Enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-d-glucose using GerB (dTDP-4-keto-6-deoxy-d-glucose aminotransferase)

Article abstract of DOI:10.1016/j.carres.2007.04.007

Over-expressed GerB (dTDP-4-keto-6-deoxy-d-glucose aminotransferase) of Streptomyces sp. GERI-155 was used in the enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-d-glucose (2) from dTDP-4-keto-6-deoxy-d-glucose (1). {A figure is presented}. Five enzymes including dTMP kinase (TMK), acetate kinase (ACK), dTDP-glucose synthase (TGS), dTDP-glucose 4,6-dehydratase (DH), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (GerB) were used to synthesize 2 on a large scale from glucose-1-phosphate and TMP. A conversion yield of up to 57% was obtained by HPLC peak integration given a reaction time of 270 min. After purification by two successive preparative HPLC systems, the final product was identified by HPLC and then analyzed by ¹H, ¹³C, ¹H-¹H COSY NMR spectrometry.

Full text of DOI:10.1016/j.carres.2007.04.007

Carbohydrate Research 342 (2007) 1412–1418
Enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose using
GerB (dTDP-4-keto-6-deoxy-D-glucose aminotransferase)
Young Soo Chung,a,b Dae Hee Kim,a Won Min Seo,a Hei Chan Lee,b
Kwangkyoung Liou,b Tae-Jin Ohb,* and Jae Kyung Sohngb
aGenechem Inc., 59-5 Jang-dong, Yuseong-gu, Daejeon 305-343, South Korea
bInstitute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University,
#100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, South Korea
Received 18 January 2007; accepted 11 April 2007
Available online 22 April 2007
Abstract—Over-expressed GerB (dTDP-4-keto-6-deoxy-D-glucose aminotransferase) of Streptomyces sp. GERI-155 was used in the
enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose (2) from dTDP-4-keto-6-deoxy-D-glucose (1).
Five enzymes including dTMP kinase (TMK), acetate kinase (ACK), dTDP-glucose synthase (TGS), dTDP-glucose 4,6-dehydratase
(DH), and dTDP-4-keto-6-deoxy-D-glucose aminotransferase (GerB) were used to synthesize 2 on a large scale from glucose-1-phos-
phate and TMP. A conversion yield of up to 57% was obtained by HPLC peak integration given a reaction time of 270 min. After
1
purification by two successive preparative HPLC systems, the final product was identified by HPLC and then analyzed by H, 13C,
1H–1H COSY NMR spectrometry.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Aminotransferase; dTDP-4-Amino-4,6-dideoxy-D-glucose; dTDP-4-Keto-6-deoxy-D-glucose; L-Glutamic acid; Streptomyces sp. Geri-155
1. Introduction
Deoxyaminosugars comprise an important class of
deoxysugar moieties synthesized by a variety of micro-
organisms including plants, fungi, and bacteria.1 Bacteria,
Abbreviations: ACK, acetate kinase; DH, dTDP-glucose 4,6-dehydra-
tase; HPLC, high performance liquid chromatography; IPTG, isopro-
pyl-D-thiogalactopyranoside; PLP, pyridoxal phosphate; SAM,
S-adenosylmethionine; TEAB, tetraethylammonium borate; TGS,
dTDP-glucose synthase; TMK, dTMP kinase
actinomycetes in particular, produce secondary metabo-
lites with modified deoxysugar moieties, which have anti-
biotic and antitumor activities.2,3 Deoxyaminosugars are
usually formed via a transamination reaction, which
results in the substitution of an amino group for a keto
*
Corresponding author. Tel.: +82 41 530 2276; fax: +82 41 544
2919; e-mail: tjoh3782@sunmoon.ac.kr
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.04.007

