Nucleotidylation of unsaturated carbasugar in validamycin biosynthesis

Article abstract of DOI:10.1039/c0ob00475h

Validamycin A is a member of microbial-derived C₇N-aminocyclitol family of natural products that is widely used as crop protectant and the precursor of the antidiabetic drug voglibose. Its biosynthetic gene clusters have been identified in seve

Full text of DOI:10.1039/c0ob00475h

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PAPER
Nucleotidylation of unsaturated carbasugar in validamycin biosynthesis†
Jongtae Yang,a Hui Xu,a,b Yirong Zhang,b Linquan Bai,b Zixin Dengb and Taifo Mahmud*a
Received 22nd July 2010, Accepted 17th September 2010
DOI: 10.1039/c0ob00475h
Validamycin A is a member of microbial-derived C7N-aminocyclitol family of natural products that is
widely used as crop protectant and the precursor of the antidiabetic drug voglibose. Its biosynthetic
gene clusters have been identified in several Streptomyces hygroscopicus strains, and a number of genes
within the clusters have been functionally analyzed. Of these genes, valB, which encodes a sugar
nucleotidyltransferase, was found through inactivation study to be essential for validamycin
biosynthesis, but its role was unclear. To characterize the role of ValB in validamycin biosynthesis, four
carbasugar phosphate analogues were synthesized and tested as substrate for ValB. The results showed
that ValB efficiently catalyzes the conversion of valienol 1-phosphate to its nucleotidyl diphosphate
derivatives, whereas other unsaturated carbasugar phosphates were found to be not the preferred
substrate. ValB requires Mg2+, Mn2+, or Co2+ for its optimal activity and uses the purine-based
nucleotidyltriphosphates (ATP and GTP) more efficiently than the pyrimidine-based NTPs (CTP,
dTTP, and UTP) as nucleotidyl donor. ValB represents the first member of unsaturated carbasugar
nucleotidyltransferases involved in natural products biosynthesis. Its characterization not only expands
our understanding of aminocyclitol-derived natural products biosynthesis, but may also facilitate the
development of new tools for chemoenzymatic synthesis of carbohydrate mimetics.
involvement of a number of unusual enzymes that exclusively
Introduction
recognize carbasugars (cyclitols) as substrates.8 The pathway leads
Carbohydrates are indispensable elements in living organisms, not
to a condensation between two cyclitol intermediates by an unde-
only as molecules for energy storage but also as mediators of many
termined coupling mechanism. The lack of clear understanding
complex biological processes.1–3 Their structural diversity enables
of this coupling reaction has led to speculation about how the
nitrogen bridge in validamycin A is formed.8–13
them to encode information for specific molecular transactions
such as cell–cell recognition and molecular targeting.1–3 Due
to their ubiquitous involvement in many biological processes,
including immune response, cancer, and inflammation, developing
new carbohydrate mimetics (e.g., carbasugars, aminosugars, etc)
can provide a new roadmap for drug discovery.4–6
Among the naturally derived carbasugars are the C7N-
aminocyclitol family of natural products, such as the a-glucosidase
inhibitor acarbose and the antifungal agent validamycin A. They
have attracted attention due to their important biomedical and
agricultural uses.7 Validamycin A is a bacterial-derived crop
protectant that is widely used to treat sheath blight disease in
rice plants. The core aminocyclitol unit, valienamine, is used as
the semi-synthetic precursor of the antidiabetic drug voglibose.
Prompted by the identification of the biosynthetic gene cluster,
investigations into validamycin biosynthesis have revealed the
It has been speculated that the core validoxylamine unit of
validamycin is formed by a condensation between a nucleotidyl
unsaturated carbasugar, such as NDP-valienol (3a) or NDP-1-
epi-valienol (3b) (Scheme 1), and an amino-bearing carbasugar.9
3a and 3b may be derived from valienol 1-phosphate (1a) or 1-epi-
valienol 1-phosphate (1b), respectively, catalyzed by a carbasugar
nucleotidyltransferase. A similar reaction has been proposed in
the acarbose pathway involving 1-epi-valienol 1,7-diphosphate
(2b) and NDP-1-epi-valienol 7-phosphate (4b) as the substrate
and the product of the putative nucleotidyltransferase AcbR.11,14
However, no biochemical evidence is available to support this
notion.
Although sugar 1-phosphate nucleotidyltransferases have been
widely studied, little is known about their involvement in car-
basugar or aminocyclitol biosynthesis. The only report that
directly implicated a sugar 1-phosphate nucleotidyltransferase
in NDP-carbasugar formation was that of the Escherichia coli
glucose 1-phosphate uridylyltransferase, which could catalyze the
conversion of carbaglucose 1-phosphate to its UDP-product,
albeit at a lower turnover rate than observed with the natural
substrate, glucose 1-phosphate.15 While it is unclear if other nu-
cleotidyltransferases are tolerant to ring oxygen substitutions, the
aDepartment of Pharmaceutical Sciences, Oregon State University, Corvallis,
OR 97331, USA. E-mail: Taifo.Mahmud@oregonstate.edu; Fax: (+1) 541-
737-3999
bLaboratory of Microbial Metabolism and School of Life Sciences &
Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, China
† Electronic supplementary information (ESI) available: Strains used in
the study; NMR spectra. See DOI: 10.1039/c0ob00475h
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Scheme 1 Proposed biosynthetic pathways to validamycin involving either valienol 1-phosphate or 1-epi-valienol 1-phosphate (A) and to acarbose
involving either valienol 1,7-diphosphate or 1-epi-valienol 1,7-diphosphate (B).
corresponding enzyme from yeast did not recognize carbaglucose
1-phosphate as substrate.15
ValB. The results not only provide insight into the role of
ValB in validamycin biosynthesis but may also facilitate the
development of new tools for the production of carbohydrate
mimetics.
A gene (valB) encoding a sugar 1-phosphate nucleotidyltrans-
ferase homologous to AcbR was identified in the validamycin
biosynthetic gene cluster of Streptomyces hygroscopicus subsp.
jinggangensis 5008.16 While the catalytic function of ValB was
unclear, its homologue (VldB, 99% identity) from another val-
idamycin producing strain was reported to convert glucose 1-
phosphate to UDP-glucose.13 It was then postulated that VldB is
responsible for the formation of pathway specific UDP-glucose,
which is important for the glucosylation of validoxylamine
A to validamycin A. As this result is inconsistent with the
notion that ValB, and also AcbR, is involved in carbasugar
nucleotidylation, we decided to investigate the actual role of
ValB in validamycin biosynthesis. The present paper reports
our results on the inactivation of valB in S. hygroscopicus
5008, complementation experiments, synthesis of all four possible
substrates (1a, 1b, valienol 1,7-diphosphate (2a), and 2b) for
ValB, as well as functional characterization of recombinant
Results
Inactivation of valB in S. hygroscopicus 5008 abolishes production
of validamycin A and validoxylamine A
Whereas Kim and co-workers have demonstrated that VldB
could convert glucose 1-phosphate to UDP-glucose,13 the question
remains as to what is the exact role of ValB in validamycin
biosynthesis. Is ValB really responsible for the formation of UDP-
glucose, the substrate for the glycosyltransferase ValG,17 or is it
involved in the formation of NDP-cyclitol, setting the stage for
a subsequent coupling reaction.9 To address this question, the
valB gene was inactivated by replacing a 936 bp internal DNA
fragment of valB with oriT-aac(3)IV in S. hygroscopicus 5008.
