamine routinely for glycosylation studies.
sequences for dozens of GTs are known, only a marginal number
Introduction
of these enzymes have been fully characterized. Unavailability
Glycosylated microbial natural products possess a wide range of
of functional GTs, or their acceptor or sugar donor substrates
pharmaceutical properties such as anticancer, antiviral, antifungal
or antibiotic.1 The sugar moieties of these molecules are often
deoxygenated to various degrees, and are appended to the
continue to contribute to the slow progress of GT studies.
Ravidomycin V (1) and deacetylravidomycin V (2) are potent
antitumor antibiotics produced by Streptomyces ravidus.18,19 Both
backbone through O- or C-, and rarely through N- or S-glycosidic
antibiotics contain a common angucyclinone-derived tetracyclic
linkages.2–4 Structural and functional studies have demonstrated
aromatic benzo[d]naptho[1,2-b]pyran-6-one core moiety, which
that the sugar moieties contribute significantly to the biological
is connected to their respective sugar residues at C-4 posi-
activities of the parent compounds.
Due to the large structural diversity of deoxysugars, and due
tion through a C–C bond (Fig. 1). Deacetylravidomycin V (2)
contains D-ravidosamine (N,N-dimethyl-3-amino-3,6-dideoxy-D-
to their huge importance for biological activities, much attention
galactose) and exhibits superior anticancer/antibacterial activities
has been given to elucidate their biosynthetic pathways.5–13 Recent
progress in understanding these pathways has proven that an
array of glycosylated natural products can be generated through
enzymatic glycodiversification.14–17 However, the successful ap-
plication of this concept requires an in-depth knowledge of
various deoxysugar biosynthetic pathway enzymes including their
catalytic activities, their preferred substrates, and their flexibility
towards unnatural substrates. Additionally, glycodiversification
of natural products also requires glycosyltransferases (GTs)
with sugar-donor substrate flexibility, allowing the attachment
of various sugar residues to target aglycones. Although gene
Department of Pharmaceutical Sciences, College of Pharmacy, University of
Kentucky, 789 South Limestone Street, Lexington, KY, 40536-0596, USA.
E-mail: jrohr2@email.uky.edu; Tel: +1 859 323 5031
Fig. 1 Structures of natural products comprising ravidosamine sugar
† Electronic supplementary information (ESI) available: Full details of
in vitro experimentation and NMR data. See DOI: 10.1039/c0ob00854k
moiety.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1799–1808 | 1799
©

View Article Online
than ravidomycin V (1) itself. The 4¢-hydroxyl group of D-
ravidosamine is acetylated in ravidomycin V. Ravidomycin M (3)
and deacetylravidomycin M (4) possess a methyl side chain at C-8
position instead of the vinyl side chain present in the major con-
geners 1 and 2. The enantiomeric sugar L-ravidosamine was found
in the anthracycline antibiotic komodoquinone A (7).20,21 The
substitution pattern specifically at the 3¢-amino and 4¢-hydroxyl
groups of D-ravidosamine are different in the other reported
ravidomycin analogues FE35A (5) and FE35B (6). Gilvocarcin V,22
chrysomycin V,23 Mer 1020 dA-D,24 BE 12406 A-B,25,26 and
polycarcin V27 are other closely related analogues, differing from
ravidomycin mainly because of their neutral deoxysugar residues.
Although the biological activities of these compounds have never
been investigated in direct comparison, their biological activity
data revealed that amino sugar containing gilvocarcin analogues
show increased antibiotic/anticancer potency when compared to
compounds with neutral or branched neutral sugar moities.28
Thus, investigation of the deoxysugar biosynthetic enzymes re-
sponsible for the production of D-ravidosamine, and character-
ization of the glycosyltransferase that attaches the sugar to the
polyaromatic backbone are particularly important. An in-depth
characterization of these enzymes may provide an opportunity
to generate new ravidomycin analogues with altered aminosugar
residues for structure–activity relationship (SAR) studies.
To understand the biosynthesis of ravidomycin V, we recently
cloned and sequenced its biosynthetic gene cluster from Strepto-
myces ravidus.29 The deoxysugar biosynthetic genes and associated
glycosyltransferase were found in the cluster spanning a 6 kb
region (Fig. 2). Bioinformatics analysis combined with heterolo-
gous expression experiments suggested that five genes, ravD, ravE,
ravIM, ravAMT and ravNMT, encode enzymes involved in the
conversion of D-glucose-1-phosphate (8) to NDP-D-ravidosamine,
the proposed sugar donor substrate for the glycosylation reaction.
Similarly, the product of ravGT has been proposed to be the C-
glycosyltransferase that links D-ravidosamine to the polyketide-
derived backbone defuco-gilvocarcin V.
pathway. We demonstrate through in vitro assays and NMR
analyses that ravD, ravE, ravIM and ravAMT encode TDP-D-
glucose synthase, TDP-D-glucose-4,6-dehydratase, TDP-4-keto-6-
deoxy-D-glucose-3,4-ketoisomerase and TDP-3-keto-6-deoxy-D-
galactose-3-aminotransferase, respectively. Similarly, the product
of ravNMT was characterized as TDP-3-amino-3,6-dideoxy-D-
galactose-N,N-dimethyltransferase. Characterization of these five
deoxysugar biosynthetic enzymes unambiguously established the
biosynthetic pathway of 17. In addition, we report a one-pot
enzymatic synthetic protocol for the production of 17. The
results presented in this study lay the foundation for the in vitro
characterization of the glycosyltransferase RavGT involved in
ravidomycin biosynthesis.
Results and discussion
Expression and characterization of RavD and RavE
Basic local sequence alignment search tool (BLAST) analysis of
RavD revealed high sequence similarity to several NDP-D-glucose
synthases from deoxysugar biosynthetic pathways. RavD has the
highest sequence homology to the proposed NDP-glucose syn-
thase ChryD (83% identity/90% similarity) from the chrysomycin
biosynthetic pathway.29 The other proteins that displayed high
amino acid homology with RavD were NDP-D-glucose synthases
GilD (75%/86%),31 MtmD (76%/86%),32 SsfS1 (74%/85%)33 and
StrD (71%/83%)34 from the gilvocarcin, mithramycin, SF2575 and
streptomycin biosynthetic pathways, respectively. These proteins
are proposed/proven to catalyze the attachment of TDP at C-
1 of glucose utilizing 8 and TTP (25). These similarity results
indicated that RavD catalyzes the conversion of 8→9. To validate
this proposed function, the enzyme was expressed in E. coli. SDS-
PAGE analysis revealed a dominant band for His6-RavD (Fig.
S1†). Incubation of the purified enzyme with 8 and 25 resulted in
the production 9 (calculated 98% product yield). The conversion
was confirmed through HPLC analysis of the products with the
addition of commercially available 9 and also through comparison
1
of the H NMR data of the product with data reported in the
literature (Fig. S6, S11†).35 Compared to TTP, there was less
than 5% turnover observed when using adenosine-5¢-triphosphate
(ATP), uracil-5¢-triphosphate (UTP), guanosine-5¢-triphosphate
(GTP) or cytidine-5¢-triphosphate (CTP) as substrates.
Fig. 2 A section of the ravidomycin gene cluster spanning the putative
TDP-D-ravidosamine (17) biosynthetic genes (ravD, E, IM, AMT and
NMT).