Y. S. Chung et al. / Carbohydrate Research 342 (2007) 1412–1418
1413
group. These deoxyaminosugars are crucial for the inter-
actions between antibiotic molecules, which appear to
play an important role in the function of the cellular
membrane of the target organism.4 Therefore, studies
on the synthesis and attachment of deoxyaminosugars
are important for the engineered biosynthesis of new
antibiotics from bacteria. Bowers et al. reported the syn-
thesis of dTDP-4-amino-4,6-dideoxy-D-glucose (2) from
galactose using a 16-step procedure.5 Alternative biosyn-
thetic pathways for the biosynthesis of deoxyaminosu-
gars have been proposed and several aminotransferases
that catalyze the installation of an amino group on a
deoxysugar have been studied. The aminotransferases
studied include N-methyl-L-glucosamine (streptomy-
cin),6 D-desosamine (erythromycin),7,8 D-desosamine
(oleandomycin),9 and D-mycaminose (tylosin).10
However, there are no reports on the enzymatic syn-
thesis of 2. We have recently isolated the mycinose gene
cluster involved in the biosynthesis of dihydrochaloco-
mycin from Streptomyces sp. KCTC 0041BP.11 The
GerB open reading frame was predicted to be an amino-
transferase involved in the biosynthesis of 2. The de-
duced amino acid sequence (426 aa) shares an identity
of roughly 65% with a number of the aminotransferases
in the GeneBank database. We report the cloning and
expression of gerB in Escherichia coli BL21 (DE3) along
with the enzymatic synthesis of 2 and its stereochemistry
1
1
via H, 13C, and H–1H COSY NMR analysis.
2. Results and discussion
2.1. Identification of dTDP-4-keto-6-deoxy-D-glucose
aminotransferase, GerB
The gerB gene is located in the region furthest upstream
in the dihydrochalocomycin biosynthetic gene cluster.
The deduced amino acid sequence of gerB showed a
high degree of similarity to other putative genes within
the GeneBank database; such as EryCIV (GeneBank
accession no. AAB84075),7 which is the aminotransfer-
ase involved in D-desosamine biosynthesis (62% iden-
tity); OleNI (GeneBank accession no. AAD55456),9
the dehydratase involved in the transamination (66%
identity); and DesI (GeneBank accession no.
AAC68684),12 the dehydrase involved in D-desosamine
biosynthesis (61% identity). These results are shown in
Figure 1. Liu and co-workers recently accomplished
the biosynthesis of dTDP-D-desosamine using
a
two-step C-4 deoxygenation in which DesI was used to
catalyze the C-4 transamination of dTDP-4-keto-6-
deoxy-D-glucose (1) to generate 2 and in which DesII
Figure 1. Multi-alignment analysis of the deduced amino acid sequence of GerB with other known aminotransferases: EryCIV, AAB84075; OleNI,
AAD55456; DesI, AAC68684. The conserved amino acids are in bold.

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Y. S. Chung et al. / Carbohydrate Research 342 (2007) 1412–1418
was used for the S-adenosylmethionine (SAM)-depen-
dent C-4 deamination rather than for a deoxygen-
ation.13–15 Further sequence analysis revealed that
GerB is similar to DesI (61% identity, Fig. 1), and that
GerQ is similar to DesII (73% identity, data not shown).
These observations suggest that two genes, GerB and
GerQ, are involved in C-4 deoxygenation in the desos-
amine biosynthetic gene cluster. The GerB protein may
be catalyzed by the C-4 transamination of 1 to generate
2 in the dihydrochalocomycin biosynthetic pathway.
plasmid pGERB. The nucleotide sequence of the cloned
gerB was confirmed by sequencing. The pGERB plas-
mid was transformed into E. coli BL21 (DE3) and ex-
pressed with 1 mM isopropyl-D-thiogalactopyranoside
(IPTG) induction to produce a 47-kDa protein compris-
ing GerB (426 aa) (Fig. 2).
2.3. Enzymatic activity of dTDP-4-keto-6-deoxy-D-
glucose aminotransferase, GerB
GerB is believed to encode for C-4 transamination in the
later stages of D-desosamine biosynthesis. The enzy-
matic reaction was carried out in 100 mM Tris–HCl buf-
fer (pH 7.5) containing 4 mM 1, 8 mM L-glutamic acid
(or L-glutamine) and 1 mM pyridoxal phosphate (PLP)
in order to investigate the activity of GerB. The reaction
was initiated by the addition of GerB during incubation
at 37 ꢁC. The mixture was screened for the transforma-
tion reaction from 1 to 2 using high performance liquid
chromatography (HPLC), as shown in Figure 3.16 The
reaction was carried out using L-glutamine or L-glutamic
acid as the source of the 4-amino group and L-glutamic
acid showed a better conversion to 2 under identical
reaction conditions, as clearly shown in the HPLC data
(Fig. 4a and b). Thus, L-glutamic acid was used as the
source of the amino group for all subsequent reactions.
The peak of the expected product (2) was observed after
the reaction started and included the intermediate peak
corresponding to 1. A conversion yield of roughly 66%
was obtained within 5 h.
2.2. Expression of dTDP-4-keto-6-deoxy-D-glucose
aminotransferase, GerB
PCR was performed using the GerB-F and GerB-R
primers and the expected PCR product of 1.3-kb was
obtained and cloned into the BamHI/HindIII site of a
pET28a expression vector to produce the expression
1
2
kDa
150 -
100 -
75 -
50 -
35 -
47-kDa
2.4. One-pot enzymatic synthesis of dTDP-4-amino-
4,6-dideoxy-D-glucose
25 -
Several nucleotide sugars, such as UDP-D-glucose,
dTDP-L-rhamnose and 1, were effectively synthesized
from glucose-1-phosphate through a series of enzymatic
reactions in our previous studies.17–19 The synthesis of 2
was performed using a one-pot reaction (Fig. 5) under
the same reaction conditions and starting materials used
in previous experiments; TMP, glucose-1-phosphate,
L-glutamic acid and PLP. The enzymatic conversion of
2 was achieved using TMK (0.05 mg/mL), ACK
(0.04 mg/mL), TGS (0.03 mg/mL), DH (0.03 mg/mL),
and dTDP-4-keto-6-deoxy-D-glucose aminotransferase
10 -
Figure 2. Identification of the over-expressed GerB by SDS–PAGE:
lane 1: marker; lane 2: soluble.
Figure 3. Conversion of dTDP-4-keto-6-deoxy-D-glucose (1) to dTDP-4-amino-4,6-dideoxy-D-glucose (2) using dTDP-4-keto-6-deoxy-D-glucose
aminotransferase (GerB).

