Cloning and in vitro characterization of dTDP-6-deoxy-l-talose biosynthetic genes from Kitasatospora kifunensis featuring the dTDP-6-deoxy-l-lyxo-4- hexulose reductase that synthesizes dTDP-6-deoxy-l-talose

Article abstract of DOI:10.1016/j.carres.2010.07.004

Kitasatospora kifunensis, the talosin producer, was used as a source for the dTDP-6-deoxy-l-talose (dTDP-6dTal) biosynthetic gene cluster, serving as a template for four recombinant proteins of RmlA_Kkf, RmlB _Kkf, RmlC_Kkf, and Tal, which complete the biosynthesis of dTDP-6dTal from dTTP, α-d-glucose-1-phosphate, and NAD(P)H. The identity of dTDP-6dTal was validated using ¹H and ¹³C NMR spectroscopy. K. kifunensis tal and tll, the known dTDP-6dTal synthase gene of Actinobacillus actinomycetemcomitans origin, have low sequence similarity and are distantly related within the NDP-6-deoxy-4-ketohexose reductase family, providing an example of the genetic diversity within the dTDP-6dTal biosynthetic pathway.

Full text of DOI:10.1016/j.carres.2010.07.004

Carbohydrate Research 345 (2010) 1958–1962
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Cloning and in vitro characterization of dTDP-6-deoxy-L-talose biosynthetic
genes from Kitasatospora kifunensis featuring the dTDP-6-deoxy-L-lyxo-4-hex-
ulose reductase that synthesizes dTDP-6-deoxy-L-talose
*
Suman Karki, Hye-Gyeong Yoo, So-Yeon Kwon, Joo-Won Suh, Hyung-Jin Kwon
Department of Biological Science, Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Kitasatospora kifunensis, the talosin producer, was used as a source for the dTDP-6-deoxy-L-talose (dTDP-
Received 11 May 2010
Received in revised form 25 June 2010
Accepted 4 July 2010
6dTal) biosynthetic gene cluster, serving as a template for four recombinant proteins of RmlAKkf, RmlBKkf
,
RmlCKkf, and Tal, which complete the biosynthesis of dTDP-6dTal from dTTP, -glucose-1-phosphate,
a
-D
and NAD(P)H. The identity of dTDP-6dTal was validated using 1H and 13C NMR spectroscopy. K. kifunensis
tal and tll, the known dTDP-6dTal synthase gene of Actinobacillus actinomycetemcomitans origin, have low
sequence similarity and are distantly related within the NDP-6-deoxy-4-ketohexose reductase family,
providing an example of the genetic diversity within the dTDP-6dTal biosynthetic pathway.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
dTDP-6-Deoxy-L-talose
Kitastospora kifunensis
Biosynthetic gene cluster
dTDP-6-Deoxy-L-lyxo-4-hexulose reductase
6-Deoxyhexoses are well-known components of cell surface
glycans and secondary metabolites in bacteria.1,2 6-Deoxyhexoses
are biosynthesized in the form of nucleotide diphosphate (NDP)-
6-deoxyhexoses, exemplified in the biosynthesis of thymidine
sugar 4-epimerases in addition to RmlABCD.7 It was proposed that
one of these epimerases synthesizes dTDP-6dTal from dTDP-Rha.