The amino acid sequence of the product of ravE displayed strong
similarities (70%–75% amino acid identities) to NDP-D-glucose-
4,6-dehydratases involved in deoxysugar biosynthetic pathways
from various secondary metabolites such as RmbB (accession no
ACR46364) from the doxorubicin, ChryE from the chrysomycin,
GilE from the gilvocarcin, MtmE from the mithramycin, StrE
from the streptomycin, and SsfS2 from the SF2575 biosynthetic
pathway. These similarity results indicated that RavE could
serve as a TDP-D-glucose-4,6-dehydratase, which catalyzes the
deoxygenation and oxidation at C-6 and C-4 positions of 9,
respectively. To test this assumption, RavE was heterologously
produced in E. coli and purified. The size of the protein band
observed on SDS-PAGE was in agreement with the calculated
value for His6-RavE. Incubation of 9 in the presence of NAD+
did not reveal any noticeable change in the HPLC profile when
compared to the control without enzyme except for the broadening
of the TDP-D-glucose peak (Fig. 3, trace b). The preparative scale
Suzuki and co-workers have developed a synthetic route for the
production of D-ravidosamine 30 Multiple synthetic steps involved
in this protocol and a low overall yield are major drawbacks of
this approach. Activation of the sugar with a suitable nucleosyl-
diphosphate (NDP) group poses another challenge towards the
synthetic preparation of the donor substrate for RavGT. Recent
development in understanding of deoxysugar biosynthetic path-
ways has made it possible to synthesize a target NDP-activated
sugar in vitro through the activities of suitable enzymes. This
approach is superior compared to the chemical total synthesis as it
is concerted, less time-consuming, stereo- and regioselective, and
offers high yields of product in an environmentally friendly proce-
dure. We herein, report the preparation of thymidine diphosphate-
activated ravidosamine (TDP-D-ravidosamine, 17) from glucose-
1-phosphate 8 utilizing enzymes of the ravidosamine biosynthetic
1800 | Org. Biomol. Chem., 2011, 9, 1799–1808
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
These results support the aforementioned FdtA-like role of RavIM
in the biosynthesis of 17.
To assign the exact functional role of RavIM, the enzyme was
heterologously produced in E. coli, and the protein was purified
using immobilized metal affinity chromatography (IMAC) (Fig.
S1†). Size exclusion chromatography analyses of RavIM with
known proteins suggested that the enzyme exists in dimeric form.
The enzyme was then subjected to a series of enzyme assays. No
significant change was observed in the HPLC chromatogram of
the assay mixture, when RavIM was incubated with 10 for 30 min.
However, the incubation of 10 with RavIM for 24 h resulted in the
consumption of 10 and the production of a new unknown product
observable in the HPLC at 2.9 min (presumably (2R,3S)-2-methyl-
3,5-dihydroxy-4-keto-2,3-dihydropyran 14), TDP (at 51 min) and
a minor product (at 43 min). The UV spectrum of the minor
compound observed at 43 min indicated a thymine chromophore.
Thus, the unknown compound peak at 43 min could be the
product (12) of RavIM. Despite several attempts we were unable
to isolate and characterize 12 due to its poor yield (< 5%)
and instability. However, these results indicate RavIM might
be catalyzing the isomerization reaction (10→12), and that
product 12 might be spontaneously decomposing to TDP (24)
and dihydromaltol derivative 14 in the absence of the follow-up
enzyme, aminotransferase RavAMT.
Fig. 3 HPLC traces demonstrating the conversion of TDP-D-glucose
(9) to TDP-3-amino-3,6-dideoxy-D-galactose (15). Trace a: 9 gener-
ated from TTP (25) and glucose-1-phosphate (8) using RavD; trace
b: reaction mixture following the incubation of 9 with RavE; trace
c: reaction mixture including 10, PLP, L-glutamate, RavIM and
RavAMT; trace d: TDP-3-amino-3,6-dideoxy-D-galactose (15) purified
through gel filtration. The compounds for 14, 23 and 24 represents
yet uncharacterized compound (preferably (2R,3S)-2-methyl-3,5-dihy-
droxy-4-keto-2,3-dihydropyran), TMP and TDP, respectively.
BLAST analyses revealed that the product of ravAMT shows
strong sequence similarity to aminotransferases involved in
the biosynthesis of D-desosamine such as EryC139 (60%/75%),
reaction was carried out by incubating 8, 25 and NAD+ with RavD
and RavE. Separation of the crude products through gel filtration
led to the isolation of 10 with 90% product yield. The structure
40
41
OleN2
(61%/73%) and DesV
(59%/71%) from the
erythromycin A, oleandomycin and pikromycin biosynthetic
pathways, respectively. All of these homologous enzymes
were pyridoxal-5-phosphate (PLP)-dependent and were pro-
posed/characterized to catalyze amino transfer from an amino
acid, preferably L-glutamate, to the carbonyl carbon of a de-
oxysugar intermediate.41,42 Therefore, we assumed that RavAMT
might serve as an aminotransferase which converts the product of
RavIM (12) to TDP-3-amino-3,6-dideoxy-D-galactose (15).
To validate the function of RavAMT, the enzyme was over-
expressed in E. coli with a His6-tag and was purified using
IMAC. Analysis of the structure by size exclusion chromatography
indicated that the enzyme exists as a dimer. Incubation of 10 with
RavIM and RavAMT in the presence of sodium glutamate and
pyridoxal-5-phosphate (PLP) resulted in the formation of a new
compound showing a peak (calculated 20% yield) at 12.8 min in
the HPLC analysis. Production of TDP (24) in minor amounts
was also noticed (Fig. 3, trace c and d). The new peak was
collected from the HPLC and desalted through gel filtration. Low
resolution ESI mass analyses revealed a molecular ion peak at
546 Daltons, which matched with the molecular weight of 15
(molecular formula C16H27N3O14P2, calcd. [M - H]- = 546.0968,
Fig. S12†).
The negative-mode high-resolution ESI-MS revealed a molec-
ular ion peak at 546.0922 which further confirmed the calculated
molecular formula of 15. The1H NMR of this compound revealed
C-5 methyl protons as a doublet at d 1.16. The proton at C-
5 position appeared as a quartet at d 4.25. The small coupling
constant (J = 3.0 Hz) observed for the C-4 proton (d 3.93)
indicated its equatorial orientation. The proton at C-3 position
appeared as a double doublet at d 3.63 with the coupling constants
J = 11.0 Hz and 3.0 Hz. This further confirmed the presence
1
of 10 was confirmed through comparison of its H NMR data
1
with those reported in the literature (see ESI for the H NMR
spectrum†). These results demonstrated that RavD and RavE
catalyze the conversion of 8→9 and 9→10, respectively, during
TDP-D-ravidosamine (17) biosynthesis.
Functional assignment of RavIM and RavAMT as isomerase and
aminotransferase
BLAST analysis of the amino acid sequence of RavIM revealed
a modest sequence similarity (40%–50% amino acid identities)
with putative enzymes belonging to cupin2-superfamily. These
include hypothetical proteins from Beggiatoa sp. PS (accession
ZP_01998770), Limnobacter sp. MED105 (ZP_01913949) and C-
terminal domains of WxcM domain-containing proteins from
various organisms. WxcM from Xanthomonas compestris was
proposed to catalyze two steps, i) isomerization of 10→12 by its
C-terminal region and ii) N-acetylation of 15 by the N-terminal
region, during the biosynthesis of the TDP-N-acetyl-3-amino-3,6-
dideoxy-D-galactose (18) subunit of surface lipopolysaccharide
(LPS)36 RavIM also showed considerable amino acid sequence
identities (42% identity/62% similarity) to the recently char-
acterized 3,4-ketoisomerase FdtA from Aneurinibacillus ther-
moaerophilus.37 FdtA catalyzes the conversion of 10→12 during
the biosynthesis of the S-layer glycan chain. Closer analysis of the
RavIM amino acid sequence revealed the presence of the char-
acteristic signature [RGXHAH(K/R)X(L/I)XQXGS] identified
in FdtA (Fig. S3†). Analyses of active-site residues in the FdtA
crystal structure and subsequent site-directed mutagenesis studies
identified several active-site residues in this signature region.38
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1799–1808 | 1801
©

View Article Online
Table 1 1H NMR (500 MHz) data of TDP-3-amino-3,6-dideoxy-D-
galactose (15) and TDP-D-ravidosamine (17) in D2O
reasoned that RavNMT serves as a dimethyltransferase catalyzing
the conversion of 15→17.
