Biosynthesis of the carbamoylated D-gulosamine moiety of streptothricins: Involvement of a guanidino-N-glycosyltransferase and an N-acetyl-D-gulosamine deacetylase

Article abstract of DOI:10.1002/anie.201412190

Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase. The dynamic duo: Two novel enzymes, StnG and StnI, have been found to be involved in the biosynthetic pathway of the carbamoylated D-gulosamine moiety in streptothricins. StnG is a GT-A fold glycosyltransferase that catalyzes the unprecedented attachment of a sugar to the imine nitrogen atom of a guanidine group; StnI catalyzes the deacetylation of the N-acetyl-D-gulosamine moiety.

Full text of DOI:10.1002/anie.201412190

Angewandte
Chemie
DOI: 10.1002/anie.201412190
Biosynthesis
Biosynthesis of the Carbamoylated d-Gulosamine Moiety of
Streptothricins: Involvement of a Guanidino-N-glycosyltransferase and
an N-Acetyl-d-gulosamine Deacetylase**
Zhengyan Guo, Jine Li, Hua Qin, Min Wang, Xun Lv, Xuebing Li, and Yihua Chen*
Abstract: Streptothricins (STNs) are atypical aminoglycosides
containing a rare carbamoylated d-gulosamine (d-GulN)
moiety, and the antimicrobial activity of STNs has been
exploited for crop protection. Herein, the biosynthetic pathway
of the carbamoylated d-GulN moiety was delineated. An N-
acetyl-d-galactosamine is first attached to the streptolidine
lactam by the glycosyltransferse StnG and then epimerized to
N-acetyl-d-gulosamine by the putative epimerase StnJ. After
carbamoylation by the carbamoyltransferase StnQ, N-acetyl-
d-GulN is deacetylated by StnI to furnish the carbamoylated
d-GulN moiety. In vitro studies characterized two novel
enzymes: StnG is an unprecedented GT-A fold N-glycosyl-
transferase that glycosylates the imine nitrogen atom of
guanidine, and StnI is the first reported N-acetyl-d-GulN
deacetylase.
S
treptothricins (STNs) are atypical aminoglycosides featur-
ing a streptolidine lactam base, a rare carbamoylated d-
gulosamine (d-GulN) sugar, and various side chains attached
to the d-GulN amino group (Scheme 1A and see Figure S1 in
the Supporting Information). The structural diversity of STNs
arises from the number of b-lysine moieties (ranging from 1 to
7
) attached to d-GulN and by the position of the carbamoy-
[
1]
lation. STNs possess antibacterial and antifungal bioactiv-
ities and are used to prevent bacterial and fungal diseases in
crops. The discovery of STN F (1) more than 70 years ago
initiated a sustained effort to elucidate the biosynthetic logic
[2]
[1a]
Scheme 1. STN structures, genes, and biosynthetic pathway. A) Struc-
tures of STNs and the d-GulNAc-containing compound gualamycin.
B) Partial stn gene cluster, with genes studied in this work denoted as
black arrows. C) A proposed biosynthetic pathway for the d-GulN
moiety of STNs. AMP=adenosine monophosphate, ATP=adenosine
triphosphate, PPi=pyrophosphate, Pi=inorganic phosphate.
[
3]
of STNs, which revealed that the streptolidine lactam and
[
+]
[+]
[+]
[
*] Dr. Z. Guo, Dr. J. Li, Dr. H. Qin, Dr. M. Wang, Prof. Y. Chen
State Key Laboratory of Microbial Resources
Institute of Microbiology, Chinese Academy of Sciences
Beijing 100101 (China)
E-mail: chenyihua@im.ac.cn
the poly-b-lysine chain originate from l-arginine and l-lysine,
Dr. X. Lv, Prof. X. Li
CAS Key Laboratory of Pathogenic Microbiology & Immunology
Institute of Microbiology
Chinese Academy of Sciences
Beijing 100101 (China)
[
4]
respectively. Recent genetic and enzymatic studies demon-
strated that the poly-b-lysine chain is formed by atypical
nonribosomal peptide synthetases and then attached to
streptothrisamine (2) or 10-de-O-carbamoyl-12-O-carba-
+
[4a]
[
] These authors contributed equally to this work.