Capsular polysaccharides of Actinobacillus actinomycetemcomi-
tans serotype
whereas 6-deoxy-
c
contain 6-deoxy-
D
L
-talan, the 6dTal polymer,
-talan
diphosphate (dTDP)-
synthesized through a series of reactions involving glucose-1-
phosphate thymidylyltransferase (RmlA), dTDP- -glucose 4,6-
dehydratase (RmlB), dTDP-6-deoxy- -xylo-4-hexulose 3,5-epimer-
ase (RmlC), and dTDP-6-deoxy- -lyxo-4-hexulose reductase (RmlD)
L-rhamnose. dTDP-L-rhamnose (dTDP-Rha) is
-talan is found in serotype a.8 6-Deoxy-
L
has also been characterized in lipopolysaccharides of Rhizobium
D
loti9 and capsular polysaccharides of Agrobacterium rubi.10 Cloning
D
of the 6-deoxy-L-talan biosynthetic gene cluster from A. actinomy-
cetemcomitans and its subsequent biochemical characterization re-
L
(Fig. 1). The committed step in the synthesis of dTDP-6-deoxyhex-
ose pathway is the RmlB reaction, and diverse dTDP-6-deoxyhex-
ose structures are generated by enzymatic modifications of the
vealed that dTDP-Rha and dTDP-6dTal are synthesized through
stereospecific 4-keto-reductions of dTDP-6-deoxy-L-lyxo-4-hexu-
lose mediated by RmlD and Tll, respectively (Fig. 1).11–13 6dTal is
also found in the surface glycopeptidolipid of Mycobacterium avium
and other Mycobacterium strains.14,15 However, a homology search
failed to reveal a tll ortholog from genomes of these strains, which
implies genetic diversity within the bacterial dTDP-6dTal biosyn-
thetic pathway. We cloned and characterized the dTDP-6dTal bio-
synthetic genes from Kitasatospora kifunensis, a rare actinomycetes,
to investigate the genetic diversity within this pathway.
product of this reaction, dTDP-6-deoxy-
6-Deoxy- -talose (6dTal) is component of the bacterial
D
-xylo-4-hexulose.3
L
a
lipopolysaccharides and capsular polysaccharides. Based on simi-
larity to the dTDP-Rha pathway, the dTDP-6dTal pathway was
hypothesized to involve either dTDP-Rha 4-epimerase activity or
dTDP-6-deoxy-
specificity that opposes that of RmlD (Fig. 1). Early cell-free exper-
iments in Escherichia coli O45 demonstrated that pair of
L-lyxo-4-hexulose reductase activity with a stereo-
a
K. kifunensis produces talosins, the genistein glycosides of
6dTal.16 It has been proposed that talosin is produced from exoge-
nous genistein in soybean meal, which is included in fermentation
medium, and that K. kifunensis has the potential to generate dTDP-
6dTal. Through PCR targeted to rmlB, we were able to clone a
9605 bp region surrounding this gene from K. kifunenesis (Fig. 2).
stereospecific 4-keto-reductions led to the formation of dTDP-
Rha and dTDP-6dTal.4 The O-antigenic polysaccharide (O-PS)
moieties of lipopolysaccharides from E. coli and Burkholderia
pseudomallei contain 6dTal5,6 and the cognate biosynthetic gene
cluster of B. pseudomallei was found to encode three nucleotide
There are 11 predicted ORFs within this region, including rmlAKkf
,
rmlBKkf, and rmlCKkf (ORF5, 6, and 7, respectively). The products of
ORF5, 6, and 7 show 76%, 62%, and 66% similarities, respectively,
* Corresponding author. Tel.: +82 31 330 6470; fax: +82 31 335 8249.
E-mail address: hjink@mju.ac.kr (H.-J. Kwon).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.07.004

S. Karki et al. / Carbohydrate Research 345 (2010) 1958–1962
1959
talosyl residue at O-2 position in O-PS of B. pseudomallei.20 Overall,
the homology analysis suggests that the region we cloned belongs
to the biosynthetic gene cluster of the extracellular glycan that
contains 6dTal or Rha.
To examine whether RmlAKkf, RmlBKkf, RmlCKkf, and Tal consti-
tute the dTDP-6dTal biosynthetic pathway, these proteins were
over-expressed and purified as hexa-histidine fusions (Supplemen-
tary data). When RmlAKkf was incubated with 1 mM thymidine tri-
α−D-glucose-1-phosphate + dTTP
RmlA
OH
O
HO
HO
dTDP-D-glucose
OH
O dTDP
phophate (dTTP) and 5 mM
quantitatively converted into a new compound, which we assigned
as dTDP- -glucose (Fig. 3a and b). When RmlBKkf was added to the
RmlAKkf reaction mixture, the dTDP- -glucose peak was reduced
and a new broad peak was observed (Fig. 3c). In the HPLC condi-
tions used, dTDP-4-keto-6-deoxy- -glucose produces broad
peak,12 suggesting that the RmlAKkf/RmlBKkf reaction successfully
generated dTDP-4-keto-6-deoxy- -glucose. When RmlCKkf and Tal
a-D-glucose-1-phosphate, dTTP was
RmlB
O
H3C
O
D
D
dTDP-6-deoxy-D-glucose-
xylo-4-hexulose
HO
OH
D
a
O
dTDP
RmlC
D
H3C
were added to the RmlAKkf/RmlBKkf reaction mixture together with
either nicotinamide adenine dinucleotide or nicotinamide adenine
dinucleotide phosphate, the formation of a new product was ob-
served at 50 min in both cases (Fig. 3d and e). We determined
the structure of this product using NMR spectroscopy.