To validate the proposed function of RavNMT, the enzyme was
heterologously produced in E. coli, and purified through IMAC
(Fig. S1†). Size exclusion chromatography analysis of RavNMT
along with standard proteins suggested that RavNMT exists as a
dimer. Incubation of 15 with RavNMT in the presence of SAM
resulted in the complete consumption of 15 with concomitant
production of a new peak (calculated ~ 90% product yield) at
4.8 min in the HPLC (Fig. 4). The compound pertaining to
the new peak was isolated and purified through HPLC and gel-
filtration. High resolution ESI mass of the isolated compound
revealed a quasi molecular ion peak of 574.1227 Dalton, which
is in agreement with the molecular formula C18H31N3O14P2 of
the proposed product 17 (calcd. MW, [M - H]- = 574.1281).
15
17
Multiplicity
(J/Hz)
Multiplicity
(J/Hz)
Position
dH [ppm]
dH [ppm]
1
2
3
4
5.54
3.88
3.63
3.93
4.25
1.16
6.28
2.36–2.28
4.55
4.10–4.16
4.10–4.16
1.87
7.66
—
dd (6.9, 3.5)
td (11.1, 3.5)
dd (11.1, 3.1)
br, d (3.1)
q (6.4)
d (6.4)
t (6.8)
m
br, m
m
m
s
br, s
—
5.64
4.21
3.60
4.21
4.25
1.19
6.33
2.29–2.38
4.60
4.10–4.16
4.10–4.16
1.91
7.70
2.94
dd (6.9, 3.6)
m
dd (11.4, 2.7)
m
q (6.6)
d (6.6)
t (6.8)
m
br, m
m
5
5-CH3
1¢
2¢
3¢
4¢
5¢
m
1
5¢¢-CH3
6¢¢
d (1.1)
d (1.1)
s
Moreover, the H NMR analysis of the compound revealed a
new singlet peak at d 2.94 indicating the presence of two N-
methyl groups. To prove that RavNMT catalyzes the transfer of
two methyl groups sequentially, the reaction was stopped after
10 min and the products subjected to MS analyses. The negative-
mode high-resolution ESI mass spectra of the reaction mixture
revealed molecular ion peaks of 574.1227, 560.1073 and 546.0922
Daltons, which corresponded to the molecular ions of 17, 16 and
15, respectively. These results showed that RavNMT catalyzes the
transfer of methyl groups from SAM to the amino nitrogen of 15
in a sequential manner.
3-N(CH3)2
of an equatorial proton at C-4 and of an axial hydrogen atom
at C-2. As observed for 10, the proton at C-2 and C-1 of 15
appeared as a double triplet at d 3.88 and a double doublet at d
5.54, respectively. These assignments were made by analyzing the
coupling constants of the 1H NMR in addition to 1H–1H COSY
correlations (Table 1). As a positive control, FdtA and FdtB were
also expressed in E. coli and the purified enzymes were used to
replace RavIM and RavAMT, respectively. Incubation of 10 with
FdtA resulted in the gradual consumption of substrate with the
concomitant production of a new compound, likely 12 (eluted at
43 min) (see Figs. S2 and S4†). This peak continuously grew for
60 min and then remained unchanged from 60 through 120 min.
However, a continuous production of TDP (24) and a new peak
(1.8 min) was observed throughout the assay period indicating
the decomposition of 12 (Fig. S5†). The specific activity of FdtA
(201 mmol mg-1 min-1) was found to be ~6-fold higher than that of
RavIM (31.3 mmol mg-1 min-1). Co-incubation of FdtA and FdtB
with TDP-4-keto-6-deoxy-D-glucose, pyridoxal-5-phosphate and
L-glutamate resulted in the production of 15 as observed earlier
through the co-assay of RavIM and RavAMT. These results also
clearly established that RavIM and RavAMT act as 3,4-keto-
isomerase and aminotransferase, respectively, which catalyze the
conversion of 10→12 and 12→15, respectively.
Fig.
4
HPLC
traces
demonstrating
the
conversion
of
TDP-3-amino-3,6-dideoxy-D-galactose (15) to TDP-D-ravidosamine (17).
Trace a: control assay mixture including 15 and S-adenosylmethionine
(SAM); trace b: products formed after addition of RavNMT with the
mixture shown in trace a; trace c: TDP-D-ravidosamine (17) purified
through gel filtration. Peaks 23 and 24 represent TMP and TDP produced
through the spontaneous decomposition of 15, respectively. The peak
marked with an asterisk was not characterized, as its UV spectrum did
not indicate a thymidine chromophore.
Heterologous production and characterization of RavNMT
The encoded amino acid sequence of ravNMT showed strong
similarity to various S-adenosylmethionine (SAM)-dependent
methyltransferases (AdoMet-MTase). These homologous en-
zymes include N-methyltransferases such as SnogX (56%/71%)
and SnogA (57%/70%) from the TDP-L-nogalamine biosynthetic
pathway.43 Similarly, N,N-dimethyltransferases such as DesVI
(55%/71%) and OleM1 (53%/68%) from the TDP-D-desosamine
42,44
pathway
represent the other homologous enzymes in the
One-pot enzymatic synthesis of TDP-D-ravidosamine
database. Both enzymes are involved in the biosynthesis of TDP-
D-desosamine, and transfer two methyl groups in an iterative
fashion from SAM to the amino nitrogen. Since the nitrogen
at C-3¢ position of ravidosamine contains two methyl groups,
and there was no other deoxysugar-N-methyltransferase encoding
candidate gene identified in the ravidomycin V gene cluster, we
One of the major goals of this study was to generate the
anticipated sugar donor substrate 17 of RavGT, necessary for
future in vitro glycosylation assays. Such assays require an easy,
reproducible and an efficient way to generate 17. Having all of the
biosynthetic enzymes in hand, we attempted to set up a protocol
1802 | Org. Biomol. Chem., 2011, 9, 1799–1808
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
to generate 17 in a large-scale one-pot setup. However, the fairly
expensive starting material TTP (25) would limit a larger-scale
production of 17. Therefore, we cloned the enzymes necessary
for the biosynthesis of TTP from E. coli following a reported
protocol.45 Thymidine monophosphate kinase (TMK) generates
24 from 23, and acetate kinase (ACK) further phosphorylates 24
to TTP (25). ATP serves as a phosphate donor thereby converting
into ADP, acetate kinase also regenerates ATP by transferring the
phosphate group of acetyl phosphate to ADP (Scheme 2).
