Characterization of l-glutamine:2-deoxy-scyllo-inosose aminotransferase (tbmB) from Streptomyces tenebrarius

Article abstract of DOI:10.1016/j.bmcl.2004.10.028

2-Deoxystreptamine (DOS)-containing aminoglycoside-aminocyclitol (AmAc) antibiotics represent the majority of clinically important AmAcs. Biosynthetic investigations of formation of DOS in actinomycetes are limited to the characterization of 2-deoxy-scyllo-inosose synthase, the first step enzyme of the DOS biosynthetic pathway. A gene encoding l-glutamine:2-deoxy-scyllo-inosose aminotransferase (tbmB) from the tobramycin producer Streptomyces tenebrarius was expressed heterologously in Escherichia coli. The conversions of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and scyllo-inosose to scyllo-inosamine with the activity of TbmB were determined in vitro. The results indicate that tbmB catalyzes the second step of the DOS biosynthetic pathway during the biosynthesis of 2-deoxystreptamine, a subunit of tobramycin, in S. tenebrarius.

Full text of DOI:10.1016/j.bmcl.2004.10.028

Bioorganic & Medicinal Chemistry Letters 15 (2005) 89–92
Characterization of L-glutamine:2-deoxy-scyllo-inosose
aminotransferase (tbmB) from Streptomyces tenebrarius
Madan K. Kharel, Bimala Subba, Hei Chan Lee, Kwangkyoung Liou and
Jae Kyung Sohng*
Institute of Biomolecule Reconstruction, Sun Moon University, 100 Kalsanri, Tangjeonmyun, Asansi, Chungnam 336-708, Korea
Received 10 August 2004; revised 6 October 2004; accepted 9 October 2004
Available online 28 October 2004
Abstract—2-Deoxystreptamine (DOS)-containing aminoglycoside–aminocyclitol (AmAc) antibiotics represent the majority of
clinically important AmAcs. Biosynthetic investigations of formation of DOS in actinomycetes are limited to the characterization
of 2-deoxy-scyllo-inosose synthase, the first step enzyme of the DOS biosynthetic pathway. A gene encoding L-glutamine:2-deoxy-
scyllo-inosose aminotransferase (tbmB) from the tobramycin producer Streptomyces tenebrarius was expressed heterologously in
Escherichia coli. The conversions of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and scyllo-inosose to scyllo-inosamine with
the activity of TbmB were determined in vitro. The results indicate that tbmB catalyzes the second step of the DOS biosynthetic
pathway during the biosynthesis of 2-deoxystreptamine, a subunit of tobramycin, in S. tenebrarius.
Ó 2004 Elsevier Ltd. All rights reserved.
Aminoglycoside–aminocyclitol (AmAc) antibiotics are
among the oldest antibiotics in human therapeutic
usage. These antibiotics are primarily produced by the
actinomycetes and have been using for the treatment
of the infections caused by Gram positive (pseudo-
monas) and Gram negative (bacillus) pathogens.
Despite the emergence of several AmAcs-resistant
pathogens, the synergistic effect of AmAcs with other
antibiotics on action has sustained their clinical utilities,
particularly for treating the nosocomial infections.1–3
Most of these compounds have an aglycon (cyclitol sub-
unit) with two amino groups. Glycosylations of hyd-
roxyl groups of this aminocyclitol with other sugar
subunits lead to the formation of AmAc antibiotics.
Based on the aminocyclitol present, these antibiotics
can be divided into two groups; one with streptamine
and the other with 2-deoxystreptamine (DOS) subunit.
The latter includes the most of the clinically important
AmAc antibiotics (gentamicin, kanamycin, ribostamy-
cin, tobramycin, neomycin, etc.) whereas the former in-
cludes spectinomycin, bluensomycin, streptomycin, etc.
the isolation of the butirosin biosynthetic gene cluster
from Bacillus circulans.4 2-Deoxy-scyllo-inosose syn-
thase (btrC) catalyzes a redox reaction resulting in the
formation of 2-deoxy-scyllo-inosose (DOI) (3, Scheme
1), and introduces the intracellular metabolite flux to
the DOS biosynthetic route in B. circulans.5 The stereo-
chemistry of carbocyclization, and the crucial role of
some amino acid residues of BtrC have been deter-
mined.6,7 BtrS transaminates the product of BtrC
(DOI) to form 2-deoxy-scyllo-inosamine (4) utilizing L-
glutamine (an amino donor) in the presence pyridoxal
phosphate.8,9 Dehydrogenation of scyllo-inosamine
and the subsequent transamination of the product are
the predicted biosynthetic reactions to generate DOS
(Scheme 1). Because the bacteria belonging to actino-
mycetes are the major AmAcs producers, we have ex-
tended the genetic and biochemical investigations of
AmAcs production to these organisms isolating biosyn-
thetic gene clusters for tobramycin from S. tenebrarius
(AJ579650), gentamicin from Micromonospora echinos-
pora (AJ575934) and kanamycin from Streptomyces
kanamyceticus (AJ582817). Recently, Yanai and Mura-
kami have also reported a cluster for kanamycin biosyn-
thesis independently.10
Genetic and biochemical investigations on the biosyn-
thesis of the DOS-containing AmAcs have begun with
Despite a good structural similarity between streptamine
and DOS, the genetic and biosynthetic investigations
of their formation to date have indicated two separate
biosynthetic routes (Scheme 1). Organization of
Keywords: Biosynthesis; scyllo-Inosamine; 2-Deoxy-scyllo-inosamine;
Streptomyces tenebrarius.
*
Corresponding author. Tel.: +82 41 530 2246; fax: +82 41 544
2919; e-mail: sohng@sunmoon.ac.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.028