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Fig. 1 Construction and analysis of valB mutant strains of S. hygroscopicus 5008. A, Schematic representation for the replacement of a 936 bp fragment
of valB with oriT-aac(3)IV. B, Gel electrophoresis of PCR amplified products of the wild-type and the mutant strains. C, Bioassay comparison between
the wild-type 5008, the valB mutant ZYR-2, and the valB mutant complemented with cloned valB (ZYR-2/valB) using Pellicularia sasakii as the indicator
strain. Growth inhibition was determined by the appearance of abnormal branching of the mycelia and the formation of a thick border. D, HPLC analysis
of the wild-type 5008, the valB mutant ZYR-2, and the valB mutant complemented with cloned valB (ZYR-2/valB). Validamycin A and validoxylamine
A are indicated with arrows.
This was achieved by using a pHZ1358-derived plasmid pJTU702
in which oriT-aac(3)IV had been inserted between a 3.2 kb left
flanking and a 1.5 kb right flanking sequence of the 936 bp
DNA fragment to be replaced (Fig. 1A). The plasmid pJTU702
was introduced into the wild-type strain of S. hygroscopicus 5008
through conjugation18 and two thiostrepton-sensitive apramycin-
resistant transformants (ZYR-2-1 and ZYR-2-2) were obtained.
Total DNA from these two mutant strains and from the wild-
type strain was used as templates for PCR analysis using two
primers, ValB-Det-F and ValB-Det-R. The two mutants gave a
1.8 kb PCR product, while the wild-type strain gave a 1.4 kb PCR
product, which confirmed that a 936 bp DNA fragment internal to
valB had been replaced by the 1384 bp oriT-aac(3)IV cassette in
these mutants (Fig. 1B). Fermentation broths of the mutants were
analyzed by bioassay and HPLC. No inhibition of the fungus
Pellicularia sasakii could be detected in the bioassay (Fig. 1C)
and no peak corresponding to validamycin A and validoxylamine
A was detected by HPLC analysis, indicating a complete loss of
production of both compounds in the valB mutants (Fig. 1D). The
lack of validoxylamine A formation in the mutant strains indicated
that ValB is involved in the formation of the core aminocyclitol
unit, rather than in UDP-glucose formation.
Restoration of validamycin A and validoxylamine A production in
ZYR-2 through complementation with cloned valB
To confirm the critical role of valB in validoxylamine A biosynthe-
sis, the PCR-amplified valB gene was cloned onto the integrative
vector pPM927 downstream of the PermE* promoter, and the
product subsequently introduced into the valB mutant ZYR-
2-1 by conjugation. Fermentation broths of the ZYR-2 strain
complemented with valB regained inhibitory activity in the
bioassay (Fig. 1C) and HPLC analysis of the fermentation extracts
clearly demonstrated that validoxylamine A and validamycin A
productions were restored to about 50% of those of the wild-type
strain (Fig. 1D).
Chemical synthesis of valienol phosphates
As the inactivation experiments suggested the involvement of ValB
in the formation of validoxylamine A, we attempted to identify
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Scheme 2 Synthesis of valienol 1-phosphate 1a, 1-epi-valienol 1-phosphate 1b, valienol 1,7-diphosphate 2a, and 1-epi-valienol 1,7-diphosphate 2b. a)
EtSH, TFA; b) DMSO, Ac2O; c) Ph3PCH3Br, t-BuOK, benzene; d) HgCl2, HgO, aq. CH3CN; e) vinylmagnesium bromide, THF, 0 ◦C; f) Grubbs’ 2nd
gen. catalyst, CH2Cl2, reflux; g) iPr2NP(OBn)2, 1H-tetrazole, CH2Cl2, m-CPBA; h) BBr3, CH2Cl2; i) ZnCl2, Ac2O, AcOH; j) NaOMe, MeOH.
the substrate for this enzyme by preparing and testing a number
of carbasugar compounds. Despite that numerous strategies have
been applied to the synthesis of carbocyclic framework of carba-
sugars, both in racemic and enantiomerically pure forms,7,19 the
synthesis of unsaturated carbasugar phosphates, such as valienol
phosphates and its analogues, has never been reported. In order to
gain access to these unsaturated carbasugar phosphates chemical
syntheses were carried out using a ring-closing metathesis strategy
as shown in Scheme 2. The key intermediate tetrabenzylated diene
7 was synthesized from commercially available 2,3,4,6-tetra-O-
benzyl-D-glucopyranose (5) in five steps according to a literature
procedure.19 Ring-closing metathesis19 of 7 mediated by Grubbs’
2nd generation catalyst20 furnished, after column chromatogra-
phy, the tetrabenzylated valienols 8a and 8b in 21% and 57%
yields, respectively (Scheme 2). Alcohols 8a and 8b were then
individually treated with dibenzyl diisopropyl phosphoramidite
in the presence of 1H-tetrazole to form the phosphite interme-
diates, which were readily oxidized by m-chloroperoxybenzoic
acid (m-CPBA) to afford phosphate compounds 9a and 9b,
respectively.21 Finally, debenzylation was achieved by treating the
compounds with BBr3 at -40 ◦C to give the desired products 1a
22
and 1b.
The synthesis of valienol 1,7-diphosphate analogues also pro-
ceeded from compounds 8a and 8b. Regioselective debenzylation
of 8a and 8b using ZnCl2, Ac2O and AcOH, followed by a
deacetylation step furnished diols 10a and 10b, respectively.23 The
two free alcohol functionalities were then phosphorylated and the
product debenzylated as described for the synthesis of 1a and
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1b to afford valienol 1,7-diphosphate (2a) and 1-epi-valienol 1,7-
diphosphate (2b).
to analyze the NDP-valienol products by HPLC or to isolate
the products by chromatography were unsuccessful. We observed
that during prolonged incubation and/or chromatography, the
products degraded to valienol. Therefore, an alternative assay for
the enzyme reaction was developed.