moyl-streptothrisamine (3; Figure 1C). However, little is
[
**] We thank Prof. Gongli Tang (Shanghai Institute of Organic
Chemistry, CAS) for providing the plasmid pTG6001, and Dr. Jinwei
Ren, Dr. Guomin Ai, and Dr. Wenzhao Wang (Institute of Micro-
biology, CAS) for MS and NMR data collection. We also thank Prof.
Geoff Horsman (Wilfrid Laurier University, Canada) for critically
reading the paper. This work was supported in part by MOST of
China (2013CB734003, 2015CB150602) and the NSFC (grants
known about the biosynthesis of the carbamoylated d-GulN
moiety except that d-glucosamine (d-GlcN) is a precursor,
based on an isotope-labeled feeding experiment. In addition
[
5]
to STNs, the d-GulN moiety also occurs in other natural
[6]
products such as gualamycin (Scheme 1A). Herein, we
elucidate the biosynthetic mechanism of the carbamoylated
d-GulN moiety and report StnG to be a GT-A fold N-
glycosyltransferase that catalyzes an unprecedented glycosy-
lation of a guanidinoimine and characterize StnI as the first N-
acetyl-d-gulosamine (d-GulNAc) deacetylase.
31170037, 31370095, 31200045). Y.C. is a scholar of “the 100 Talents
Project” of CAS.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201412190.
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
We previously reported the cloning of a silent STN
that StnG catalyzes the glycosylation of 4, the identity of the
sugar substrate was required to probe the activity of StnG
in vitro.
biosynthetic gene cluster from Streptomyces sp. TP-A0356
[7]
and its expression in Streptomyces coelicolor (Scheme 1B).
Bioinformatics analyses revealed that the stnG and stnQ
genes were the two best candidates for biosynthesis of the
carbamoylated d-GulN moiety. StnG is a putative type-A
glycosyltranferase (GT) that displays marginal homology
with characterized GTs in the GT-2 family (e.g. 16.1%
StnQ is a putative carbamoyltransferase sharing 53%
identity with TobZ, a well-characterized carbamoyltransfer-
[11]
ase involved in tobramycin biosynthesis. The DstnQ mutant
CIM1007 was also constructed by allelic replacement (see
Figure S3B in the Supporting Information) and like CIM1006
it lost its ability to produce STNs. However, CIM1007
accumulated a new metabolite (5; Figure 1) with the chemical
[
8]
identity to SpsL), and the DXD motif for binding divalent
metal ions in GT-A family enzymes is well conserved in StnG
[
9]
+
(
see Figure S2 in the Supporting Information). The DstnG
formula C H N O (HR-ESI-MS m/z 374.1666 [M+H] ,
1
4
23
5
7
mutant CIM1006 was constructed by replacing stnG with
a kanamycin resistance cassette (see Figure S3A in the
Supporting Information), which abolished STN production
but resulted in the accumulation of a new metabolite (4)
calcd 374.1676). NMR analysis of 5 revealed that its
streptolidine lactam group is conjugated with a b-d-GulNAc
through an N-glycosidic bond. The small coupling constants
of JH8H9 (2.9 Hz), JH9H10 (< 3.5 Hz), and JH10H11 (< 6.0 Hz)
indicate both H-9 and H-10 are in equatorial configura-
(
Figure 1). Notably, the DstnG mutant CIM1006 and the three
[
1f,g]
tions.
The acetyl group was assigned by its NMR signals
(
(
d : 2.00 ppm, d : 22.3 ppm (C-12 methyl) and d : 174.2 ppm
H
C
C
C-13)) and its attachment to 8-NH was shown by HMBC
2
correlation from H-8 (d : 4.23 ppm) to C-13 (see Table S4 and
H
Figure S7 in the Supporting Information). The absence of
a carbamoyl group in 5 implies that StnQ is a carbamoyl-
transferase decorating the d-GulNAc moiety, while the
surprising presence of the N-acetyl group suggests that
deacetylation is required prior to poly-b-lysine installation
at the 8-NH group en route to fully elaborated STNs.