Y. S. Chung et al. / Carbohydrate Research 342 (2007) 1412–1418
1415
Figure 4. HPLC diagram for the conversion of dTDP-4-keto-6-deoxy-D-glucose (1) to dTDP-4-amino-4,6-dideoxy-D-glucose (2) by GerB.
(a) L-glutamine; (b) L-glutamic acid.
2Pi
O
N
HO
HO
CH3
OH
dTDP-
glucose
4,6-dehydratase
dTDP-
glucose
Synthase
H3C
O
O
O
NH
OH
OH
O
O
P
O
P
2-
OTDP
OPO3
O
O
O
O
OH
OH
O
OH
OH
OH
O-
O-
Glucose-1-phosphate
dTDP-D-glucose
TTP
OH
dTDP-4-keto-6-deoxy-D-glucose (1)
Acetate
Glutamic
acid
Acetate Kinase
GerB
PLP
Acetyl-Pi
α-ketoglutarate
TDP
O
N
CH3
OH
H3C
O
NH
ADP
Acetyl-Pi
Acetate
O
P
O
P
Acetate
Kinase
O
O
O
O
NH2
dTMP Kinase
O
OH
O-
O-
ATP
OH
TMP
dTDP-4-amino-4,6-dideoxy-D-glucose (2)
Figure 5. Enzymatic pathway for the one-pot synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose (2) from glucose-1-phosphate.
(GerB, 0.04 mg/mL). A conversion yield of approxi-
mately 57% was obtained by HPLC peak integration
given a reaction time of 270 min. These results con-
firmed the role of GerB in catalyzing the C-4 transami-
nation of 2 via an amino sugar intermediate in the
biosynthesis of D-desosamine.

1416
Y. S. Chung et al. / Carbohydrate Research 342 (2007) 1412–1418
1
1
2.5. Purification and identification of dTDP-4-amino-4,6-
dideoxy-D-glucose
product was subjected to H, 13C, and H–1H COSY
NMR with D2O rather than CDCl3 as in Bowers
et al.’s report.5 The chemical shifts observed in the proton
NMR are closely related to those in the published data
Two successive preparative HPLC purifications were
performed using mixed solvent systems to isolate 2.
Water and 1.0 M tetraethylammonium borate (TEAB)
aqueous solution were used as eluents varying the later
concentration from 0% to 80%. Acetonitrile was then
used as an eluent and 10 mM TEAB aqueous solution
was used to vary the concentration of acetonitrile from
0% to 100%. The purified product was analyzed by ana-
lytical HPLC after lyophilization as described in the
experimental section. HPLC peak integration proved
that 2 was more than 99% pure (Fig. 6). The purified
00
with minor shifts down-field. The peak patterns for H3
and H4 were double doublets in the previously reported
data; however, those observed in our NMR were triplets
00
00 00
00 00
since the coupling constants (J2 3 ¼ 9:9, J3 4 ¼ 9:6, and
J4 5 ¼ 9:8) are nearly equivalent. The 1H NMR data are
00 00
summarized in Table 1. Electrospray-MS: m/z 546.3
[MꢀH+]ꢀ calcd for C16H26N3O14P2: 546.34.
Based on the results of our study, GerB is thought to
be a dTDP-4-keto-6-deoxy-D-glucose aminotransferase
enzyme responsible for the production of a key interme-
Figure 6. HPLC diagram for the conversion of dTDP-4-keto-6-deoxy-D-glucose (1) to dTDP-4-amino-4,6-dideoxy-D-glucose (2) using a one-pot
reaction. (a) After 150 min; (b) after 270 min; and (c) after two purifications with preparative HPLC.