Preparation of recombinant ValB and initial characterization of
ValB with different valienol phosphates and NTPs
With the potential substrates for the enzyme in hand, the
recombinant ValB protein was then prepared by cloning the
1.1 kb valB gene into expression vector pRSET B (Invitrogen)
and the resulting plasmid was introduced into E. coli BL21
Gold(DE3)/pLysS by heat-pulse transformation. Gene expression
was induced by isopropyl b-D-1-thiogalactopyranoside (IPTG)
and the recombinant protein was purified through a Ni-NTA
column and dialyzed against 60 mM Tris-HCl pH 8 to give >80%
pure 45 kDa protein (Fig. 2A).
Preliminary enzymatic reactions of ValB were carried out
using the four different substrates (1a, 1b, 2a, and 2b) and
all five naturally occurring NTPs (ATP, GTP, CTP, dTTP, and
UTP). Analysis of the products by TLC and MS revealed that
only 1a was converted to the corresponding NDP-valienols.
Similar experiments with glucose 1-phosphate also gave NDP-
glucose products, suggesting that ValB has somewhat relaxed
substrate specificity. However, due to their instability, attempts
Coupled colorimetric assays of ValB
Because the conversion of valienol 1-phosphate and a nucleotidyl
triphosphate to the corresponding NDP-valienol produces inor-
ganic pyrophosphate (PPi), measuring the net amount of PPi in
the reaction mixture provides an indirect measure of the NDP-
valienol being produced. Therefore, a coupled colorimetric assay
using EnzChek Pyrophosphate Assay Kit24 (Molecular Probes)
was used to measure the amount of PPi in the reactions (Scheme
3). The system includes the enzymes inorganic pyrophosphatase
and purine nucleoside phosphorylase (PNP), in which the former
enzyme catalyzes the conversion of PPi into two equivalents
of inorganic phosphate (Pi), and the latter, in the presence
of Pi, converts 2-amino-6-mercapto-7-methylpurine ribonucleo-
side (MESG) to ribose 1-phosphate and 2-amino-6-mercapto-7-
methylpurine. Enzymatic conversion of MESG results in a shift in
Fig. 2 Preparation and characterization of recombinant ValB. A, SDS PAGE Analysis of ValB (M = protein marker, W = whole cell extract, S = soluble
protein, P = purified protein). B–C, Relative activity of ValB based on coupled colorimetric assays using four different valienol phosphate substrates (B)
and five different NTPs (C). D–E, MS analysis of ValB using four different valienol phosphate substrates (D) and five different NTPs (E).
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Scheme 3 Coupled colorimetric assays of ValB. Enzymatic conversion of MESG to ribose 1-phosphate and 2-amino-6-mecapto-7-methylpurine results
in a shift in absorbance maximum from 330 nm to 360 nm.
absorbance maximum from 330 nm for the substrate to 360 nm
for the product.
Metal ion cofactors for ValB
Most sugar 1-phosphate nucleotidyltransferases require metal
ions for their activity.25,26 To assess the metal ion dependence
of ValB activity, the reactions were carried out using EDTA-
treated protein in the presence of various metal ions. However,
light absorption at 360 nm by some metal ions precluded the use
of the colorimetric assay for these experiments. Therefore, TLC
analysis with detection under UV light at 254 nm (Fig. 3A) and MS
(Fig. 3B) were used to analyze the reaction products. The results
confirmed that metal ions are indeed essential for ValB activity, as
reactions using EDTA-treated ValB alone did not give any product
at a detectable level. Of eight metal ions tested, Mg2+, Mn2+, and
Co2+ were able to rescue the activity of the EDTA-treated enzyme,
whereas Fe2+, Cu2+, Ca2+, Zn2+, and Ni2+ were inactive (Fig. 3). The
results suggest that ValB adopts a similar metal ion requirement
as sugar 1-phosphate nucleotidyltransferases commonly found in
biological systems.
The substrate specificity of ValB was then reevaluated using this
assay, in which 1a, 1b, 2a, and 2b were tested in the presence of
GTP. After 1 h incubation, EnzChek solution was added and the
reaction mixtures were incubated in a 96-well plate for 30 min
at 22 ◦C. The change in absorbance at 360 nm was recorded in
triplicate. Consistent with the preliminary results, the colorimetric
assay and MS provided compelling evidence that 1a is the preferred
substrate for ValB (Fig. 2B and 2D).
Utilization of various nucleotidyltriphosphates by ValB
BLAST analysis of the amino acid sequence of ValB revealed
that besides having high sequence identities with other putative
carbasugar nucleotidyltransferases, AcbR (from the acarbose
pathway in Actinoplanes sp., 64%), GagR (from the acarbose
pathway in Streptomyces glaucescens, 67%), and SalF (from the
salbostatin pathway in Streptomyces albus, 68%), ValB is also
similar to the glucose 1-phosphate adenylyltransferases (GlgC,
27–41%). The result was inconsistent with the report by Kim
and co-workers, as it indicated that VldB is a uridylyltransferase
enzyme.13 To identify the preferred nucleotidyl donor(s) in ValB
reaction, the enzyme was incubated with valienol 1-phosphate
in the presence of different NTPs. Colorimetric assay of the
products indicated that ValB more efficiently utilized the purine-
based nucleotidyltriphosphates, ATP and GTP, than pyrimidine-
based NTPs as nucleotidyl donor (Fig. 2C). These results were
unambiguously supported by strong signals for both ADP-
valienol (m/z 583.93) and GDP-valienol (m/z 599.93) products
in the mass spectra (Fig. 2E). Interestingly, ValB also used CTP
as nucleotidyl donor, converting valienol 1-phosphate to CDP-
valienol (m/z 559.93). On the other hand, only low-level activity
of ValB was observed in the colorimetric assay with dTTP or
UTP. However, the amounts of the products seemed to be below
the MS-detectable levels.
Discussion
Sugar nucleotides are ubiquitous in Nature. They play critical
roles in primary and secondary metabolism as substrates for many
important enzymes, particularly the glycosyltransferases. They are
normally derived from sugar 1-phosphates and NTPs catalyzed
by sugar 1-phosphate nucleotidyltransferases, also referred to as
sugar nucleotide pyrophosphorylases (EC 2.7.7.-). In bacteria and
plants, sugar 1-phosphate nucleotidyltransferases are involved in
the biosynthesis of starch, peptidoglycans, and lipopolysaccha-
rides, and their participation in natural product biosynthesis has
been fully appreciated. In addition, many genes encoding sugar
1-phosphate nucleotidyltransferases have been found in natural
products biosynthetic gene clusters, including those of the C7N-
aminocyclitol natural products, such as acarbose,11 validamycin,16
and salbostatin.27 However, their roles in the biosynthesis of these
compounds had not been carefully studied.