Ten milliliters of the RmlAKkf/RmlBKkf/RmlBKkf/Tal reaction sam-
ple were processed by HPLC and subsequently used for NMR mea-
surements (Fig. 4). The 1H and 13C NMR spectral assignments are
presented in Table 1. The 1H NMR data are in good agreement with
the previously reported data of dTDP-6-dTal,12 indicating that Tal
has the same catalytic role as Tll (Table 1). Small coupling con-
stants for H-300 and H-400 splitting, which were calculated as ꢀ3.0
and 3.1 Hz, respectively, are indicative of dTDP-6dTal. H-600 ap-
pears to be correlated with H-400 and H-500 in the NOESY spectra,
O
dTDP-6-deoxy-L-glucose-
O dTDP
lyxo-4-hexulose
O
HO
OH
RmlD
H3C
Tll or Tal
O
H3C
HO
HO
O
O dTDP
O dTDP
HO
HO
epimerase ?
OH
OH
dTDP-6-deoxy-L-talose
dTDP-L-rhamnose
Figure 1. Biosynthetic pathways of both dTDP-
L
-rhamnose and dTDP-6-deoxy-L-
talose (dTDP-6dTal). The formation of dTDP-6dTal by Tll reductase was previously
described12 while the Tal-mediated formation of dTDP-6dTal was validated in this
study. The epimerization reaction is speculative and lacks experimental support.
to the dTDP-Rha biosynthetic enzymes of Mycobacterium tuberculo-
sis H37Rv. In addition, this region contains ORFs for glycosyltrans-
ferases (ORF1, ORF8, and ORF11, which is N-terminally truncated),
an NDP-hexose 4-ketoreductase (ORF9), an O-antigen acetyltrans-
ferase (ORF10), and four hypothetical proteins. The product of
ORF9 (named tal) is similar (percentage similarity, amino acids
that overlap) to the NDP-4-keto-6-deoxyhexose reductases of
Streptomyces, including those of med-ORF14 (57%, 317),17 hedN
(57%, 297),18 and dnmV (50%, 314),19 while showing low similarity
to Tll (37%, 265) and RmlD proteins (37%, 222, with StrL from Strep-
tomyces griseus). The predicted functions of Med-ORF14 and HedN
are dTDP-3-amino-4-keto-2,3,6-trideoxy-
tase in the biosynthesis of dTDP-
-angolosamine.17,18 DnmV is
dTDP-3-amino-4-keto-2,3,6-trideoxy- -glucose 4-ketoreductase in
dTDP-
-daunosamine biosynthesis.19 Notably, these enzymes dis-
play the same stereospecificity as Tll in the 4-keto-reduction
although their substrates differ in - and -configurations. ORF10
D-glucose 4-ketoreduc-
D
L
Figure 3. HPLC profiles of the enzyme reactions for dTDP-6-deoxy-
synthesis. A: authentic dTTP. B: RmlAKkf with dTTP and -glucose-1-phosphate.
C: RmlAKkf and RmlBKkf with dTTP and -glucose-1-phosphate. D: RmlAKkf
RmlBKkf RmlCKkf and Tal with dTTP, -glucose-1-phosphate, and NADH. E:
RmlAKkf RmlBKkf RmlCKkf and Tal with dTTP, -glucose-1-phosphate, and
NADPH. closed circle: dTTP, open circles: dTDP- -glucose, closed triangles:
dTDP-4-keto-6-deoxy- -glucose, open triangles: dTDP-6-deoxy- -talose, an open
rectangle: NAD+, a closed rectangle: NADP+.