2
Deacetylation may be catalyzed by the stnI gene, which
encodes a protein that displays moderate homology with
[12]
LmbE-like deacetylases (e.g. 23.0% identity to TT1542).
StnI possesses the HXDD motif for metal-ion binding and
catalysis that is conserved in LmbE-like deacetylases (see
[
13]
Figure S8 in the Supporting Information). Allelic replace-
ment generated the DstnI mutant CIM1008 (see Figure S3C in
the Supporting Information), which accumulated 6 as the
main metabolite (Figure 1). HR-ESI-MS analysis of 6 showed
+
an [M+H] ion at m/z 417.1727, consistent with the chemical
formula C H N O (calcd 417.1734; see Figure S9 in the
1
5
24
6
8
Supporting Information), and the mass difference between 5
and 6 indicates that 6 is a carbamoylated derivative of 5. The
NMR spectra of 6 confirmed the presence of a carbamoyl
group (d : 159.0 ppm (C-15)) connected to the 12-OH group
through an ester linkage by the HMBC correlation signal
C
Figure 1. HPLC metabolite profiles of the stn gene inactivation
mutants. CIM1001, S. coelicolor M145 harboring the stn cluster, which
produces STN F (1) together with N-acetyl-STN B (10) and N-acetyl-
STN A (11; Figure 1).
from H-12 (d : 4.22 ppm) to C-15 (see Table S5 and Figure S9
H
in the Supporting Information). These data suggest that StnI
functions as a deacetylase to remove the N-acetyl group of 6
to form 3. The equatorial configurations of H-9 and H-10 in
the d-GulNAc moiety were determined by the small coupling
constants of JH8H9 (2.8 Hz), JH9H10 (< 3.5 Hz), and JH10H11
(< 3.0 Hz). Interestingly, the isolation of 12-carbamoylated 6
as a main product from CIM1008 indicates that the carba-
moyltransferase StnQ prefers the 12-OH position. We
previously showed that the DstnO mutant CIM1004 accumu-
lated both 2 (10-O-carbamoylated) and 3 (12-O-carbamoy-
lated) in a ratio of about 2:5, which implies that StnQ may
also carbamoylate at the 10-OH position. Moreover, the
existence of both 10- and 12-carbamoylated STNs produced
by STN producers (see Figure S1 in the Supporting Informa-
stn mutants described below were all complemented success-
fully by expressing the replaced gene in trans, therefore
excluding the possibility of a polar effect (see Figure S4 in the
Supporting Information). The chemical formula of the water-
soluble compound 4 was determined to be C H N O by
6
10
4
2
high-resolution electrospray ionization mass spectrometry
+
(
HR-ESI-MS; m/z 171.0872 [M+H] , calcd 171.0877). Anal-
ysis of its 1D and 2D NMR data revealed that 4 has the same
planar structure as the streptolidine lactam (see Table S3 and
[
10]
Figure S5 in the Supporting Information),
and its
(
2S,3S,4R) configuration was confirmed by electronic circular
[1d,2]
dichroism (ECD) analysis (see Figure S6 in the Supporting
Information). In summary, although the results demonstrated
tion)
further supports a certain degree of promiscuous
StnQ regiospecificity.
2
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
As d-GulNAc deacetylase activity has not previously
and UDP-d-GlcNAc as substrates. To our delight, although
been observed, we sought to characterize StnI in vitro. We
the reaction was slow, a new product was detected by HPLC
analysis of an overnight assay. HR-ESI-MS analysis revealed
purified the N-His -tagged StnI (see Figure S10 in the
6
+
Supporting Information) and tested its deacetylase activity
by incubating the enzyme with 5 or 6. HPLC analyses
revealed no conversion of 5, but a new product was detected
when using 6 as a substrate (Figure 2A). The chemical
its chemical formula is C H N O (m/z 374.1675 [M+H] ,
1
4
23
5
7
calcd 374.1676), consistent with the expected product 8
(Figure 2B and see S13B in the Supporting Information).