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Fig. 3 Identification of metal ion cofactors for ValB. A, TLC analysis of ValB products with different metal ions under UV light at 254 nm (M-, with
no metal ion; BE, with boiled enzyme; AG, ADP-glucose standard; thick arrows show ADP–valienol products). B, MS analysis of ValB products with
different metal ions. All reaction mixtures contained EDTA.
Whereas VldB, a ValB homologue (99% identity) from another
validamycin producing strain, was reported to serve as a glucose
1-phosphate uridylyltransferase,13 results from our in vitro and
in vivo studies suggest otherwise. First, ValB, while able to
recognize glucose 1-phosphate as substrate, efficiently converts
valienol 1-phosphate to its corresponding nucleotidyl derivatives.
Interestingly, the 1-epi- and the 1,7-diphosphate-analogues are not
preferred substrates. Secondly, ValB most efficiently utilizes ATP
and GTP, instead of UTP, as nucleotidyl donor. This, in fact, is
consistent with the relatively high sequence identity between ValB
(also VldB) and the glucose 1-phosphate adenylyltransferases
(GlgC). Furthermore, inactivation of valB in S. hygroscopicus
5008 abolished the production of both validoxylamine A and
validamycin A, which provided compelling evidence for the critical
role of ValB in validoxylamine A biosynthesis, and not in the
conversion of validoxylamine A to validamycin A. In other words,
ValB is most likely responsible for the synthesis of ADP- or GDP-
valienol, setting the stage for a coupling reaction leading to the
bis-cyclitol core unit formation. In addition, our previous study
has shown that the glycosyltransferase ValG, which catalyzes the
conversion of validoxylamine A to validamycin A, most efficiently
uses UDP-glucose, not ADP- or GDP-glucose, as sugar donor.16,17
Therefore, as ValB prefers ATP and GTP as nucleotidyl donor, it
is less likely that this enzyme plays a critical role in supplying
UDP-glucose for ValG reaction.
Viewed together, the present study not only clarifies the catalytic
function and role of ValB in validamycin A biosynthesis but also
provides insight into the pathway directly upstream of valienol
1-phosphate (Scheme 4). It now can be postulated that the conver-
sion of valienone 7-phosphate, the product of the cyclitol kinase
ValC,28 to valienol 1-phosphate would involve a stereoselective
reduction of C-1 followed by phosphate transfer from C-7 to C-
1. The latter step may be catalyzed by a phosphohexomutase,
possibly ValO.
As a valienol 1-phosphate nucleotidyltransferase, ValB repre-
sents the first member of the unsaturated carbasugar nucleotidyl-
transferase family involved in natural product biosynthesis. Other
members of this class of enzymes may include AcbR, GacR, and
SalF.11,14,27 Although AcbR, GacR, and SalF have been annotated
as putative 1-epi-valienol 1,7-diphosphate adenylyltransferases,
their natural substrate may very likely be the same as that of ValB.
Therefore, careful characterization of those enzymes is warranted
to reveal their actual role in acarbose and salbostatin biosynthesis.
Conclusion
The present study provides compelling evidence for the critical role
of ValB in validamycin biosynthesis. ValB catalyzes the conversion
of the unsaturated carbasugar phosphate valienol 1-phosphate
to NDP-valienol. This enzyme requires Mg2+, Mn2+, or Co2+ for
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Scheme 4 Biosynthetic pathway to NDP-valienol. Conversion of valienone 7-phosphate to valienol 1-phosphate requires stereoselective reduction of
C-1 ketone and transfer of the phosphate group.
its activity and utilizes the purine-based nucleotidyltriphosphates
ATP and GTP more efficiently than the pyrimidine-based NTPs
(CTP, dTTP, and UTP) as nucleotidyl donor. ValB represents the
first characterized unsaturated carbasugar nucleotidyltransferase
involved in natural products biosynthesis. Members of this
class of enzymes may include AcbR/GacR, and SalF from the
acarbose and the salbostatin pathways, respectively.11,14,27 Results
of the present study not only expand our understanding of the
biosynthesis of carbasugar-derived natural products, but may
also facilitate the development of new tools for chemoenzymatic
synthesis of carbohydrate mimetics.
residual solvent signals as the internal standard (CDCl3: dC
77.16), or with sodium 2,2-dimethylsilapentane-5-sulfonate (DSS)
(d 0.0) as an external standard. Phosphorus NMR spectra were
recorded on a Bruker 300 (112 MHz) spectrometer with complete
proton decoupling. Phosphorus chemical shifts are reported in
ppm (d) relative to an 85% H3PO4 (d 0.0) external standard.
Low-resolution electrospray ionization (ESI) mass spectra were
recorded on a ThermoFinnigan liquid chromatograph-ion trap
mass spectrometer, and High-resolution electrospray mass spectra
were recorded on Waters/Micromass LCT spectrometer. Ion-
exchange column chromatography was carried out on Dowex
1 ¥ 8 (Aldrich) and size exclusion chromatography was done on
Sephadex LH-20 (Pharmacia). Restriction enzymes, T4 DNA lig-
ase and Taq polymerase were purchased from various companies
(New England Biolabs, Takara, MBI Fermentas, and Toyobo).
Synthesis of oligonucleotide primers and DNA sequencing of PCR
products were performed by Shanghai Sangon and Invitrogen Co.,
Ltd. Gel Recovery Kit (Tiangen) was used for DNA purification
from agarose gels. TSBY liquid medium (per litre: TSB 30 g, yeast
extract 10 g, sucrose 103 g [pH 7.2]) was used for the growth
of mycelia and the isolation of total DNA. SFM medium (2%
agar, 2% mannitol, 2% soybean powder [pH 7.2]) was used for
sporulation and conjugation. YMG liquid medium (0.4% yeast
extract, 1% malt extract, 0.4% glucose [pH 7.3]) was used for
the fermentation of S. hygroscopicus 5008 and its derivatives. For
Streptomyces, apramycin and thiostrepton were used at 30 mg ml-1
and 25 mg ml-1, respectively, both in SFM agar and in liquid media.
E. coli strains were cultured as described elsewhere.29
Experimental
General
All chemical reactions were performed under an argon or nitrogen
atmosphere employing oven-dried glassware. Analytical thin-layer
chromatography (TLC) was performed using silica gel plates
˚
(60 A) with a fluorescent indicator (254 nm), which were visualized
with a UV lamp and ceric ammonium molybdate (CAM) solution.