L-talose
L
a-D
a
a-D
-
D
,
,
,
,
,
D
L
,
a-D
D
is homologous to wbiA (53%, 346), which is a member of the O-
A
PS biosynthetic gene cluster from B. pseudomallei.7 The wbiA locus
D
L
was shown to be responsible for the acetylation of the 6-deoxy-L-
Figure 2. Restriction map and gene organization of the 9605-bp DNA region isolated from Kitasatospora kifunensis. Coverage of the two sequenced plasmid clones is shown.
Deduced function of each gene product is provided below the map.

1960
S. Karki et al. / Carbohydrate Research 345 (2010) 1958–1962
Figure 4. 1H NMR spectra of dTDP-6-deoxy-
L
-talose from the RmlAKkf/RmlBKkf/RmlCKkf/Tal reaction in the presence of dTTP, a-D-glucose-1-phosphate, and NADH. (A) The
spectra were obtained in pure D2O. Panel B is an expansion of the H-200–H-500 region.
further supporting the identity of dTDP-6dTal (Supplementary
data).
CSGGSGSSGCSGGSTTCATCGG-30
(forward)
and
50-
GGGWRCTGGYRSGGSCCSTAGTTG-30 (reverse).22 A distinct product
of the predicted size (0.55 kb) was amplified and cloned into
pGEM-T vector. The rmlB fragment was used as a probe in Southern
blot experiment with K. kifunensis genomic DNA which was di-
In conclusion, Tal performs the same chemical reaction as Tll in
the biosynthesis of dTDP-6dTal despite the fact that these proteins
share a low similarity. We speculate that Tal and Tll took separate
evolutionary tracks that functionally converged. Phylogenetic anal-
ysis supports the idea that Tal and Tll belong to discrete phyloge-
netic clades (Fig. 5). The absence of a tll homolog in the O-PS
biosynthetic gene cluster from B. pseudomallei7 led to the proposal
that B. pseudomallei synthesizes dTDP-6dTal through C00-4 epimer-
ization of dTDP-Rha, while A. actinomycetemcomitans utilizes Tll
activity for that purpose.12 Although any of the three putative
epimerases (WbiB, WbiG, or WbiI) predicted in the O-PS biosyn-
thetic gene cluster does not show a strong homology to Tal, phylo-
genetic analysis indicates that these putative epimerases are more
Table 1
NMR spectroscopy identification of dTDP-6-deoxy-L-talose from the RmlA/RmlB/
RmlC/Tal reaction
O
7
CH3
3
HN
1
6
O
N
6"
H3C
5"
O-
O
O
O-
1"
5'
closely related to Tal than UDP-
shown). Our results highlight a possibility that the dTDP-6-
deoxy- -lyxo-4-hexulose reductase gene involved in the biosynthe-
D-glucose 4-epimerases (data not
O
P
P
O
4"
O
O
O
1'
4'
HO
L
HO
OH
dc
sis of dTDP-6dTal is encoded in the O-PS biosynthetic gene cluster
of B. pseudomallei. As such, our results provide new insight into the
analysis of the glycan biosynthetic genes of bacterial genomes.
OH
Atom
dH (number of H, splittinga, J)
2
4
5
6
153.5
168.2
113.4
139.0
13.5
86.6
40.2
72.6
86.7
67.4
97.7
71.8
69.8
72.0
74.0
17.3
1. Experimental
7.71 (1H, s)
1.93 (3H, s)
6.35 (1H, m, Jd = 6.7)
2.38 (2H, m, Jd = 5.8)
4.60 (1H, m)
7
1.1. Bacterial strains
10
20
30
40
50
100
200
300
400
500
600
K. kifunensis MJM341 was a provision of Extract Collection of
Useful Microorganisms (ECUM) at Myongji University, Yongin, Kor-
ea. K. kifunensis was maintained in Bennett agar and cultured in
TSB (10 mM MgCl2, 0.5% glycine) for total DNA isolation. E. coli
4.19 (1H, m)
4.19 (1H, m), 4.15 (1H, m)
5.14 (1H, d, J = 8.8)
4.02 (1H, d, J = 2.5)
3.82 (1H, dd, J1 = ꢀ3.0, J2 = ꢀ3.0)
3.68 (1H, d, J = 3.1)
3.74 (1H, m, J1b = ꢀ6.4)
1.29 (3H, d, J = 6.5)
DH5a was used for general subcloning.