We then examined StnG activity towards UDP-d-GulNAc
(synthesized as described in the Supporting Information) and
UDP-N-acetyl-d-galactosamine (UDP-d-GalNAc).
Although no reaction occurred when using the former
substrate, which indicates that the STN d-GulNAc moiety is
formed after glycosyl transfer, the effective conversion of 4
into a new product 9 was observed with the latter substrate
(
Figure 2B). Compound 9 shares the same chemical formula
+
C H N O as 5 and 8 (HR-ESI-MS m/z 374.1664 [M+H] ,
1
4
23
5
7
calcd 374.1676; see Figure S13D in the Supporting Informa-
tion), but could be clearly differentiated by careful HPLC
analysis (see Figure S16D in the Supporting Information).
The reaction conditions of StnG were then optimized using 4
and UDP-d-GalNAc as substrates (see Figure S14 in the
Supporting Information). Steady-state kinetic analyses under
the optimized conditions (pH 8.0, 308C with 0.05 mm Mn )
revealed Michaelis–Menten behavior for all substrates, and
the resulting steady-state kinetic constants clearly demon-
strate a strong preference of UDP-d-GalNAc over UDP-d-
GlcNAc as the sugar donor for StnG (Table 1 and see
Figure 2. HPLC analysis of representative assays of StnI (A) and StnG
(
B). i) 6 standard; ii) 6 + StnI; iii) 6 + denatured StnI; iv) 4 standard;
v) 4 + UDP-GlcNAc + StnG; vi) 4 + UDP-GalNAc + StnG; vii) 4 +
UDP-GulNAc + StnG; viii) 4 + UDP-GalNAc + denatured StnG.
Detection at 190 nm.
2
+
formula of the new product was C H N O , the same as 3, as
1
3
22
6
7
+
determined by HR-ESI-MS (m/z 375.1607 [M+H] , calcd
75.1623; see Figure S11 in the Supporting Information).
3
Table 1: StnG kinetic parameters for different substrates.
These results suggest that StnQ first carbamoylates the d-
GulNAc moiety of 5 to form 6, which is then deacetylated by
StnI to generate 3. Significantly, as all the previously
characterized LmbE family deacetylases catalyze the deace-
[
a]
Toward UDP-GlcNAc and UDP-GalNAc as Donors
À1
À1 À1
Donor
Km [mm]
kcat [min
]
kcat/Km [s m ]
UDP-GlcNAc
UDP-GalNAc
1.15Æ0.12
0.27Æ0.03
3.2Æ0.31
72.2Æ6.9
46.4Æ4.8
3
[13]
4.46ꢀ10 Æ412
tylation of N-acetyl-d-glucosamine (d-GlcNAc),
expands the substrate spectrum of this enzyme family.
StnI
[
b]
Given that the d-GulNAc moiety exists in both 5 and 6,
we speculated that d-GlcNAc, the acetylated derivative of the
identified precursor d-GlcN, is a precursor of STN biosyn-
Toward 4 as an Acceptor
À1
À1 À1
Acceptor
Km [mm]
kcat [min
]
kcat/Km [s
m ]
3
4
0.42Æ0.04
86.7Æ8.5
3.44ꢀ10 Æ347
1
3
thesis. To validate this hypothesis, N-[1,2- C ]-d-GlcNAc was
2
[a] With saturating 4 (6 mm) as the acceptor. [b] With saturating UDP-
fed to the DstnI mutant CIM1008. As anticipated, N-[1,2-
GalNAc (5 mm) as the donor.
1
3
C ]-d-GlcNAc was incorporated into 6 efficiently when
2
analyzed by MS (see Figure S12 in the Supporting Informa-
tion), thereby proving the intermediacy of d-GlcNAc in the
biosynthesis of the d-GulNAc moiety.