Chromatographic purification of products was performed on silica
˚
gel (60 A, 72–230 mesh) column. Optical rotations were measured
on a Jasco P1010 polarimeter (100 mm cell was used) at the sodium
D line. Proton NMR spectra were recorded on Bruker 300 or
400 MHz spectrometers. Proton chemical shifts are reported in
ppm (d) relative to the residual solvent signals as the internal
standard (CDCl3: dH 7.26; D2O: dH 4.79). Multiplicities in the
1H NMR spectra are described as follows: s = singlet, br s =
broad singlet, d = doublet, br d = broad doublet, t = triplet, q =
quartet, m = multiplet; coupling constants are reported in Hz.
Carbon NMR spectra were recorded on Bruker 300 (75 MHz) or
400 (100 MHz) spectrometers with complete proton decoupling.
Carbon chemical shifts are reported in ppm (d) relative to the
Bacterial strains and plasmids
Bacterial strains and plasmids used in this study are listed in
Table S1.† S. hygroscopicus 5008, the wild-type producer of
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validamycin, was used for validamycin isolation, bioassay, and
generation of mutant strains. E. coli DH10B was used as the
cloning host, and E. coli BW25113 was used as the host for l-
Red-mediated recombination.30 pMD-T vector (Takara) was used
for the cloning of PCR amplified fragments. pHZ1358 (an E. coli
and Streptomyces shuttle vector) was used for gene replacement
in 5008.31 pJTU96832 was used as a transitional cloning vector.
pPM927 was used for mutant complementation.33
The extracts were loaded onto Agilent ZORBAX SB-C18 column
(5 mm, 4.6 ¥ 250 mm) for HPLC analysis (Waters 2690). The
mobile phase (5 mM sodium phosphate buffer–methanol, 98 : 2,
v/v) was applied with the flow rate of 0.6 mL min-1 at room
temperature. The eluate was monitored at 210 nm with Waters
996 photodiode array detector and the data was analyzed with a
Waters Millennium Chromatography Manager.
Synthesis of valienol 1-phosphate, 1-epi-valienol 1-phosphate,
valienol 1,7-diphosphate, and 1-epi-valienol 1,7-diphosphate
Construction of pJTU702 for targeted replacement of a 936 bp
DNA fragment internal to valB
Dibenzyl (1S,4R,5S,6R)-4,5,6-tris(benzyloxy)-3-[(benzyloxy)-
methyl]cyclohex-2-en-1-yl phosphate (9a). A solution of 8a
(740 mg, 1.38 mmol) in CH2Cl2 (5 mL) was added to a solution of
dibenzyl N,N-diisopropylphosphoramidite (944 mg, 2.76 mmol)
and 1H-tetrazole (290 mg, 4.14 mmol) in CH2Cl2 (25 mL), which
had been stirred for 40 min at room temperature under Ar,
and the reaction mixture was further stirred for 4 h at room
temperature. The reaction mixture was cooled to -78 ◦C and
m-CPBA (595 mg, 3.45 mmol) was added. After the cooling
bath was removed, stirring of the reaction mixture was continued
for an additional 40 min at room temperature. The solvent
was removed using a rotary evaporator, and the residue was
redissolved in EtOAc (150 mL). The organic layer was washed
with 10% aqueous Na2SO3 (100 mL), saturated aqueous NaHCO3
(100 mL), and brine (100 mL). The organic layer was dried
over MgSO4, filtered and concentrated under reduced vacuum.
Column chromatography (silica gel, Hex : EtOAc = 2 : 1) yielded
the title compound (963 mg, 88%); [a]2D3 +43.2 (c 1.0 in CHCl3);
dH(300 MHz; CDCl3) 3.62 (1 H, ddd, J 2.1, 3.3, 9.8), 3.92 (1 H,
d, J 13.3), 4.12 (1 H, dd, J 7.4, 9.7), 4.18 (2 H, bd, J 11.2), 4.46
(2 H, dd, J 11.9, 14.6), 4.64–4.68 (1 H, m), 4.70 (2 H, d, J 11.3),
4.83 (1 H, d, J 11.0), 4.88 (1 H, d, J 11.5), 4.96 (1 H, d, J 11.0),
4.99–5.11 (4 H, m), 5.18–5.23 (1 H, m), 5.89 (1 H, dd, J 1.1, 5.7),
7.23–7.41 (30 H, m); dC(75 MHz; CDCl3) 69.23 (d, Jc-p 4.8), 69.30
(d, Jc-p 4.8), 70.0, 71.2 (d, Jc-p 5.9), 72.4, 72.9, 74.6, 75.2, 78.3 (d, Jc-p
5.1), 79.8, 80.3, 121.3 (d, Jc-p 2.8), 127.74, 127.76, 127.79, 127.82,
127.85, 128.0, 128.31, 128.34, 128.46, 128.49, 128.55, 128.63, 136.1
(d, Jc-p 7.4), 136.2 (d, Jc-p 7.7), 138.01, 138.05, 138.5, 138.7, 143.1;
m/z (ESI) 819.3073 ([M + Na]+. C49H49O8PNa requires 819.3063).
A 5.8 kb BamHI fragment from pHZ2229 was cloned into
the BamHI site of pHZ1358 to generate pJTU700. Using the
gel-purified 1384-bp EcoRI/HindIII fragment from pIJ773
as template,30 a 1.4 kb disruption cassette was obtained
by PCR amplification with the primers ValB-PCR-F (5¢-
ATGGGGCCGCTGGGACGCGGCAGGCTCAAGCCGCTG-
GTGattccggggatccgtcgacc-3¢, nucleotides of pIJ773 are in
lowercase) and ValB-PCR-R (5¢- GATGGCCGACGCCAGGT-
GTGTTCCGTCCGGTACCTGGGCtgtaggctggagctgcttc
-3¢,
nucleotides of pIJ773 are in lowercase). Amplification was
performed with Taq DNA polymerase in a 50 ml reaction
with 100 ng template DNA, 10 mM dNTPs, 50 pmol each
primer, and 5% DMSO in a thermocycler (BioRad). After initial
denaturation at 94 ◦C for 2 min, 10 cycles were performed
with denaturation at 94 ◦C for 45 s, annealing at 50 ◦C for
45 s, and extension at 72 ◦C for 90 s, followed by 15 cycles
with the annealing temperature increased to 55 ◦C. A last
◦
elongation step was done at 72 C for 5 min. This fragment was
used to replace the 936 bp DNA internal to valB in pJTU700
through the l-Red-mediated recombination. The resulting
plasmid pJTU702 was used for targeted replacement of a 936 bp
DNA fragment internal to valB with the 1.4 kb oriT-aac(3)IV
cassette in the wild-type strain 5008. Oligonucleotide primers
ValB-Det-F (5¢-TGCTCCACCTGCCTGTA-3¢) and ValB-Det-R
(5¢-CACCTCTTCCCGCTCC-3¢) were used for the confirmation
of the valB mutant. PCR amplification was performed with
Taq DNA polymerase under similar conditions. The PCR
product was analyzed by gel electrophoresis and enzymatic
digestion.