90
M. K. Kharel et al. / Bioorg. Med. Chem. Lett. 15 (2005) 89–92
TbmX,
OPO 2-
3
H2N
H2N
H2N
O
O
TbmB HO
HO
TbmA HO
OH
HO
TbmB?
TbmC, ?
HO
OH
NH
2
OH
HO
HO
HO
HO
HO
OH
OH
OH
OH
O
OH
2
3
4
5
6
I
HO
2-
HO
OPO2
Tobramycin
OH
HO
HO
Spectinomycin
OH
1
SpeA
II
HO
H2N
H2N
O
HO
HO
OH
OH
HO
OH
OH
HO
OH
SpcS2
OH
OH
SpeB
NH
2
HO
HO
HO
OH
OH
OH
OH
8
7
9
10
Scheme 1. Proposed pathways for the biosyntheses of 2-deoxy-streptamine (pathway I) and streptamine (pathway II). Tobramycin and
spectinomycin are the typical antibiotics with 2-deoxystreptamine and actinamine (methylated streptamine) subunits. 1: myo-Inositol-6-phosphate; 2:
glucose-6-phosphate; 3: 2-deoxy-scyllo-inosose; 4: 2-deoxy-scyllo-inosamine; 5: 3-amino-2-deoxy-scyllo-inosose; 6: 2-deoxystreptamine; 7: myo-
inositol; 8: scyllo-inosose; 9: scyllo-inosamine; 10: streptamine.
streptamine biosynthetic genes in a cluster has been re-
ported in spectinomycin producer Streptomyces specta-
bilis.11 Biosynthesis of streptamine begins with the
formation of myo-inositol-1-phosphate (1) from G-6-P
(2) by myo-inositol-1-phosphate synthase. The product
undergoes dephosphorylation to yield myo-inositol (7)
with the activity of myo-inositol monophosphatase
(SpeA). Dehydrogenation of myo-inositol with myo-
inositol dehydrogenase (SpeB) to form scyllo-inosose
(8) and its subsequent transamination by an amino-
transferase (SpcS2) have been determined as actinamine
(a methylated derivative of streptamine) biosynthetic
steps. Transamination of scyllo-inosose with StsC from
Streptomyces griseus has been determined as one of
the streptidine biosynthetic steps.12 However, the bio-
synthesis of DOS in actinomycetes has been largely
circumstantial and is limited to the characterization of
DOI synthase in S. tenebrarius.13 In this communica-
tion, we report the characterization of 2-deoxy-scyllo-
inosose aminotransferase (tbmB) from the tobramycin
producer S. tenebrarius.
SDS-PAGE by inducing the culture (at OD600 0.5) with
0.4mM IPTG at 20°C for 20h. Expression of btrC and
enzyme assay of the product were carried out as previ-
ously described.9 Coupled assays of TbmB with BtrC
and myo-inositol dehydrogenase (Sigma) were carried
out in 1mL phosphate buffer (50mM, pH7.5) contain-
ing 5mM G-6-P, 2.5mM NAD+, 5mM L-glutamine
and 0.6mM pyridoxal 50-phosphate (PLP). The reaction
was initiated by the addition of 6mg of each enzyme and
quenched by heating at 90°C following the incubation at
37°C for 20h. The protein was removed by centrifuga-
tion and the resulting mixture was acidified to pH3 with
50mM H2SO4. An aliquot (50lL) was taken for the
ESI-MS analysis. Water was used in place of G-6-P
for a reference reaction. Two distinct peaks were ob-
served in the reaction mixture as expected. A peak (m/z
164) displayed the MW of the expected 2-deoxy-
scyllo-inosamine (4, Scheme 2), while the other corre-
sponded to the formation of a-ketoglutarate (m/z
146.3) according to the Scheme 2 (data is not shown).
To confirm the formation of 2-deoxy-scyllo-inosamine,
the pH of the reaction mixture was raised to 6, passed
through the ion exchange column (IRC-50), washed
the impurities with water, and the compound was eluted
with 5mL of 2M ammonia solution. The solvent was
evaporated and concentrated the sample to 1mL under
reduced pressure. UV–vis derivative of 2-deoxy-scyllo-
inosamine (13) was prepared following the previous re-
port of Stead and Richards with slight modifications
(Scheme 2).14 To the concentrated effluent (500lL), 9-
fluorenylmethyl chloroformate (FMOCl) (20lL, 20lg/
mL) was added and heated the mixture at 37°C for
3h. The mixture was filtered through a membrane
(0.25lm pore diameter) and taken for HPLC analysis
at 260nm. A new peak was detected while separating
the components in C-18 column (Mightysil RP-18 Gp,
Japan) with a linear gradient of acidified water (with
0.1% trifluoroacetic acid) and acetonitrile from 100%
to 0%, and comparing the chromatogram with that of
the reference sample. LC–MS analysis revealed peaks
with MW 385.6 and 386.6, which correspond to the
[M+] and [M+H+] for the amide derivative of 2-deoxy-
scyllo-inosamine (13) (Fig. 1).
The deduced amino acid sequence encoded by tbmB is
74% and 60% identical to putative 2-deoxy-scyllo-inos-
ose aminotransferases from S. kanamyceticus (KanB,
protein_id CAE46938.1) and M. echinospora (GtmB,
protein_id CAE06513.1), respectively. Lesser amino
acid identities of TbmB to recently characterized BtrS
(BAC41204.1) from B. circulans were also found. The
latter protein represents the sole 2-deoxy-scyllo-inosose
aminotransferase with the functional identification re-
ported to date.8,9
To determine tbmB encodes a 2-deoxy-scyllo-inosose
aminotransferase, the gene was amplified by PCR with
a set of primers (AMTT1: 50-AGGAATTCTCGAC-
CATGCCCGTCCA-30 and AMTT2: 50-CAAAGCTT-
GGAGCGGGCGCTCAG-30) and cloned into EcoRI
and HindIII site of pET32a(+) vector to form pTBMB.
The cloned DNA was transformed into competent E.
coli BL21(DE3) cells following the standard protocol.
The transformant was cultured in Luria Bertani (LB)
medium. Overexpression of the tbmB was observed on