Both proteins were overexpressed in E. coli and purified
through IMAC (Fig. S1†). Incubation of 23 with acetyl phosphate
and catalytic amount of ATP resulted in the production of
24 and 25 in quantitative yields. Since these reactions were
reversible, the reaction was driven forward by adding TDP-D-
glucose synthase and 8 to the assay mixture (Fig. 5). TDP-
D-glucose synthase from Salmonella typhimurium (RfbA) was
heterologously produced in E. coli and was used in place of
relatively unstable RavD. Considering the fact that 24 can serve as
an inhibitor for the downstream pathway enzymes, overall TDP-
ravidosamine (17) biosynthesis was divided into two stages in
the same pot. In the first stage, 23, 8 and ATP were incubated
with ACK, TMK, RfbA and RavE/RfbB/RmlB that resulted
in the production of 10 efficiently (~95% yield) in 5 min (for
details, see Experimental section). Because of its instability RavE
was substituted by the TDP-D-glucose-4,6-dehydratase (RfbB)
from Salmonella typhimurium. RfbB has been routinely used
for an efficient conversion of 9→10.8,35,46 However, following
several batches of protein production, the recombinant RfbB was
expressed mostly as insoluble protein. Therefore, an additional
TDP-D-glucose-4,6-dehydratase (encoded by the gene rmlB) was
cloned from E. coli, and the protein was expressed and purified.
MS analysis of the products revealed that addition of RmlB in
the assay mixture resulted in the complete conversion of 9→10
within five minutes. Following the complete conversion of 8→10,
the enzymes were removed from the assay mixture and the second
stage enzymes (RavIM, RavAMT, and RavNMT) along with their
co-factors (PLP, L-glutamate, and S-adenosyl-methionine (SAM))
were then added to the assay mixture to complete the synthesis
of 17. More than 95% of 10 was consumed in 65 min (Fig. 6)
with the concomitant productions of 15 and a mixture of 16
and 17. The presence of 16 and 17 in the assay mixture was
confirmed through HR-MS analysis. However, we were unable to
determine their exact quantities, as both compounds were eluted
at identical retention times in the HPLC. The production of these
compounds remained unchanged from 60 to 180 min. This was a
clear indication of the lack of SAM in the assay mixture. Addition
of excess SAM resulted in a dramatic increase in production of 17
with the consumption of 15 as well as of 16 in additional 60 min,
1
as evident from H NMR analyses of the products. The overall
yield of 17 generated was estimated to be 85%.
Fig. 5 Enzymatic total synthesis of TDP-D-ravidosamine. Trace a:
Thymidine monophosphate (TMP, 23) incubated with acetate kinase
(ACK), thymidine monophosphate kinase (TMK) and adenosine triphos-
phate (ATP), 24 = TDP, 25 = TTP; trace b: assay mixture of trace a incu-
bated with glucose-1-phosphate (G-1-P) and RavD, 9 = TDP-D-glucose;
trace c: assay mixture of trace b supplemented with RavE, 10 =
TDP-4-keto-6-deoxy-D-glucose; trace d: assay mixture of trace c sup-
plemented with RavIM, RavAMT, L-glutamate and pyridoxal-phosphate
(PLP), 15 = TDP-3-amino-3,6-dideoxy-D-galactose; trace e: assay mix-
ture of trace d supplemented with S-adenosyl methionine (SAM) and
RavNMT, 17 = TDP-D-ravidosamine. Reactions were carried out for
1 h prior to the HPLC analyses. The compounds on the HPLC traces
for a, b, c and d remained unchanged when the commercial TTP was
used in place of the TTP generated in situ by ACK, TMK, acetyl
phosphate, TMP (23) and ATP. The peaks with numbers 9, 10, 15, 17,
23, 24 and 25 represent TDP-D-glucose, TDP-4-keto-6-deoxy-D-glucose,
TDP-3-amino-3,6-dideoxy-D-galactose, TDP-D-ravidosamine; TMP, TDP
and TTP, respectively. The peak marked with an asterisk was not charac-
terized, as its UV spectrum did not indicate a thymidine chromophore.
SAM represents S-adenosyl methionine.
Fig. 6 Conversion profile of TDP-4-keto-6-deoxy-D-glucose (10) to
TDP-D-ravidosamine (17) at the second stage. Lines with empty circles,
solid triangles and empty squares represent the concentration profile
of TDP-4-keto-6-deoxy-D-glucose (10), TDP-3-keto-6-deoxy-D-galactose
(12), and TDP-3-amino-3,6-dideoxy-D-galactose (15), respectively. The
line with filled squares from
0 through 180 min represents
concentrations of the mixture of TDP-D-ravidosamine (17) and
TDP-3-N-methyl-3-amino-3,6-dideoxy-D-galactose (16). Further addition
of SAM at 180 min (arrow) resulted in the complete conversion of 15 and
16→17 in 240 min.
Discussion
Bioactive glycosylated natural products containing amino sug-
ars have the advantage that they can be solubilized through
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1799–1808 | 1803
©

View Article Online
Scheme 1 Biosynthetic pathways to TDP-D-ravidosamine (17), TDP-D-mycaminose (22), and TDP-3-N-acetamido-3,6-dideoxy-D-galactose (18).
formulation as salts, or packaged into nano-liposome delivery
vehicles.47–49 Salt forms of such products often exhibit better
bioavailability in addition to their increased hydrophilicity. There-
fore, the study of dedicated glycosyltransferases which utilize
amino sugars is promising for the generation of new pharmaceuti-
cally useful natural products through glycodiversification. C-GTs
which establish stable C–C bonds between sugar and aglycone are
considered special, since C-glycosylated compounds cannot be de-
stroyed by hydrolysis through glycosidases. Although dozens of C-
GTs have been identified from secondary metabolite biosynthetic
pathways, none of those that utilize deoxyaminosugars has been
characterized in vitro. Deacetylravidomycin V (2) is of particular
interest as it contains a C-glycosidically linked aminosugar and
is one of the most potent drugs among the gilvocarcin family of
anticancer compounds. It was our hope that by producing the
anticipated sugar donor substrate for RavGT, presumably TDP-
D-ravidosamine (17), we would be able to further investigate the
glycosylation activity of RavGT in vitro.
We had previously demonstrated that expression of the cosmid
cosRav32 in S. lividans TK64 accumulates 2 and deacetyl-
ravidomycin M (4). This indicated that the hypothesized enzymes
RavD, RavE, RavIM, RavAMT and RavNMT are sufficient
to biosynthesize 17. Biosynthesis of all deoxysugars begins
with the activation of hexose-1-phosphate utilizing a particular
NTP. Although TTP is most commonly used by NDP-hexose
synthases, there are also examples where GTP or CTP are utilized
for activation.50–53 Therefore, various NTPs were tested for the
activation of 8. In vitro assays clearly demonstrated that RavD
preferentially utilizes TTP (25) to generate 9 which converts
into 10 through the activity of RavE. These two steps are
common to a variety of deoxysugar biosynthetic pathways, such
as TDP-D-olivose,54 TDP-L-olivose,55 TDP-D-mycarose,14 TDP-L-
56
mycarose,46 TDP-D-fucose and TDP-L-rhamnose.57 Isomeriza-
tion of 10→12, catalyzed by RavIM, was the unique step of the
TDP-ravidosamine (17) biosynthetic pathway. Such a conversion
was further confirmed by the formation of an identical product in
a parallel assay involving RavIM’s homologue FdtA. To the best
of our knowledge, no such isomerase has been previously reported
from secondary metabolite biosynthetic pathways. FdtA from
Aneurinibacillus thermoaerophillus L420-91 was the only enzyme
characterized to catalyze such a transformation during the biosyn-
thesis of3-acetamido-3,6-dideoxy-D-galactose (18) during cell wall
biosynthesis.37,38 The TDP-D-mycaminose (22) biosynthetic path-
way is identical to the ravidosamine (17) pathway except for the
isomerization step where Tyl1a converts 10→19 with an equatorial
hydroxyl group at the C-4 position (Scheme 1).35 Incorporation of
ravidosamine in place of the mycaminose moiety of a pikromycin
derivative through the expression of FdtA in the Tyl1a-deletion
mutant has demonstrated a close resemblance of these pathways.58
Comparative studies of RavIM and FdtA revealed that the former
enzyme is less active compared to the latter (specific activity of
RavIM appeared to be ~6.4-fold less than that of FdtA). Such
a small turnover of the recombinant RavIM hindered us from
studying the isomerization step in detail. Therefore, FdtA was
used to monitor the 3,4-ketoisomerization reaction.