Dibenzyl (1R,4R,5S,6R)-4,5,6-tris(benzyloxy)-3-[(benzyloxy)-
methyl]cyclohex-2-en-1-yl phosphate (9b). In a manner analo-
gous to the preparation of 9a from 8a, 8b gave 9b in 80% yield.
[a]2D4 -42.9 (c 1.0 in CHCl3); dH(300 MHz; CDCl3) 3.77–3.80 (2
H, m), 3.88 (1 H, d, J 12.4), 4.20 (1 H, d, J 12.5), 4.30 (1 H, d,
J 5.3), 4.46 (2 H, dd, J 4.9, 11.5), 4.71 (1 H, d, J 10.8), 4.79 (1
H, d, J 11.1), 4.83 (2 H, d, J 10.8), 4.88 (1 H, d, J 11.0), 4.95 (1
H, d, J 10.8), 4.97–5.03 (4 H, m), 5.07 (1 H, m), 7.20–7.35 (30 H,
m); dC(75 MHz; CDCl3) 69.36 (d, Jc-p 5.7), 69.45 (d, Jc-p 5.7), 72.6,
75.1, 75.7, 78.87 (d, Jc-p 6.3), 79.7, 82.67 (d, Jc-p 6.5), 83.95 (d, Jc-p
1.7), 124.1, 127.7, 127.8, 127.9, 127.9, 128.0, 128.1, 128.4, 128.5,
128.6, 128.7, 135.88 (d, Jc-p 2.0), 135.97 (d, Jc-p 2.1), 138.1, 138.37,
138.41, 138.44; m/z (ESI) 819.3120 ([M + Na]+. C49H49O8PNa
requires 819.3063).
Construction of pJTU918 for the complementation of ZYR-2 with
full-length cloned valB
A 1.1-kb NdeI/EcoRI fragment from the expression plas-
mid pJTU707 harboring a full-length valB was ligated to the
NdeI/EcoRI-digested pJTU968 to give pJTU915. Then the 1.4 kb
MfeI/EcoRI fragment, containing PermE* promoter and valB,
was cleaved from pJTU915 and ligated into EcoRI-digested
pPM927 to give pJTU918. The plasmid was introduced into ZYR-
2 through conjugation as previously described.18 The thiostrepton-
resistant exconjugants were selected.
HPLC analysis of validamycin A
For HPLC analysis, the strains were cultured in 50 mL YMG
liquid medium in 250 mL baffled flasks at 37 ◦C and 220 rpm for
6 days. The fermentation broth was acidified with oxalic acid and
applied to a Dowex 50 W column (25 mL) as previously reported.34
Valienol 1-phosphate (1a). BBr3 (195 mL of 1.0 M solution
in CH2Cl2, 0.195 mmol) was added to a solution of 9a (21 mg,
0.0263 mmol) in CH2Cl2, and the reaction mixture was stirred
◦
for 45 min at -35 C. Water (0.7 mL) and MeOH (0.7 mL) were
446 | Org. Biomol. Chem., 2011, 9, 438–449
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added to the reaction mixture at the same temperature, and the
pH of the solution was adjusted to weak basic using saturated
aqueous NaHCO3 solution. The reaction mixture was diluted
with H2O (3 mL) and CH2Cl2 (3 mL), and the aqueous layer
was further extracted with CH2Cl2 (3 mL). The aqueous layer was
subjected to a Dowex 1 ¥ 8 anion-exchange column (Cl-, 1.5 ¥
7.5 cm), washed with H2O (30 mL), and the product was eluted
with a NaCl gradient (30 mL of 0.1 M, 0.2 M, 0.3 M, and 0.4
M solution). The product fractions were pooled and lyophilized.
The resulting white solid was dissolved in H2O and desalted by
passing it through a Sephadex LH-20 column using H2O as an
eluent. The product fractions were combined and lyophilized to
give the title compound (4.9 mg, 62%); [a]2D0 +62.3 (c 0.13 in H2O);
dH(300 MHz; D2O) 3.56 (1 H, dd, J 3.8, 9.9), 3.80 (1 H, dd, J 7.8,
10.5), 4.02 (1 H, d, J 8.1), 4.12 (1 H, d, J 14.4), 4.20 (1 H, d, J
13.8), 4.66 (1 H, m), 5.91 (1 H, d, J 4.5); dC(75 MHz; D2O; DSS)
64.0, 72.2 (d, Jc-p 4.9), 73.3 (d, Jc-p 4.1), 74.8, 75.8, 124.6 (d, Jc-p
2.5), 144.7; dP(122 MHz; D2O; H3PO4) 0.27; m/z (ESI) 255.0264
([M - H]-. C7H12O8P requires 255.0270).
0.367 mmol) and 1H-tetrazole (45 mg, 0.643 mmol) in CH2Cl2
(4 mL), which had been stirred for 40 min at room temperature
under Ar, and the reaction mixture was further stirred for 4 h at
room temperature. The reaction mixture was cooled to -78 ◦C and
m-CPBA (79 mg, 0.456 mmol) was added. After the cooling bath
was removed, stirring of the reaction mixture was continued for an
additional 40 min at room temperature. The solvent was removed
using a rotary evaporator, and the residue was redissolved in
EtOAc (15 mL). The organic layer was washed with 10% aqueous
Na2SO3 (10 mL), saturated aqueous NaHCO3 (10 mL), and brine
(10 mL). The organic layer was dried over MgSO4, filtered and
concentrated under reduced vacuum. After two successive column
chromatography (silica gel, Hex : EtOAc = 1 : 2 and toluene–
EtOAc = 3 : 1) the title compound was obtained (58.1 mg, 66%);
[a]2D3 +26.0 (c 1.0 in CHCl3); dH(300 MHz; CDCl3) 3.47–3.52 (1 H,
m), 3.99–4.07 (2 H, m), 4.40 (1 h, dd, J 6.3, 12.6), 4.53–4.61 (3 H,
m), 4.64 (1 H, d, J 11.1), 4.77 (1 H, d, J 10.8), 4.82 (1 H, d, J 11.7),
4.91 (1 H, d, J 11.1), 4.94–5.04 (8 H, m), 5.04–5.09 (1 H, m), 5.74 (1
h, d, J 5.4), 7.20–7.39 (35 H, m); dC(75 MHz; CDCl3) 67.0 (d, Jc-p
5.3), 69.30 (d, Jc-p 5.8), 69.36 (d, Jc-p 6.1), 69.51 (d, Jc-p 5.6), 69.56 (d,
Jc-p 5.5), 70.8 (d, Jc-p 6.2), 72.5, 74.7, 75.1, 78.1 (d, Jc-p 4.8), 78.8,
80.0, 122.5, 127.75, 127.79, 128.0, 128.06, 128.11, 128.3, 128.4,
128.49, 128.53, 128.58, 128.69, 128.76, 135.79, 135.88, 135.94,
136.04, 136.10, 136.20, 138.0, 138.2, 138.6, 140.5, 140.6; m/z (ESI)
989.3180 ([M + Na]+. C56H56O11P2Na requires 989.3196).