M. K. Kharel et al. / Bioorg. Med. Chem. Lett. 15 (2005) 89–92
91
NH2
NH2
O
O
O
H2N
HO
HO
OH FMOCl
PLP
TbmB
OH
O
HO
+
+
HO
OH
OH
HN
O
COOH
COOH
4
3
HO
O
H2N
OH
HO
OH
11
12
13
NH2
NH2
O
O
O
H2N
O
HO
HO
OH
OH
OH
OH
FMOCl
PLP
TbmB
HO
HO
+
+
HN
O
OH
OH
HO
OH
OH
COOH
COOH
9
8
HO
H2N
O
OH
11
12
14
Scheme 2. Reactions catalyzed by TbmB. 3: 2-deoxy-scyllo-inosose; 4: 2-deoxy-scyllo-inosamine; 8: scyllo-inosose; 9: scyllo-inosamine; 11:
L-glutamine; 12: a-keto-glutamate; 13: amide derivative of 4; 14: amide derivative of 9; FMOCl: 9-flourenylmethyl chloroformate; PLP: pyridoxal
50-phosphate.
Figure 1. ESI-MS of the amide derivative of 2-deoxy-scyllo-inosamine (15) where the peaks at 385.6 and 386.6 stand for the corresponding [M+] and
[M+H+] values.
TbmB also displays 55% identity of its amino acid resi-
dues to StsC (CAA70012.1) from S. griseus. The latter
protein catalyzes the transamination of scyllo-inosose
to scyllo-inosamine utilizing L-glutamine in the presence
of PLP. To clarify whether TbmB accepts the substrate
of StsC or not, a coupled assay was carried out using
myo-inositol dehydrogenase (mdh) (Sigma, USA) in
1mL incubation mixture where BtrC and G-6-P were re-
placed with 5mM of myo-inositol and 2-unit of mdh,
respectively. The LC-MS analyses of the FMOCl-deri-
vatized products revealed a peak with MW of 401.5
and 402.6, which correspond to the [M+] and [M+H+]
for the amide derivative of scyllo-inosamine (14), respec-
tively (Fig. 2).
inosamine, an intermediate in the DOS biosynthetic
pathway, in the S. tenebrarius. Here, it is emphasized
that this is the first report of identification of an
aminotransferase involved in DOS biosynthesis from
Streptomyces. Conversion of scyllo-inosose to scyllo-
inosamine with the TbmB is the other interesting fea-
ture. Walker and co-workers have given an indirect
evidence for the involvement of an aminotransferase in
the both of the transamination reactions of the DOS
biosynthetic pathway by carrying out the mimic reac-
tions using a pure enzyme isolated from cell-free extract
of M. purpurea.15 Instead, the presences of tbmB and
tbmX, gtmB and gtmD, and kanB and kanD in tobramy-
cin, gentamicin, and kanamycin biosynthetic gene
clusters support for Ôone enzyme one step transamina-
tionÕ hypothesis for DOS biosynthetic steps. However,
it is yet to be seen whether TbmB catalyzes both
The results clearly indicate that tbmB encodes a protein
(TbmB) that involves in the formation 2-deoxy-scyllo-