Y. S. Chung et al. / Carbohydrate Research 342 (2007) 1412–1418
1417
Table 1. 1H NMR data for the dTDP-4-amino-4,6-dideoxy-D-glucose
(2) in D2O
GGA TCC ACC ATG AAA CGC AAT GTC-30) and
the reverse GerB-R (50-CCG CAA GCT TTT CCC
TGA TCA GGG GGC-30) sequences were designed
for PCR amplification of the GerB coding region (the
restriction sites, BamHI and HindIII, are indicated by
underlined letters). PCR was conducted under the fol-
lowing conditions: 1 cycle for 7 min at 97 ꢁC; 30 cycles
for 1 min at 95 ꢁC, 1 min at 60 ꢁC, and 1 min at 72 ꢁC;
1 cycle for 7 min at 72 ꢁC. The PCR products of the ex-
pected size (1.2-kb) were cloned into the BamHI/HindIII
site of the pET28a expression vector to generate the
expression plasmid pGERB.
Residue
Proton Chemical shift Coupling
(d, ppm)
constant (J)
Thymidine
Ribose
H-2
H-7
s, 7.77
s, 1.97
t, 6.38
m, 2.41
m, 4.66
m, 4.22
m, 4.22
dd, 5.60
dt, 3.60
t, 3.69
H-10
H-20
H-30
H-400
H-500
H-100
00 00
4-Amino-4,6-
dideoxy-D-glucose H-200
JP 6.9, J1 2 3:6
00 00
J2 3 9:9
00 00
H-3
J3 4 9:6
00 00
J4 5 9:8
H-400
H-500
H-600
t, 2.60
m, 4.02
d, 1.32
3.3. Expression of GerB
00 00
J5 6 6:0
E. coli BL21 (DE3) cells harboring pGERB were grown
aerobically in 50 mL of Luria broth (LB) using a
250 mL shaking flask at 37 ꢁC with agitation at
200 rpm until an OD600 of 0.6 was obtained and was fol-
lowed by the addition of 1 mM IPTG for induction in
order to express GerB. The cultures were then harvested,
and pelleted after incubation for 18 h at 20 ꢁC. Crude
extracts were obtained by sonicating in 50 mM Tris–
HCl buffer (pH 7.5) containing 1 mM (DTT), 10% glyc-
erol, 1 mM (EDTA), 2 mM MgCl2Æ6H2O and 1 mM
(PMSF). The lysate was then centrifuged at 15,000g
for 30 min at 4 ꢁC; the precipitate was discarded.
diate, 2, in the biosynthesis of deoxyaminosugar (D-des-
osamine). The results also show that although L-gluta-
mine does show substrate activity in transamination,
L-glutamic acid is a better source of the 4-amino group
in the conversion to 2. Moreover, our study indicates
that 2 can be synthesized on a preparative scale from
glucose-1-phosphate and TMP using five recombinant
enzymes without any unknown effects caused by the
use of enzymes. This report is the first to describe
the preparative enzymatic synthesis of 2. Therefore,
the findings of this study can be used to aid in the syn-
thesis of any other interesting dideoxysugar intermedi-
ates including unusual amino sugars based on the
generation of an in vitro metabolic pathway.
3.4. Enzyme assay and enzymatic synthesis
GerB was assayed using 4 mM 1, 8 mM L-glutamic acid
(or L-glutamine), 1 mM PLP and 100 mM Tris–HCl
(pH 7.5). The activities of GerB were identified by com-
paring the conversion of 1 to 2. The in vitro enzymatic
reactions were conducted in 200 mL of reaction mixture
containing 10 mM TMP, 0.25 mM ATP, 20 mM MgCl2,
50 mM acetyl-phosphate, 40 mM glucose-1-phosphate,
20 mM L-glutamic acid, 0.2 mM PLP, 50 mM Tris–
HCl (pH 7.5) and enzyme extracts in order to accom-
plish a one-pot enzymatic synthesis. All other enzyme
activities were determined in 50 mM Tris–HCl buffer
(pH 7.5) at 37 ꢁC. The activities of TMK, ACK, TGS,
and DH were measured as previously described.17 The
reaction was initiated with the addition of approxi-
mately 20 mL of TMK (0.05 mg/mL), 20 mL of ACK
(0.04 mg/mL), 20 mL of TGS (0.03 mg/mL), 20 mL of
DH (0.03 mg/mL), and 20 mL of dTDP-4-keto-6-
deoxy-D-glucose aminotransferase (GerB, 0.04 mg/
mL). The reaction was carried out for 270 min at
37 ꢁC and was terminated upon heating the reaction
mixture at 100 ꢁC for 1 min. The reaction mixture was
diluted with the same buffer (1:5), and the reactions were
monitored by HPLC. The final product was purified
using SHIMADZU SPD-10Avp (source Q15 resin
200 mL, FineLine Pilot 35 column, Amersham biosci-
ences) and an ODS-AP 300-S10/20 30U · 500 mm
3. Materials and methods
3.1. Materials
All chemical reagents used were purchased from Sigma
Chemical Co. (St. Louis, MO). The dTDP-4-keto-6-
deoxy-D-glucose used in this study was provided by
the GenChem Co. (Daejeon, Korea). The enzymes used
for DNA manipulations in cloning were obtained from
Takara (Shiga, Japan). E. coli XL1-Blue (MRF) (Strat-
agene) was utilized as a host cell for the preparation of
DNA manipulation, and E. coli BL21 (DE3) (Strata-
gene) was used in the expression of the 6 · His-fusion
protein. The pET28a vector was purchased from Nova-
gen (West Palm Beach, FL) for use in gene expression.
The cloning and transformation techniques used in this
study were adapted from those described by Sambrook
et al.20
3.2. Construction of the expression vector
GerB from S. sp. Geri-155 was cloned into the expres-
sion vector using the polymerase chain reaction. A set
of modified primers harboring the GerB-F (50-GAA