1-epi-Valienol 1-phosphate (1b). In a manner analogous to the
preparation of 1a from 9a, 9b gave 1b in 41% yield. [a]2D0 -50.4
(c 1.0 in H2O); dH(300 MHz; D2O) 3.55–3.60 (2 H, m), 4.08 (1
H, d, J 14.1), 4.17–4.21 (2 H, m), 4.59 (1 H, m), 5.70 (1 H, s);
dC(75 MHz; D2O; DSS) 63.8, 74.4, 77.1 (d, Jc-p 4.0), 78.1, 78.3 (d,
J
c-p 4.9), 126.6 (d, Jc-p 3.6), 141.5; dP(122 MHz; D2O; H3PO4) 2.85;
m/z (ESI) 255.0268 ([M - H]-. C7H12O8P requires 255.0270).
(1R,4R,5S,6S)-4,5,6-Tris(benzyloxy)-3-(hydroxymethyl)cycloh-
ex-2-en-1-ol (10b). In a manner analogous to the preparation of
10a from 8a, 8b gave 10b in 78% yield. [a]2D0 -77.6 (c 1.0 in CHCl3);
dH(300 MHz; CDCl3) 1.90 (1 H, t, J 6.3), 2.14 (1 H, d, J 4.2), 3.55
(1 H, dd, J 7.2, 9.9), 3.86 (1 H, dd, J 7.2, 9.9), 4.08 (2 H, d, J 5.7),
4.33 (2 h, m), 4.72 (1 H, d, J 11.1), 4.73 (1 H, d, J 11.4), 4.82 (1
H, d, J 11.1), 4.88 (1 H, d, J 11.1), 4.98 (2 H, d, J 11.1), 5.67 (1
H, s), 7.28–7.38 (15 H, m); dC(75 MHz; CDCl3) 64.0, 71.5, 74.8,
75.2, 75.3, 80.5, 83.8, 84.2, 126.7, 127.97, 128.04, 128.13, 128.16,
128.19, 128.3, 128.67, 128.75, 128.83, 138.1, 138.29, 138.34, 138.5;
m/z (ESI) 469.2010 ([M + Na]+. C28H30O5Na requires 469.1991).
(1S,4R,5S,6S)-4,5,6-Tris(benzyloxy)-3-(hydroxymethyl)cycloh-
ex-2-en-1-ol (10a). ZnCl2 (240 mg, 1.84 mmol) was added to a
solution of 8a (99 mg, 0.184 mmol) in Ac2O–AcOH (2 : 1; 2 mL),
and the reaction mixture was stirred for 2 h at room temperature.
The reaction mixture was diluted with water (5 mL) and EtOAc
(10 mL), and the organic layer further washed with water (5 mL)
and saturated aqueous Na2CO3 solution (3 ¥ 5 mL). The organic
layer was dried over MgSO4, filtered and concentrated under
reduced vacuum to give a crude sample. The crude sample was
dissolved in MeOH (2.5 mL) and NaOMe (0.8 mL of 30%
solution in MeOH) was added to the reaction mixture. The
reaction mixture was stirred for 4 h at room temperature and
quenched with water (2 mL) and saturated aqueous NH4Cl
(10 mL). The reaction mixture was diluted with EtOAc (5 mL),
and the organic layer further washed with water (5 mL) and brine
(5 mL). The organic layer was dried over MgSO4, filtered and
concentrated under reduced vacuum. Column chromatography
(silica gel, Hex : EtOAc = 1 : 2) yielded the title compound (51 mg,
62%); [a]2D4 +2.3 (c 0.23 in CHCl3); dH(300 MHz; CDCl3) 1.73
(1 H, t, J 6.3), 2.58 (1 H, d, J 3.3), 3.60 (1 H, dd, J 3.9, 9.2),
4.05–4.13 (3 H, m), 4.16 (1 H, d, J 6.9), 4.30 (1 H, q, J 4.2), 4.67
(1 H, d, J 11.4), 4.69 (1 H, d, J 11.7), 4.75 (1 H, d, J 11.1), 4.80 (1
H, d, J 11.4), 4.83 (1 H, d, J 11.1), 4.92 (1 h, d, J 11.1 Hz), 5.85 (1
H, dd, J 1.5, 4.8), 7.29–7.39 (15 H, m); dC(75 MHz; CDCl3) 64.0,
65.2, 73.1, 74.2, 74.9, 79.1, 79.28, 79.33, 124.1, 127.90, 128.1,
128.2, 128.6, 128.70, 128.72, 138.1, 138.3, 138.6, 141.7; m/z (ESI)
469.1978 ([M + Na]+. C28H30O5Na requires 469.1991).
Dibenzyl (1R,4R,5S,6R)-4,5,6-tris(benzyloxy)-3-(([bis(benzy-
loxy)phosphoryl]oxy)methyl)cyclohex-2-en-1-yl phosphate (11b).
In a manner analogous to the preparation of 11a from 10a, 10b
gave 11b in 70% yield. [a]D23 -33.0 (c 1.0 in CHCl3); dH(300 MHz;
CDCl3) 3.65–3.78 (2 H, m), 4.21 (1 H, bd, J 7.2), 4.41 (1 H,
dd, J 5.9, 12.6), 4.56 (1 H, d, J 8.4), 4.62 (1 H, d, J 10.8), 4.73
(1 H, d, J 11.1), 4.78 (1 H, d, J 11.1), 4.79 (1 H, d, J 11.4),
4.83 (1 H, d, J 11.4), 4.92 (2 H, d, J 12.0), 4.97–5.03 (8 H, m),
7.19–7.33 (35 H, m); dC(75 MHz; CDCl3) 69.35, 69.49, 69.53,
69.57, 69.61, 75.2, 75.5, 75.6, 78.4 (d, Jc-p 6.3), 78.7, 82.4 (d, Jc-p
6.4), 83.9, 125.3, 127.7, 127.86, 127.94, 127.96, 128.05, 128.06,
128.11, 128.45, 128.56, 128.59, 128.67, 128.72, 128.77, 135.8,
135.9, 136.4, 136.5, 138.1, 138.27, 138.31; m/z (ESI) 989.3163
([M + Na]+. C56H56O11P2Na requires 989.3196).