Isolation and characterization of 12 would provide direct
evidence for the activity of FdtA and RavIM. However, the
newly formed compound was spontaneously decomposed to TDP
(24) and presumably (2R,3S)-2-methyl-3,5-dihydroxy-4-keto-2,3-
dihydropyran (14); however, the structure of the latter is yet to be
1804 | Org. Biomol. Chem., 2011, 9, 1799–1808
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 2 Enzymatic synthesis of thymidine-triphosphate (TTP).
confirmed. In a time course study, a continuous production of 24
with the consumption of 10 was observed, but the concentration
of a new peak pertaining to 12 remained unchanged after 55 min.
This suggested an existence of two parallel reactions: (i) the
enzyme-catalyzed isomerization 10→12 and (ii) the spontaneous
decomposition of 12→24 and 14. The product 12 could easily
undergo keto–enol tautomerism leading to the spontaneous
decomposition as shown in Scheme 1. There is precedence for
such a transformation, observed in the D-mycaminose pathway
where the Tyl1a product 19 spontaneously degraded to TDP
synthesis described here is also likely the better method to mass-
produce ravidosamine derivatives in general.
In summary, all of the proposed ravidosamine biosynthetic
enzymes were characterized in vitro. The results unambiguously
established a pathway for the biosynthesis of the sugar moiety of
deacetylravidomycin V (2) and deacetylravidomycin M (4). The
one-pot enzymatic synthesis protocol developed in this study is
a valuable tool for producing TDP-D-ravidosamine (17) in large
scale for in vitro glycosylation studies involving RavGT as well as
other suitable GTs.
and
(20).35
(2R,3R)-2-methyl-3,5-dihydroxy-4-keto-2,3-dihydropyran
Experimental
Comparable conversions of 10→15 using RavIM-RavAMT
or FdtA-FdtB were in agreement with previous reports using
Tyl1a-TylB, where the isomerization reaction is reversible and
transamination drives the equilibrium forward.35 The conversion
of 15 to 17 through the activity of RavNMT unambiguously
proved the enzyme to be an N,N-dimethyltransferase. Identi-
fication of both 17 and 16 in the assay mixture suggested a
sequential transfer of methyl groups from SAM to the amino
nitrogen. DesVI and TylM1 from desosamine and mycaminose
biosynthetic pathways, respectively, are other examples of such
N,N-dimethyltransferases which catalyze dimethylation of a sugar
amino group in a sequential manner.44,59
Along with a high demand of NDP-activated sugars for in vitro
glycosylation studies, reports on one-pot enzymatic syntheses of
NDP-sugars are rapidly growing.46,57,60 With all of the functional
biosynthetic enzymes in hand, we also devised a one-pot enzymatic
synthesis protocol for the quick and efficient production of TDP-
D-ravidosamine (17) in a cost-efficient manner. Instead of using
expensive commercial TTP (25), the compound was generated
in situ from TMP and acetyl phosphate using acetate kinase
(ACK) and thymidine monophosphate kinase (TMK) from E.
coli. An identical strategy was applied earlier for the enzymatic
synthesis of TDP-L-rhamnose,57 TDP-4-keto-6-deoxy-D-glucose
(10),45 and TDP-4-amino-4,6-dideoxy-D-glucose.60 In the first
stage, intermediate 10 was produced in satisfactory yields (~95%).
Further incubation of the first-stage product with the remaining
biosynthetic enzymes resulted in the efficient production of TDP-
D-ravidosamine (17), with ~85% yield for the second-stage process.
Suzuki et al. had developed an elegant chemical synthesis of
ravidosamine and some of its derivatives.30 However, like the
syntheses of other deoxysugars, particularly when TDP-activated,
the reported procedure involved lengthy synthetic steps with
overall poor yields. In addition, the published ravidosamine
synthesis lacks the TDP-activation, and is therefore unsuited to
provide 17 for glycosyltransferase studies. The enzymatic total
Cloning and preparation of expression constructs
Oligonucleotide primers used in this study are summarized in
Table S1†. Strain E. coli XL1 Blue MRF was used for the routine
amplification of plasmid DNA. Genomic DNA of Salmonella
enterica serovar. typhimurium LT2, E. coli XL1 Blue and Aneurini-
bacillus thermoaerophillus L420-91 were prepared following a
standard protocol.61 Nucleotide sequences for the genes encoding
TDP-D-glucose-4,6-dehydratase (rmlB), acetate kinase (ack) and
thymidine monophosphate kinase (tmK) were amplified from
the genomic DNA of E. coli. Similarly, the genes encoding
TDP-D-glucose thymidyltransferase (rfbA), TDP-D-glucose-4,6-
dehydratase (rfbB), and TDP-4-keto-6-deoxy-D-glucose-3,4-keto-
isomerase (fdtA) and aminotransferase fdtB were amplified from
the genomic DNA of Salmonella typhimurium LT2 and Aneurini-
bacillus thermoaerophillus L420-91, respectively. All of the TDP-
D-ravidosamine biosynthetic genes were amplified from cosmid
cosRav32 29
Expression and purification of proteins
Pfu polymerase (Stratagene) was used for PCR amplifications,
and the obtained products were cloned into the ZeroBlunt-TOPO
vector (Invitrogen). Sequencing of the TOPO clones were carried
out to ascertain the fidelity of the PCR amplifications. Nucleotide
fragments covering ack, ravD, ravE, ravIM, ravAMT, ravNMT
and rfbA genes were isolated from their respective TOPO-clones
using NdeI and EcoRI restriction enzymes and subsequently
ligated to identical sites of the pET28a(+) expression vector
(Novagen). Similarly, gene sequences for tmk, rfbB and rmlB were
ligated at EcoRI/HindIII, BamHI/HindIII and BamHI/XhoI
sites of pET28a(+), respectively. The generated constructs were
then transformed into E. coli BL21(DE3). A single colony was
inoculated into 20 mL of LB liquid supplemented with kanamycin
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1799–1808 | 1805
©

View Article Online
sulfate and was grown for 4 h to prepare a seed culture. One liter
of LB (100 mL ¥ 10 flasks) was inoculated with 10 mL of the seed
culture and grown at 37 ◦C until OD600 reached ~0.5. The culture
was then grown at 18 ◦C for 12 h following addition of IPTG
(0.5 mmol final concentration). The cell pellets were collected by
centrifugation (4000 ¥ g, 15 min) and were washed twice with
20 mL of lysis buffer (50 mmol KH2PO4, 300 mmol NaCl, 10 mmol
imidazole, pH 7.6). Disruption of the pellets was carried out using
a French Press (Thermo electron corporation), and the crude
soluble enzyme fraction was collected through centrifugation
(17 000 ¥ g). N-terminal His-tagged enzymes were purified through
immobilized metal affinity chromatography (IMAC). The purified
proteins were further desalted using a Profinia protein purification
system (Bio-Rad). The Bradford protein assay method was used
to determine the concentration of the enzymes.62 The purities of
the enzymes were evaluated using dodecylsulfate–polyacrylamide
gel electrophoresis (SDS-PAGE). The yields of purified proteins
(mg per L culture) which slightly varied from batch to batch are
as follows: ACK, 8–10; TMK, 18–20; RfbA, 18–20; RmlB, 21–
24; FdtA, 6–8; FdtB, 10–13; RavD, 18–20; RavE, 8–10; RavNMT,
12–15; RavAMT, 12–15 and RavIM, 4–6.
and 5 mmol RavD/RfbA. The mixture was incubated for 1 h, and
the reaction was quenched by heating the mixture at 90 ◦C for
5 min. The mixture was centrifuged at 10 000 ¥ g for 10 min and
the supernatant was lyophilized. The dry powder was dissolved in
5 mL of water andthe solutionwas loadedintothe pre-equilibrated
Bio-Gel P2 gel filtra◦tion column 100 cm ¥ 25 mm. Separation
was carried out at 4 C with water at flow rate of 0.2 mL min-1.