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Y. S. Chung et al. / Carbohydrate Research 342 (2007) 1412–1418
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and its purity was analyzed by HPLC.
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Various nucleotides and 2 were analyzed by HPLC
using a strong anion exchange column (Hypersil ODS
4.6 · 250 mm, 5 lm particle size) with 100 mM potas-
sium phosphate buffer (pH 7.0):methanol = 95:5 (v/v).
The eluted nucleotides were monitored by absorbance
at 270 nm at a flow rate of 1.0 mL/min. ESI mass spec-
trometry analysis was performed in negative ion mode
at a voltage of 5 V using a Thermo Finnigan AQA mass
spectrometer (Thermo Finnigan) equipped with the
standard Thermo ESI source. The purified products
were dissolved in 0.70 mL D2O (99.9 atom % D) and
used for NMR analysis in order to identify the final
product. H, H COSY and 13C NMR spectra were re-
corded using a Varian UNITY-INOVA 300 MHz
NMR spectrometer (Varian) at a probe temperature of
298 K, and the chemical shifts were expressed in ppm
relative to the internal HOD signal (4.80).
1
1
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Acknowledgement
16. Kharel, M. K.; Subba, B.; Lee, H. C.; Liou, K.; Sohng, J.
K. Bioorg. Med. Chem. Lett. 2005, 15, 89–92.
17. Oh, J.; Lee, S.-G.; Kim, B.-G.; Sohng, J. K.; Liou, K.; Lee,
H. C. Biotechnol. Bioeng. 2003, 84, 452–458.
18. Lee, H. C.; Lee, S.-D.; Sohng, J. K.; Liou, K. J. Biochem.
Mol. Biol. 2004, 37, 503–506.
This study was supported by the 21C Frontier Microbial
Genomics and Application Center Program, the Minis-
try of Science and Technology (Grant MG02-0301-004-
2-3-1), Republic of Korea.
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J. K.; Lee, H. C.; Liou, K.; Kim, B.-G. Biotechnol. Bioeng.
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