Valienol 1,7-diphosphate (2a). BBr3 (437 mL of 1.0 M solution
in CH2Cl2, 0.437 mmol) was added to a solution of 11a (47 mg,
0.049 mmol) in CH2Cl2, and the reaction mixture was stirred for
90 min at -50 ◦C. Water (1.0 mL) was added to the reaction
mixture at the same temperature, and the pH of the solution was
adjusted to weak basic using saturated aqueous NaHCO3 solution.
The reaction mixture was diluted with H2O (5 mL) and CH2Cl2
Dibenzyl (1S,4R,5S,6R)-4,5,6-tris(benzyloxy)-3-(([bis(benzoxy)-
phosphoryl]oxy)methyl)cyclohex-2-en-1-yl phosphate (11a). A so-
lution of 10a (41 mg, 0.92 mmol) in CH2Cl2 (2 mL) was added to
a solution of dibenzyl N,N-diisopropylphosphoramidite (125 mg,
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(5 mL), and the aqueous layer was further extracted with CH2Cl2
(5 mL). The aqueous layer was subjected to Dowex 1 ¥ 8 anion-
exchange column (Cl-, 2.5 ¥ 8.0 cm), washed with H2O (80 mL),
and the product was eluted with a NaCl gradient (80 mL of 0.1
M, 0.2 M, 0.3 M, and 0.4 M solution). The product fractions were
pooled and lyophilized. The resulting white solid was dissolved in
H2O and desalted by passing it through a Sephadex LH-20 column
using H2O as an eluent. The product fractions were combined and
lyophilized to give the title compound (3.1 mg, 19%); [a]2D5 +57.3 (c
0.12 in H2O); dH(300 MHz; D2O) 3.76 (1 H, bd, J 10.8), 3.89–3.96
(1 H, m), 4.21 (1 H, d, J 7.8), 4.49 (1 H, dd, J 12.9), 4.63 (1 H,
dd, J 13.2, 10.2), 4.85 (1 H, m), 6.12 (1 H, d, J 4.2); dC(75 MHz;
D2O; DSS) 67.5 (d, Jc-p 4.7), 72.8 (d, Jc-p 6.2), 73.1 (d, Jc-p 5.5),
74.4, 75.3, 125.3, 143.0 (d, Jc-p 6.4); dP(122 MHz; D2O; H3PO4)
1.22, 1.23; m/z (ESI) 334.9946 ([M - H]-. C7H13O11P2 requires
334.9933).
dialyzed for 14 h against 1 litre of dialysis buffer (60 mM Tris-
HCl pH 8, 10 mM MgCl2, and 0.05 mM DTT).
Enzymatic assays. The ValB enzymatic reaction was assayed
by measurement of inorganic pyrophosphate (PPi) production,
which was detected by the coupled colorimetric assay using
the EnzChek Pyrophosphate Assay Kit (Molecular Probes).
The purified ValB protein solution (20 mL, 0.35 mg mL-1) was
incubated with different valienol phosphates (0.2 mM), NTPs
(0.2 mM), MgSO4 (0.2 mL), and Tris·HCl pH 7.5 buffer (25 mL) in
a 50 mL total volume for 30 min. ValB enzymatic reaction solution
was added to the EnzChek solution (150 mL) in a 96-well plate.
The plate was then immediately placed into a spectrophotometric
microplate reader and incubated for 30 min at 22 ◦C. The change in
absorbance at 360 nm was obtained in triplicate after subtracting
the background absorbance, and Pi contamination in valienol
phosphates and NTPs.
Enzymatic assays for electrospray mass spectroscopy and TLC
analyses were carried out using ten times concentrations of
substrates and metal ions. The mixtures were incubated for 1 h
at 30 ◦C and the products were checked by TLC (254 nm) and
mass spectroscopy (ESI, negative ion mode).
1-epi-Valienol 1,7-diphosphate (2b). In a manner analogous to
the preparation of 2a from 11a, 11b gave 2b in 53% yield. [a]D25
-39.1 (c 0.07 in H2O); dH(300 MHz; D2O) 3.60–3.72 (2 H, m), 4.38
(1 H, dd, J 6.6, 13.8), 4.51–4.57 (1 H, m), 4.67 (1 H, m), 5.86 (1
H, s); dC(75 MHz; D2O; DSS) d 67.2 (d, Jc-p 4.6), 74.0, 77.3 (d, Jc-p
3.8), 77.9, 78.3 (d, Jc-p 5.0), 128.1 (d, Jc-p 4.4), 138.9 (d, Jc-p 6.3);
dP(122 MHz; D2O; H3PO4) 0.96, 1.38; m/z (ESI) 334.9928 ([M -
H]-. C7H13O11P2 requires 334.9933).
Acknowledgements
The project described was supported by Award Number
R01AI061528 from the National Institute of Allergy and Infec-
tious Diseases and the Herman Frasch Foundation. The content
is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institute of Allergy and
Infectious Diseases or the National Institutes of Health. Work at
Shanghai Jiaotong University was supported by grants from the
973 and 863 Programs of the Ministry of Science and Technology,
the Natural Science Foundation of China, and Shanghai Leading
Academic Discipline Project B203. HX was in part supported by
the exchange program from China Scholarship Council and the
U.S. National Institutes of Health.
Cloning and expression of the valB gene in E. coli. The
1.1-kb valB gene was amplified by PCR (primers: ValB-F, 5¢-
GGATCCACATATGGACGGAGTGCGTGCC-3¢, ValB-R, 5¢-
¯
¯
¯
GAATTCACAGCGCCACCTCTTCCCGCTC-3¢)and ligated to
¯ ¯
pMD T vector (Takara) and sequenced. The confirmed gene
was cut out with BamHI and EcoRI and inserted into pRSET
B expression vector (Invitrogen). The valB plasmid DNA was
introduced into E. coli BL21 Gold(DE3)/pLysS by heat-pulse
transformation. The mixture was then plated onto LB agar plates
containing ampicillin (100 mg mL-1) and chloramphenicol (25
◦
mg mL-1), and incubated at 37 C for overnight. Single colonies
were transferred into test tubes containing LB medium with the
above antibiotics and incubated on a rotary shaker (220 rpm)
at 37 ◦C for overnight. Seed cultures in LB medium (10 mL)
containing the antibiotics were inoculated with 100 mL of the
test tube culture and were grown at 37 ◦C for overnight. The
production cultures in LBBS medium (50 mL) containing the
antibiotics were inoculated with seed cultures (5 mL), and were
incubated at 37 ◦C to an OD600 of 0.60. Isopropyl b-D-1-
thiogalactopyranoside (IPTG) (0.2 mM) was then added, and the
incubation was continued at 28 ◦C to an OD600 of 1.04. The
cells were harvested by centrifugation at 5000 rpm for 15 min and
stored frozen at -80 ◦C until further use.
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