Aliquots of 4 mL were collected and the purity of compounds was
monitored by HPLC at 267 nm. Pure fractions were combined,
lyophilized and analyzed by NMR. About 80 mg of pure 9 was
obtained along with an additional 125 mg of 9 with minor TMP
(23) and TDP (24) impurities. The purity 9 was assayed through
HPLC and 1H NMR (Fig. S6†). 1H NMR of 9 (500 MHz, D2O): d
1.90 (3H, s, 5¢¢-Me), 2.31–2.38 (2H, m, 2¢-H), 3.43 (1H, t, J 9.8 Hz,
3-H), 3.51 (1H, td, J 9.8, 3.5 Hz, 2-H), 3.72–3.78 (2H, m, 4-H, 5-
H), 3.81–3.90 (2H, m, 6-H2), 4.12–4.16 (1H, m, 4¢-H), 4.12–4.16
(2H, m, 5¢-H), 4.62 (1H, m, 3¢-H), 5.58 (1H, dd, J 7.4, 3.5 Hz,
1-H), 6.33 (1H, t, J 6.98 Hz, 1¢-H), 7.72 (1H, s, br, 6¢¢-H).
Enzyme assay of RavE and preparation of
TDP-4-keto-6-deoxy-D-glucose
To carry out TDP-D-glucose-4,6-dehydratase assays, the enzymes
(4 mmol RavE or 3 mmol RfbB or 3 mmol RmlB) were added to
200 mL solution composed of phosphate buffer (50 mmol, pH.
7.5), MgCl2 (20 mmol), NAD+ (2 mmol) and 9 (5 mmol). The
mixture was incubated for 30 min and the reaction was stopped.
An enzyme-free solution was analyzed by HPLC. For large-scale
production, a one-pot enzymatic synthesis was carried out in
50 mL volume composed of phosphate buffer (50 mmol, pH 7.5),
MgCl2 (20 mmol), 8 (150 mmol), TMP (23, 98.3 mmol), acetyl
phosphate (400 mmol), ATP (1 mmol), ACK (0.8 mmol), TMK
(2.1 mmol) and RfbA (2.0 mmol). The mixture was incubated for 1 h
at 37 ◦C, and then RavE (final concentration of 2.73 mmol) or RfbB
(2.3 mmol) and NAD+ (2 mmol) were added and incubation was
continued for an additional 2 h. Surprisingly, when RavE and RfbB
was expressed for additional reactions they appeared exclusively
in the form of inclusion bodies. Therefore, these enzymes were
replaced by RmlB (6 mmol) in the following batches. The reaction
mixture was heated to denature the enzymes. The precipitated
fractions were removed by centrifugation. The crude product was
lyophilized to powder and dissolved in 10 mL water. Separation of
the products was carried out in Bio-gel P2 following the protocol
described above. The purity of each fraction was analyzed by
HPLC. Pure fractions were combined and lyophilized to powder.
This afforded 700 mg of TDP-4-keto-6-deoxy-D-glucose (10) with
~95% purity and an additional 630 mg of 10 with >70% purity.
The purity and identity of the compound was further confirmed
through the comparison of 1H NMR data reported in the literature
Substrates and co-factors
Thymidine-5¢-phosphate (TMP, 23), thymidine-5¢-diphosphate
(TDP, 24), thymidine-5¢-triphosphate, (TTP, 25), uridine-5¢-
triphosphate (UTP), guanosine-5¢-triphosphate (GTP), cytidine-
5¢-triphosphate (CTP), adenosine-5¢-triphosphate (ATP), glucose-
1-phosphate (8), TDP-D-glucose (9), acetyl phosphate, pyridoxal-
5¢-phosphate (PLP), L-glutamic acid and S-adenosyl methionine
(SAM) were purchased from Sigma–Aldrich.
Enzyme assay of RavD and preparation of TDP-D-glucose
Enzyme assays for TDP-D-glucose synthases (RavD or RfbA) were
carried out in 200 mL mixture composed of 50 mmol phosphate
buffer (pH 7.5), 20 mmol MgCl2, 5 mmol glucose-1-P (8), 5 mmol
of each NTP (ATP, UTP, TTP, GTP and CTP, respectively) and
3 mmol enzyme. The mixture was incubated for 30 min in a 37 ◦C
water bath. The reaction was stopped by heating the mixture
at 90 ◦C for 3 min. The precipitated enzymes were removed by
centrifugation and the clear supernatant fractions were passed
through an amicon filter (MW cut-off 3000 Da). HPLC analyses of
the products were carried out at 25 ◦C using Waters HPLC system
equipped with photodiode array detector. Compounds were
monitored at 267 nm wavelength. The filtrate (50 mL) was injected
in the HPLC. CarboPackTM PA1 (4 ¥ 250 mm) column (Dionex)
was used to separate compounds following the previously reported
procedure.35 A gradient of ammonium acetate (500 mmol, solvent
B) and water (solvent A) was used to separate the assay products:
5–20% solvent B from 0–15 min, 20–60% solvent B from 15–
35 min, 60–100% solvent B from 35–37 min, 100% solvent B from
37–40 min, 100–5% solvent B from 40–45 min and 5% solvent B
from 45–60 min. A control sample without enzyme and standard
TDP-D-glucose (9) was also injected for the identification of the
product peak. For large-scale production of 9, TMP (5 mmol),
8 (5.1 mmol), acetyl phosphate (20 mmol) and ATP (5 mmol)
were mixed in a 20 mL solution composed of 50 mmol phosphate
buffer (pH 7.5), 20 mmol MgCl2, 4 mmol ACK, 6 mmol TMK
1
(Fig. S7†). H NMR of 10 (500 MHz, D2O): d 1.06 (3H, d, J
6.50 Hz, 5-Me), 1.77 (3H, s, J 0.97 Hz, 5¢¢-Me), 2.23 (2H, dd, J
6.90, 4.85 Hz, 2¢-H2), 3.51 (1H, td, J 10.07, 3.3 Hz, 2-H), 3.63 (1H,
d, J 10.07 Hz, 3-H), 3.93 (1H, q, J 6.5, 5-H), 3.99–4.06 (3H, m,
4¢-H, 5¢-H), 4.43–4.47 (1H, m, 3¢-H), 5.40 (1H, dd, J 6.8, 3.3 Hz,
1-H), 6.19 (1H, t, J 6.90 Hz, 1¢-H), 7.57 (1H, d, 6¢¢-H).