92
M. K. Kharel et al. / Bioorg. Med. Chem. Lett. 15 (2005) 89–92
Figure 2. ESI-MS of the amide derivative of scyllo-inosamine (14) where the peaks at 385.6 and 386.6 stand for the corresponding [M+] and [M+H+]
values.
4. Ota, Y.; Tamegai, H.; Kudo, F.; Kuriki, H.; Koike-
Takeshita, A.; Eguchi, T.; Kakinuma, K. J. Antibiot. 2000,
53, 1158–1167.
5. Kudo, F.; Tamegai, H.; Fujiwara, T.; Tagami, U.;
Hirayama, K.; Kakinuma, K. J. Antibiot. 1999, 52, 559–
571.
6. Nango, E.; Eguchi, T.; Kakinuma, K. J. Org. Chem. 2004,
69, 593–600.
7. Kakinuma, K.; Ogawa, Y.; Sasaki, T.; Seto, H.; Otake, N.
J. Antibiot. 1989, XLII-6, 926–933.
transaminations or not. Provided that an idiotroph of
M. echinospora requiring exogenous DOS to produce
gentamicin yielded a mixture of hydroxygentamicin
derivatives with broad-spectrum activities when the cul-
ture was fed with streptamine,16 construction of a gene
cassette expressing DOS and its subsequent transforma-
tion into streptamine-containing AmAcs producers
would generate new DOS-containing AmAc derivatives.
In this particular aspect, the substrate flexibility of
TbmB can be exploited for replacing DOS with strepta-
mine and vice versa to generate unusual aminocyclitols.
8. Tamegai, H.; Nango, E.; Kuwahara, M.; Yamamoto, H.;
Ota, Y.; Kuriki, H.; Eguchi, T.; Kakinuma, K. J. Antibiot.
2002, 55, 707–714.
9. Huang, F.; Li, Y.; Yu, J.; Spencer, J. B. Chem. Commun.
2002, 23, 2860–2861.
Acknowledgements
10. Yanai, K.; Murakami, T. J. Antibiot. 2004, 57, 351–354.
11. Jo, Y. Y.; Kim, S. H.; Yang, Y. Y.; Kang, C. M.; Sohng, J.
K.; Suh, J. W. J. Microbiol. Biotechnol. 2003, 13, 906–911.
12. Ahlert, J.; Distler, J.; Mansauri, K.; Pipersberg, W. Arch.
Microbiol. 1997, 168, 102–113.
The work was supported by medium-term strategic tech-
nology development (to J.K.S.), Republic of Korea.
13. Kharel, M. K.; Basnet, D. B.; Lee, H. C.; Liou, K.; Woo,
J. S.; Kim, B.-G.; Sohng, J. K. FEMS Microbiol. Lett.
2004, 230, 185–190.
References and notes
14. Stead, D. A.; Richards, R. M. E. J. Chromatogr. 1996,
675, 295–302.
15. Lucher, L. A.; Chen, Y.-C.; Walker, J. B. Antimicrob.
Agents Chemother. 1989, 33, 452–459.
16. Daum, S. J.; Rosi, D.; Goss, W. A. J. Antibiot. 1977,
XXX-1, 98–105.
1. Dworkin, R. J. Drug Resist. Updates 1999, 2, 173–179.
2. Livermore, D. M. Clin. Infect. Dis. 2002, 34, 634–640.
3. Zembower, T. R.; Noskin, G. A.; Postelnick, M. J.;
Nguyen, C.; Perterson, L. R. Int. J. Antimicrob. Agents
1998, 10, 95–105.