Figure S15 in the Supporting Information). UDP-d-GalNAc
is also abundant in Streptomyces, and normally originates
from UDP-d-GlcNAc through UDP-d-GlcNAc 4-epimerase
(e.g. SCO3137 and SCO2988 in S. coelicolor M145). Conse-
quently, we propose that StnG attaches d-GalNAc to the
guanidine of 4 to form 9, which is then converted into 5 by an
epimerase.
As sugars are typically activated to NDP sugars by
a nucleotidyltransferase prior to GT-catalyzed installation
[
14]
on an aglycone, we proposed that StnG recuits an NDP
sugar from primary metabolism because: 1) no nucleotidyl-
transferase-encoding gene exists in the stn cluster; and 2) the
stn cluster can be expressed in different heterologous hosts
Most GTs involved in secondary metabolite biosynthesis
[7]
[15]
including S. coelicolor and Streptomyces albus, which sug-
gests that the substrate of StnG is an NDP sugar common in
Streptomyces. The identification of d-GlcNAc as a precursor
of STNs implicates UDP-d-GlcNAc as a potential substrate,
which is one component of the ubiquitous peptidylglycoside
comprising the cell wall of the Gram-positive bacteria
Streptomyces.
belong to the GT-B family, which possess one domain for
NDP-sugar binding and another for aglycone recognition. In
contrast, the GT-A family enzymes are single-domain pro-
[
15]
teins, and to our knowledge ValG is the only characterized
GT-A fold GT involved in secondary metabolite biosynthesis.
While ValG is an O-GT that adds d-glucose to validoxyl-
[
16]
amine to form validamycin,
StnG represents the first
To verify the proposed activity of StnG toward UDP-d-
reported GT-A fold N-GT involved in secondary metabolite
biosynthesis, and furthermore catalyzes the unprecedented
attachment of a sugar to the imine nitrogen atom of
GlcNAc, we purified N-His -tagged StnG (see Figure S10 in
6
the Supporting Information) and incubated the enzyme with 4
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
guanidine. Considering that the guanidine moiety is wide-
spread in biological molecules such as proteins (e.g. Arg) and
nucleic acids (e.g. guanine), we speculate that this unusual N-
glycosylation may not be limited to natural products. Intrigu-
ingly, glycosylation of Arg residues in proteins has recently
been shown to be required for bacterial colonization of
[
1] a) S. A. Waksman, J. Bacteriol. 1943, 46, 299 – 310; b) V.
Zbinovsky, W. K. Hausmann, E. R. Wetzel, D. B. Borders,
E. L. Patterson, Appl. Microbiol. 1968, 16, 614 – 616; c) Y.
Kido, T. Furuie, K. Suzuki, K. Sakamoto, Y. Yokoyama, M.
Uyeda, J. Kinjyo, S. Yahara, T. Nohara, M. Shibata, J. Antibiot.
1987, 40, 1698 – 1706; d) M. Gan, X. Zheng, L. Gan, Y. Guan, X.
Hao, Y. Liu, S. Si, Y. Zhang, L. Yu, C. Xiao, J. Nat. Prod. 2011, 74,
[17]
enteropathic E. coli in a mouse model,
but the exact
1
142 – 1147.
2] Z. Ji, M. Wang, J. Zhang, S. Wei, W. Wu, J. Antibiot. 2007, 60,
39 – 744.
position and mechanism of glycosylation remains unknown.
Our finding therefore provides a potential GT-catalyzed
route to this post-translational modification.
[
[
7
3] a) Y. Sawada, S. Nakashima, H. Taniyama, Y. Inamori, Chem.
Pharm. Bull. 1977, 25, 1478 – 1481; b) T. K. Thiruvengadam, S. J.
Gould, D. J. Aberhart, H. J. Lin, J. Am. Chem. Soc. 1983, 105,
5470 – 5476; c) M. D. Jackson, S. J. Gould, T. M. Zabriskie, J.
Org. Chem. 2002, 67, 2934 – 2941.