Assay of RavIM alone or coupled with RavAMT
RavIM (20 mmol final concentration) was incubated at 37 ◦C
with 10 (1 mmol) in a 500 mL solution composed of phosphate
1806 | Org. Biomol. Chem., 2011, 9, 1799–1808
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
buffer (50 mmol, pH 7.5) and MgCl2 (20 mmol) for 60 min. An
aliquot (60 mL) was drawn from the incubation mixture and flash-
frozen in dry ice–acetone. The enzyme was removed by further
heating the mixture at 90 ◦C for 3 min. The solution was filtered
through a centricon (MW cut-off 3000 Da) and the filtrate (50 mL)
was analyzed by HPLC. In the coupled enzyme assays, RavAMT
(30 mmol), L-glutamate (10 mmol) and PLP (60 mmol) were added
in the RavIM assay mixture and progress of the reaction was
monitoreduntil 60 min. Anew peakappeared inthe coupledassays
at 12.8 min which was collected and analyzed by high-resolution
ESI-MS. Similarly, FdtA and FdtA-FdtB coupled assays were also
carried out in parallel as positive controls. For the preparative scale
production of TDP-3-amino-3,6-dideoxy-D-galactose (15), the
substrate and enzyme concentrations were doubled and the assay
volume was increased to 20 mL. After 6 h of incubation, enzymes
were removed and the volume was reduced to 1 mL. Separation
of the compounds was carried out in analytical-scale HPLC. The
compound eluted at 12.8 min as before and was collected and
separated from the ammonium acetate buffer using a Bio-Gel
P2 column. The pure fractions were combined and lyophilized.
Overall, 12 mg powder of 15 was purified. The structure of 15 was
confirmed through MS and NMR data analyses.
(0.48 mmol), ATP (45 mmol), acetyl phosphate (2 mmol), ACK
(5 mmol), TMK (10 mmol), RfbA (8 mmol), RmlB (5.8 mmol),
phosphate buffer (50 mmol, pH 7.5) and MgCl2 (20 mmol) was
incubated at 37 ◦C for 30 min. HPLC analyses of the assay mixture
revealed the production of 10 (0.47 mmol) in the assay mixture.
◦
Enzymes were precipitated by heating the solution to 95 C and
removed through centrifugation. In the second stage, RavIM
(2.3 mmol), AMT (3.9 mmol), RavNMT (4.5 mmol), L-glutamate
(7 mmol), PLP (42 mmol) and SAM (0.7 mmol) were then added
to the assay mixture and incubation was continued for 5 h.
Considering the fact that SAM degrades in water, an additional
0.7 mmol (final concentration) of SAM was supplied after 3 h.
The reaction was monitored using HPLC as discussed above. A
calibration curve was generated varying the concentration of 23
from 62 nM to 15 mmol. The concentrations of TDP-D-hexoses
were determined using this calibration curve. The reaction was
stopped by heating at 90 ◦C for 4 min. The denatured protein
was removed through centrifugation and the liquid fraction was
lyophilized. The peak corresponding to 17 was separated through
gel filtration. The fractions including 17 were lyophilized and then
analyzed by NMR spectroscopy.
Acknowledgements
HPLC analyses of FdtA-catalysed isomerization reaction
This work was supported by the US National Institutes of Health
(grant CA 102102 to JR). The NMR and mass spectrometry
core facilities of the University of Kentucky are acknowledged
for the access to their instruments. Dr Guy Carter, Wyeth, is ac-
knowledged for providing the ravidomycin producer Streptomyces
ravidus.
TDP-4-keto-6-deoxy-D-glucose (10, 1 mmol) was incubated at
37 ◦C with 24.9 mmol enzyme in 400 mL solution containing
50 mmol phosphate buffer and 20 mmol magnesium chloride.
Aliquots (60 mL) were drawn from the assay mixture at various
time points and quenched in a dry ice–acetone bath. The samples
were boiled for 4 min and centrifuged to remove the precipitated
proteins. Theclear supernatant (50 mL) was injected into the HPLC
and the peak areas under the curve were normalized (Fig. S4 and
S5†).
References
1 D. A. Johnson and H.-W. Liu, Occurrence, genetics, and mechanisms
of biosynthesis, in Comprehensive Natural Products Chemistry, ed.
D. H. R. Barton and K. Nakanishi, Elsevier, Amsterdam, vol. 3, pp.
311–365, 1999.
In vitro assay of RavNMT
2 T. Hermann, Curr. Opin. Struct. Biol., 2005, 15, 355–366.
3 V. Kren and L. Martinkova, Curr. Med. Chem., 2001, 8, 1303–1328.
4 A. C. Weymouth-Wilson, Nat. Prod. Rep., 1997, 14, 99–110.
5 G. Blanco, E. P. Patallo, A. F. Brana, A. Trefzer, A. Bechthold, J. Rohr,
C. Mendez and J. A. Salas, Chem. Biol., 2001, 8, 253–263.
6 X. M. He and H. W. Liu, Curr. Opin. Chem. Biol., 2002, 6, 590–597.
7 X. M. M. He and H. W. Liu, Annu. Rev. Biochem., 2002, 71, 701–754.
8 L. Hong, Z. Zhao, C. E. Melancon 3rd, H. Zhang and H. W. Liu, J. Am.
Chem. Soc., 2008, 130, 4954–4967.
9 C. J. Thibodeaux, C. E. Melancon and H. W. Liu, Angew. Chem., Int.
Ed., 2008, 47, 9814–9859.
10 A. Trefzer, J. A. Salas and A. Bechthold, Nat. Prod. Rep., 1999, 16,
283–299.
For an analytical scale assay, a 200 mL assay mixture containing
50 mmol phosphate buffer, 20 mmol MgCl2, 1 mmol of 15,
4.0 mmol SAM and 10 mmol RavNMT were incubated for
two hours. An enzyme-free solution was prepared as discussed
earlier and injected in an HPLC. A new peak appeared at 4.8 min
and was collected and analyzed by HR-ESI mass spectrometry. For
large-scale production, a coupled assay was carried out in 20 mL
using 10 (2 mmol), L-glutamate (14 mmol), PLP (80 mmol), SAM
(4.0 mmol) RavIM (4.8 mmol), RavAMT (6.5 mmol) and RavNMT
(8.0 mmol). After two hours of incubation, the enzymes were
removed and the solution was lyophilized. The dried powder was
dissolved in 1 mL water and separated by HPLC. The compound
eluted at 4.8 min and was collected and desalted using a Bio-gel-
P2 column. Lyophilization of the pure fractions afforded 5 mg of
TDP-D-ravidosamine (17) and 8 mg of a 1 : 1 mixture of TDP-3-N-
methyl-3-amino-3,6-dideoxy-D-galactose (16) and 17, respectively.
The structure of 17 was determined through NMR and HR-MS
analyses.
11 A. Trefzer, G. Blanco, L. Remsing, E. Kunzel, U. Rix, F. Lipata, A. F.
Brana, C. Mendez, J. Rohr, A. Bechthold and J. A. Salas, J. Am. Chem.
Soc., 2002, 124, 6056–6062.
12 A. Trefzer, S. Pelzer, J. Schimana, S. Stockert, C. Bihlmaier, H. P. Fiedler,
K. Welzel, A. Vente and A. Bechthold, Antimicrob. Agents Chemother.,
2002, 46, 1174–1182.
13 C. E. Melancon 3rd and H. W. Liu, J. Am. Chem. Soc., 2007, 129,
4896–4897.
14 M. Perez, I. Baig, A. F. Brana, J. A. Salas, J. Rohr and C. Mendez,
ChemBioChem, 2008, 9, 2295–2304.
15 S. Blanchard and J. S. Thorson, Curr. Opin. Chem. Biol., 2006, 10,
263–271.
Two-stage one-pot enzymatic synthesis of TDP-D-ravidosamine
16 T. Liu, M. K. Kharel, L. Zhu, S. Bright and J. Rohr, ChemBioChem,
2009, 10, 278–286.
Overall enzymatic synthesis of 17 was divided into two stages.