1.2. Gene cloning procedures
a
Degenerate PCR primers were used to clone an internal frag-
Observed splitting pattern.
Coupling constant for quartet.
ment of rmlB from K. kifunenesis. The primer pairs are: 50-
b

S. Karki et al. / Carbohydrate Research 345 (2010) 1958–1962
1961
GenBank
accession number no.
Protein
Amino acids
Source organism
Sequence source
RmlD_E. coli
299
301
Escherichia coli
Biochemically characterized
Genome search
ADB02812
ABY97288
RmlD_P. putida
Pseudomonas putida
Streptomycin biosynthetic gene
cluster (strL), function predicted
RmlD_S. griseus
304
307
Streptomyces griseus
CAA44443
AAM94769
Calicheamicin biosynthetic gene
cluster (calS2), function predicted
RmlD_M. echinospora
Micromonospora echinospora
Actinobacillus
actinomycetecomitans
6-Deoxy-L-talan biosynthetic
gene cluster11,12
Tll_A. actinomycetecomitans
270
BAA28138
Tll_L. goodfellowii
Tll_A. putredinis
279
279
Leptotrichia goodfellowii
Alistipes putredinis
Genome search
Genome search
EEY35364
EDS02198
Bifidobacterium
pseudocatenulatum
Tll_B. pseudocatenulatum
KR_S. griaeoruber
KR_S. peucetius
288
321
307
325
Genome search
EEG70112
AAP85346
AAB63047
BAC79033
Hedamycin biosynthetic gene
cluster (hedN), function
predicted18
Streptomyces griaeoruber
Streptomyces peucetius
Streptomyces sp. AM-7161
Daunorubicin biosynthetic gene
cluster (dnmV), genetic evidence19
Medermycin biosynthetic gene
cluster (med-orf14), function
predicted17
KR_S. sp. AM-7161
Unknown polyketide biosynthetic
gene cluster
KR_S. erythermus
Tal_K. kifunensis
333
318
Streptomyces erythermus
Kitasatospora kifunensis
ABV49596
HM132058
This study
Figure 5. Phylogenetic analysis of NDP-4-keto-6-deoxyhexose reductase proteins (including Tll and Tal). Branch length is proportional to the number of substitutions per
site. The branch support values are shown. Identities of the proteins are given in the table below. Phylogenetic tree was generated in the Phylogeny.fr platform.21 The
phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PHYML program (v3.0 aLRT).
gested with BamHI and NheI. The Southern blot gave rise to a
hybridization signal at 6-kb region in agarose gel separation. The
6-kb region was purified and used to construct a mini-library in
pGEM-11zf. Three hundred colonies were screened with colony
blot analysis, giving rise to pHJK-2001. A 4.6 kb NotI fragment,
which overlapped with the 6-kb BamHI/NheI fragment, was also
isolated through genomic Southern blot and cloned into pBlue-
script-SK, giving rise to pHJK-2002. pHJK-2001 and pHJK-2002
were sequenced and the resulting sequence was deposited in Gen-
Bank (http://www.ncbi.nlm.nih.gov/Genbank) under the accession
number HM132058. Southern hybridization was performed with
the digoxigenin-labeled DNA following the manufacturer’s proce-
dure (Roche Diagnostics, Pleasanton, CA).
underlined) are 50-attcatATGCGTGGAATCCTCTTGGCC-30 and 50-at-
taagcttTCACTTGGCCGCCTCCTC-30 (rmlAKkf), 50-attcatATGCGCATCC
TCGTCACCGGC-30
and 50-attaagcttTCATCGCAGCGCGGCCTT-30
(rmlBKkf), 50-attcatATGAAGTTCCGCGAACTGTCC-30 and 50-attaag
cttTCAGTTGGTGCGCAGGCC-30 (rmlCKkf), 50-attcatATGACCGCTCCG-
CACACGGCA-30 and 50-attaagcttTTATGCGACCGGCGCGGT-30 (tal).