The stnJ gene is the only candidate encoding an epimerase
in the stn cluster. StnJ is a putative bifunctional protein with
its N-terminus showing moderate homology with dehydro-
[
18]
genases (e.g. 20.7% identity with WIbA) and C-terminus
showing moderate homology with NAD-dependent epimer-
[
4] a) C. Maruyama, J. Toyoda, Y. Kato, M. Izumikawa, H. Takagi,
K. Shin-ya, H. Katano, T. Utagawa, Y. Hamano, Nat. Chem. Biol.
[
19]
ases (e.g. 20.2% identity with GalE). The DstnJ mutant
CIM1009 (see Figure S3D in the Supporting Information)
accumulated a metabolite possessing the same HPLC reten-
tion time as 9 (Figure 1 and see Figure S16D in the Supporting
Information). As expected, the chemical formula of the
accumulated compound is C H N O , the same as 9, when
2012, 8, 791 – 797; b) C. Y. Chang, S. Y. Lyu, Y. C. Liu, N. S. Hsu,
C. C. Wu, C. F. Tang, K. H. Lin, J. Y. Ho, C. J. Wu, M. D. Tsai,
T. L. Li, Angew. Chem. Int. Ed. 2014, 53, 1943 – 1948; Angew.
Chem. 2014, 126, 1974 – 1979.
[5] V. A. Palaniswamy, S. J. Gould, J. Chem. Soc. 1988, 8, 2283 –
286.
[6] K. Tsuchiya, S. Kobayashi, T. Kurokawa, T. Nakagawa, N.
Shimada, H. Nakamura, Y. Iitaka, M. Kitagawa, K. Tatsuta, J.
Antibiot. 1995, 48, 630 – 634.
2
1
4
23
5
7
+
analyzed by HR-ESI-MS (m/z 374.1672 for [M+H] and
96.1488 for [M+Na] , calcd 374.1676 and 396.1490, respec-
+
3
tively; see Figure S16C in the Supporting Information), which
indicates that StnJ catalyzes the epimerization of the 9-OH of
[
7] J. Li, Z. Guo, W. Huang, X. Meng, G. Ai, G. Tang, Y. Chen, Sci.
China Life Sci. 2013, 56, 619 – 627.
9
to form 5.
[8] T. J. Pollock, W. A. van Workum, L. Thorne, M. J. Mikolajczak,
Based on these results, we propose a biosynthetic pathway
M. Yamazaki, J. W. Kijne, R. W. Armentrout, J. Bacteriol. 1998,
1
80, 586 – 593.
9] a) N. Tarbouriech, S. J. Charnock, G. J. Davies, J. Mol. Biol. 2001,
14, 655 – 661; b) L. L. Lairson, B. Henrissat, G. J. Davies, S. G.
Withers, Annu. Rev. Biochem. 2008, 77, 521 – 555.
for the carbamoylated d-GulN moiety of STNs. The unpre-
cedented guanidino-N-glycosyltransferase StnG attaches
UDP-d-GalNAc to 4 to generate 9, which is converted into
[
3
5
by the putative bifunctional dehydrogenase/epimerase StnJ;
StnQ then decorates 5 by adding the carbamoyl group to its
2-OH group to generate 6; finally, StnI catalyzes the
[
10] M. Kinoshita, Y. Suzuki, Bull. Chem. Soc. Jpn. 1977, 50, 2375 –
2378.
1
[11] C. Parthier, S. Gorlich, F. Jaenecke, C. Breithaupt, U. Brauer, U.
Fandrich, D. Clausnitzer, U. F. Wehmeier, C. Bottcher, D. Scheel,
M. T. Stubbs, Angew. Chem. Int. Ed. 2012, 51, 4046 – 4052;
Angew. Chem. 2012, 124, 4122 – 4128.
12] N. Handa, T. Terada, Y. Kamewari, H. Hamana, J. R. Tame, S. Y.
Park, K. Kinoshita, M. Ota, H. Nakamura, S. Kuramitsu, M.
Shirouzu, S. Yokoyama, Protein Sci. 2003, 12, 1621 – 1632.