For the first stage, a 20 mL solution containing 8 (0.5 mmol), 23
17 C. S. Zhang, Q. Fu, C. Albermann, L. J. Li and J. S. Thorson,
ChemBioChem, 2007, 8, 385–390.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1799–1808 | 1807
©

View Article Online
18 J. A. Findlay, J. S. Liu and L. Radics, Can. J. Chem., 1983, 61, 323–
327.
39 N. Dhillon, R. S. Hale, J. Cortes and P. F. Leadlay, Mol. Microbiol.,
1989, 3, 1405–1414.
40 L. M. Quiros, I. Aguirrezabalaga, C. Olano, C. Mendez and J. A. Salas,
Mol. Microbiol., 1998, 28, 1177–1185.
19 J. A. Findlay, J. S. Liu, L. Radics and S. Rakhit, Can. J. Chem., 1981,
59, 3018–3020.
20 T. Itoh, M. Kinoshita, S. Aoki and M. Kobayashi, J. Nat. Prod., 2003,
66, 1373–1377.
41 L. S. Zhao, N. L. S. Que, Y. Q. Xue, D. H. Sherman and H. W. Liu,
J. Am. Chem. Soc., 1998, 120, 12159–12160.
21 T. Itoh, M. Kinoshita, H. Wei and M. Kobayashi, Chem. Pharm. Bull.,
2003, 51, 1402–1404.
42 C. Olano, A. M. Rodriguez, J. M. Michel, C. Mendez, M. C. Raynal
and J. A. Salas, Mol. Gen. Genet., 1998, 259, 299–308.
43 S. Torkkell, T. Kunnari, K. Palmu, P. Mantsala, J. Hakala and K.
Ylihonko, Mol. Genet. Genomics, 2001, 266, 276–288.
44 C. W. Chang, L. H. Zhao, H. Yamase and H. W. Liu, Angew. Chem.,
Int. Ed., 2000, 39, 2160–2163.
22 K. Y. Takahashi, M. Tomita and F. Shirahata, J. Antibiot., 1981, 43,
271–275.
23 U. Weiss, K. Yoshihira, R. J. Highet, R. J. White and T. T. Wei,
J. Antibiot., 1982, 35, 1194–1201.
24 T. Nakashima, F. Tadashi, S. Kazuya, S. Tomohiro, K. Hiroyuki and
Y. Takeo, US Pat. 09/308710, 1999.
25 S. Nakajima, K. Kojiri, H. Suda and M. Okanishi, J. Antibiot., 1991,
44, 1061–1064.
45 J. Oh, S. Q. Lee, B. G. Kim, J. K. Sohng, K. Liou and H. C. Lee,
Biotechnol. Bioeng., 2003, 84, 452–458.
46 H. Takahashi, Y. N. Liu and H. W. Liu, J. Am. Chem. Soc., 2006, 128,
1432–1433.
26 K. Kojiri, H. Arakawa, F. Satoh, K. Kawamura, A. Okura, H. Suda
and M. Okanishi, J. Antibiot., 1991, 44, 1054–1060.
27 Y. Q. Li, X. S. Huang, K. Ishida, A. Maier, G. Kelter, Y. Jiang, G.
Peschel, K. D. Menzel, M. G. Li, M. L. Wen, L. H. Xu, S. Grabley,
H. H. Fiebig, C. L. Jiang, C. Hertweck and I. Sattler, Org. Biomol.
Chem., 2008, 6, 3601–3605.
47 J. Zhang, S. Li and X. Li, Recent Pat. Nanotechnol., 2009, 3, 225–231.
48 J. Y. Fang, Chang Gung Med. J., 2006, 29, 358–362.
49 V. Lassalle and M. L. Ferreira, Macromol. Biosci., 2007, 7, 767–783.
50 G. Weitnauer, A. Muhlenweg, A. Trefzer, D. Hoffmeister, R. D.
Sussmuth, G. Jung, K. Welzel, A. Vente, U. Girreser and A. Bechthold,
Chem. Biol., 2001, 8, 569–581.
51 N. Palaniappan, S. Ayers, S. Gupta, S. Habibel and K. A. Reynolds,
Chem. Biol., 2006, 13, 753–764.
52 J. F. Aparicio, P. Caffrey, J. A. Gil and S. B. Zotchev, Appl. Microbiol.
Biotechnol., 2003, 61, 179–188.
28 S. Rakhit, C. Eng, H. Baker and K. Singh, J. Antibiot., 1983, 36, 1490–
1494.
29 M. K. Kharel, S. E. Nybo, M. D. Shepherd and J. Rohr, ChemBioChem,
2010, 11, 523–532.
30 D. S. Hsu, T. Matsumoto and K. Suzuki, Synlett, 2005, 801–804.
31 C. Fischer, F. Lipata and J. Rohr, J. Am. Chem. Soc., 2003, 125, 7818–
7819.
32 L. L. Remsing, J. Garcia-Bernardo, A. Gonzalez, E. Kunzel, U. Rix,
A. F. Brana, D. W. Bearden, C. Mendez, J. A. Salas and J. Rohr, J. Am.
Chem. Soc., 2002, 124, 1606–1614.
53 J. S. Thorson, S. F. Lo and H. W. Liu, J. Am. Chem. Soc., 1993, 115,
5827–5828.
54 M. Perez, F. Lombo, I. Baig, A. F. Brana, J. Rohr, J. A. Salas and C.
Mendez, Appl. Environ. Microbiol., 2006, 72, 6644–6652.
55 L. Rodriguez, I. Aguirrezabalaga, N. Allende, A. F. Brana, C. Mendez
and J. A. Salas, Chem. Biol., 2002, 9, 721–729.
33 L. B. Pickens, W. Kim, P. Wang, H. Zhou, K. Watanabe, S. Gomi and
Y. Ta n g , J. Am. Chem. Soc., 2009, 131, 17677–17689.
34 M. Stockmann and W. Piepersberg, FEMS Microbiol. Lett., 1992, 90,
185–189.
35 C. E. Melancon, L. Hong, J. A. White, Y. N. Liu and H. W. Liu,
Biochemistry, 2007, 46, 577–590.
36 F. J. Vorholter, K. Niehaus and A. Puhler, Mol. Genet. Genomics, 2001,
266, 79–95.
37 A. Pfoestl, A. Hofinger, P. Kosma and P. Messner, J. Biol. Chem., 2003,
278, 26410–26417.
56 Y. Yoshida, Y. Nakano, T. Nezu, Y. Yamashita and T. Koga, J. Biol.
Chem., 1999, 274, 16933–16939.
57 Y. B. Kang, Y. H. Yang, K. W. Lee, S. G. Lee, J. K. Sohng, H. C. Lee,
K. Liou and B. G. Kim, Biotechnol. Bioeng., 2006, 93, 21–27.
58 C. E. Melancon and H. W. Liu, J. Am. Chem. Soc., 2007, 129, 4896.
59 H. W. Chen, H. Yamase, K. Murakami, C. W. Chang, L. S. Zhao, Z. B.
Zhao and H. W. Liu, Biochemistry, 2002, 41, 9165–9183.
60 Y. S. Chung, D. H. Kim, W. M. Seo, H. C. Lee, K. Liou, T. J. Oh and
J. K. Sohng, Carbohydr. Res., 2007, 342, 1412–1418.
61 J. Sambrook and D. W. Russell, Molecular Cloning: a laboratory manual
(3rd edn), Cold Spring Harbor Laboratory Press, New York, 2001.
62 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
38 M. L. Davis, J. B. Thoden and H. M. Holden, J. Biol. Chem., 2007, 282,
19227–19236.
1808 | Org. Biomol. Chem., 2011, 9, 1799–1808
This journal is
The Royal Society of Chemistry 2011
©