PCR products were digested with NdeI and HindIII and cloned into
the same sites of pUC18. From the resulting plasmids, the inserts
were recovered as NdeI and HindIII fragments and ligated into
pET-28b, giving rise to pHJK-2011 (rmlAKkf), pHJK-2012 (rmlBKkf),
pHJK-2013 (rmlCKkf), and pHJK-2014 (tal). Each plasmid was trans-
formed into E. coli BL21 (DE3). Overnight cultures were used to
inoculate 50 mL of LB medium supplemented with 50 lg/mL kana-
mycin. The cultures were grown at 37 °C until they reached an
OD600-reading of 0.6. The cultures were then induced with 1 mM
IPTG and grown for an additional 4 h at 28 °C (for rmlAKkf and
rmlBKkf) or 37 °C (for rmlCKkf and tal). The 6xhistidine-tagged pro-
teins were isolated using a Ni-NTA agarose resin, following the
1.3. Protein expression and purification
PCR amplification was used to prepare the expression con-
structs. The PCR primers (with engineered NdeI and HindIII sites

1962
S. Karki et al. / Carbohydrate Research 345 (2010) 1958–1962
manufacturer’s protocol (Qiagen, Valencia, CA). The protein con-
tent was determined using the Bradford assay. Purified protein
yields were 11 (RmlAKkf), 1.0 (RmlBKkf), 747 (RmlCKkf), and 16
(Tal) mg/L from the culture. For the recombinant RmlBKkf, 500 mL
LB culture was further processed to obtain 1 mg of protein. The
concentrations of the protein solutions were 32 (RmlAKkf), 54
Acknowledgments
This work was supported by National Research Foundation of
Korea Grant funded by the Korean Government (MOEHRD, Basic
Research Promotion Fund) (KRF-2008-331-C00244 and 2009-
0074283), and a grant (20070401-034023) from the BioGreen 21
Program of the Rural Development Administration, Korea.
(RmlBKkf), 150 (RmlCKkf), and 49 (Tal) lM.
1.4. Enzyme reaction
Supplementary data
The formation of dTDP-6dTal was monitored in a two-step, one-
pot reaction with recombinant RmlAKkf, RmlBKkf, RmlCKkf, and Tal
proteins. RmlAKkf/RmlBKkf reaction mixture contained 1 mM thy-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.07.004.
midine triphosphate (dTTP), 5 mM
10 mM MgCl2, 50 mM KH2PO4 (pH 7.5), 0.8
a
-
D
-glucose-1-phosphate,
M RmlAKkf, 1.4
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dTDP-6-Deoxy-L-talose collected by HPLC was used for NMR
analysis. The reaction mixture was loaded on an HPLC column after
ultrafiltration (molecular weight cut-off of 30,000 kDa). The HPLC
elution was lyophilized after removal of the excess phosphate as
previously described.12 The fraction from each run was cooled on
ice and pooled with four volumes of cold ethanol to remove the ex-
cess phosphate. The ethanol solution was dried under reduced
pressure and dissolved in one tenth volumes of water to be lyoph-
ilized. NMR experiments such as 1H NMR, 13C NMR, 1H–1H COSY,
NOESY, 1H–13C HMQC, and 1H–13C HMBC were conducted in pure
D2O at 23 °C using a Bruker AVANCE 600-MHz spectrometer. 1H
NMR spectra were also recorded with an inclusion of trimethylsilyl
propionate to obtain the chemical shift data. The parameters for 1H
NMR spectral recording were as follows: pulse angle, 30°; sweep
width, 10776 Hz; acquisition time, 3.04 s; relaxation delay, 1 s;
scans number, 64; digital resolution, 0.16 Hz/point.
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