[13] A. Deli, D. Koutsioulis, V. E. Fadouloglou, P. Spiliotopoulou, S.
Balomenou, S. Arnaouteli, M. Tzanodaskalaki, K. Mavromatis,
M. Kokkinidis, V. Bouriotis, FEBS J. 2010, 277, 2740 – 2753.
14] C. J. Thibodeaux, C. E. Melancon 3rd, H.-w. Liu, Angew. Chem.
Int. Ed. 2008, 47, 9814 – 9859; Angew. Chem. 2008, 120, 9960 –
deacetylation of the carbamoylated d-GulNAc moiety to
form 3 (Scheme 1C). In the proposed pathway, an unexpected
acetylated sugar UDP-d-GalNAc is used as the precursor and
the N-acetyl group is detached just prior to installation of the
poly-b-lysine chain, in a process reminiscent of the protecting
group chemistry employed during the chemical synthesis of
amino sugars. Interestingly, similar protecting group logic has
[
[20]
been used in nature for the biosynthesis of teicoplanin.
[
Finally, the availability of several new STN intermediates
motivated experiments to test the antibiotic activity of 4, 5,
and 6 against Staphylococcus aureus and Pseudomonas
aeruginosa. No inhibition was observed (see Table S6 in the
Supporting Information), thus indicating that the poly-b-
lysine chain is indispensible for the antibiotic activity of STNs.
In summary, we have elucidated the biosynthetic mech-
anism of the carbamoylated d-GulN moiety of STNs. Two
novel enzymes, StnG and StnI, were characterized as a GT-A
fold GT adding a sugar to the guanidino-imine group and an
LmbE-like d-GulNAc deacetylase, respectively.
10007.
[
15] a) Y. Hu, S. Walker, Chem. Biol. 2002, 9, 1287 – 1296; b) S. Singh,
G. N. Phillips, Jr., J. S. Thorson, Nat. Prod. Rep. 2012, 29, 1201 –
1237.
[16] H. Xu, K. Minagawa, L. Bai, Z. Deng, T. Mahmud, J. Nat. Prod.
008, 71, 1233 – 1236.
2
[
17] S. Li, L. Zhang, Q. Yao, L. Li, N. Dong, J. Rong, W. Gao, X. Ding,
L. Sun, X. Chen, S. Chen, F. Shao, Nature 2013, 501, 242 – 246.
18] J. B. Thoden, H. M. Holden, Biochemistry 2011, 50, 1483 – 1491.
19] H. Sakuraba, T. Kawai, K. Yoneda, T. Ohshima, Arch. Biochem.
Biophys. 2011, 512, 126 – 134.
[
[
[
20] a) J. Y. Ho, Y. T. Huang, C. J. Wu, Y. S. Li, M. D. Tsai, T. L. Li, J.
Am. Chem. Soc. 2006, 128, 13694 – 13695; b) A. W. Truman, L.
Robinson, J. B. Spencer, ChemBioChem 2006, 7, 1670 – 1675.
Received: December 19, 2014
Published online: && &&, &&&&
Keywords: aminoglycoside · biosynthesis · deacetylation ·
.
d-gulosamine · glycosylation
4
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Biosynthesis
Z. Guo, J. Li, H. Qin, M. Wang, X. Lv, X. Li,
Y. Chen*
&&&& — &&&&
Biosynthesis of the Carbamoylated d-
Gulosamine Moiety of Streptothricins:
Involvement of a Guanidino-N-
glycosyltransferase and an N-Acetyl-d-
gulosamine Deacetylase
The dynamic duo: Two novel enzymes,
StnG and StnI, have been found to be
involved in the biosynthetic pathway of
the carbamoylated d-gulosamine moiety
in streptothricins. StnG is a GT-A fold
glycosyltransferase that catalyzes the
unprecedented attachment of a sugar to
the imine nitrogen atom of a guanidine
group; StnI catalyzes the deacetylation of
the N-acetyl-d-gulosamine moiety.
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!