Biosynthetic Origin of the Atypical Stereochemistry in the Thioheptose Core of Albomycin Nucleoside Antibiotics

Article abstract of DOI:10.1021/jacs.8b12565

Albomycins are peptidyl thionucleoside natural products that display antimicrobial activity against clinically important pathogens. Their structures are characterized by a thioheptose with atypical stereochemistry including a d-xylofuranose ring modified with a d-amino acid moiety. Herein it is demonstrated that AbmH is a pyridoxal 5′-phosphate (PLP)-dependent transaldolase that catalyzes a threo-selective aldol-type reaction to generate the thioheptose core with a d-ribofuranose ring and an l-amino acid moiety. The conversion of l-to d-amino acid configuration is catalyzed by the PLP-dependent epimerase AbmD. The d-ribo to d-xylo conversion of the thiofuranose ring appears according to gene deletion experiments to be mediated by AbmJ, which is annotated as a radical S-adenosyl-l-methionine (SAM) enzyme. These studies establish several key steps in the assembly of the thioheptose core during the biosynthesis of albomycins.

Full text of DOI:10.1021/jacs.8b12565

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Communication
Biosynthetic Origin of the Atypical Stereochemistry in the
Thioheptose Core of Albomycin Nucleoside Antibiotics
Richiro Ushimaru, and Hung-wen Liu
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 23 Jan 2019
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Journal of the American Chemical Society
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Biosynthetic Origin of the Atypical Stereochemistry in the Thioheptose
Core of Albomycin Nucleoside Antibiotics
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Richiro Ushimaru and Hung-wen Liu*
Department of Chemistry, and Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, University of Texas at
Austin, Austin, Texas 78712, United States
Supporting Information Placeholder
in nature. Whether these unusual stereochemistries are in-
herited directly from the biosynthetic precursors or intro-
duced after the core has been assembled is unknown.
ABSTRACT: Albomycins are peptidyl thionucleoside natural
products that display antimicrobial activity against clinically
important pathogens. Their structures are characterized by a
thioheptose with atypical stereochemistry including a D-
xylofuranose ring modified with a D-amino acid moiety. Herein
it is demonstrated that AbmH is a pyridoxal 5′-phosphate
NH
N
2
NH
N
2
4
AmbH
OH
OH
5
'
OH
S
N
L-Thr (
9
)
N
H N 6'
2
S
Unknown
precursors
O
O
5'
O
(PLP)-dependent transaldolase that catalyzes a threo-selective
H
PLP
H
H
OH
CO
2
OH
aldol-type reaction to generate the thioheptose core with a D-
ribofuranose ring and an L-amino acid moiety. The conversion
of L- to D-amino acid configuration is catalyzed by the PLP-
dependent epimerase AbmD. The D-ribo to D-xylo conversion
of the thiofuranose ring appears according to gene deletion ex-
periments to be mediated by AbmJ, which is annotated as a
radical S-adenosyl-L-methionine (SAM) enzyme. These studies
establish several key steps in the assembly of the thiohep-
tose core during the biosynthesis of albomycins.
8
10
NH
N
2
tailoring
and/or
degradation
ornithine x 3
and serine
OH
H
N
OH
OH
O
6'
S
N
ferrichrome
N
5
'
O
ATP
Fe(III)
H
O
H
2
CO H
OH
1
1
Base
S
7
albomycin δ
Me albomycin ε (
O
2
(
1
) X = NCONH
2
Me
Me
O
O
O
FeIII
2
) X = NH
OH
albomycin δ1 (3)
X = O
N
N
N
X
O
O
HO
3'
D-xylo
OH
OH
N Me
O
S
O
O
OH
S
H
N
H
6'
N
N
N
H
N
H
5'
5'
6'
H
2
N
HO
L-erythro
Albomycins (1–3) are sulfur-containing sideromycin
O
O
H
3'
RHN
2
CO H
OH
antibiotics isolated from several species of Streptomy-
ferrichrome-Fe(III) (5)
L-Ser
2
CO H
1
D-amino
cetes. The chemical structures of the albomycins consist of
4
acid
a 6'-amino-4'-thioheptose nucleoside (4) and an iron-
chelating ferrichrome siderophore (5) that are connected
via amide linkages to a serine residue (see Scheme 1). This
2
SB-217452 (6, X = NCONH )
2
Scheme 1. Proposed biosynthetic pathway of albomy-
cins
allows albomycins to be actively transported via the bacte-
3
rial iron uptake system into bacterial cells, where they are
The a-amino-b-hydroxy acid moiety at C5'-C6' of 4 is
found in several peptidyl nucleoside natural products such
as the liposidomycins, caprazamycins and muraymycins. It
has been shown that this functional group is constructed in
an aldol-type C–C bond forming reaction catalyzed by a
PLP-dependent serine hydroxymethyl transferase homo-
hydrolyzed by host peptidases liberating the thioheptose
containing SB-217452 moiety (6) and the ferrichrome 5.4
6
SB-217452 (6) has been established as an inhibitor of bac-
terial seryl-tRNA synthetase due to its resemblance to seryl
4
adenylate. Hence, albomycins have potent antimicrobial
activities against many Gram-negative and Gram-positive
bacteria.
7
logue using L-threonine and an aldehyde precursor as sub-
8
strates. A similar enzymatic conversion is also operative in
9
The gene cluster responsible for albomycin production in
the biosynthesis of obafluorin. Based on BLAST analysis,
5
Streptomyces sp. ATCC 700974 has been sequenced. Alt-
abmH in the albomycin gene cluster exhibits sequence sim-
ilarity to genes encoding L-threonine:aldehyde transal-
dolases. Hence, the gene product AbmH was hypothesized
hough several enzymes responsible for the tailoring modi-
fications have been identified, little is known about how the
5
6
'-amino-4'-thioheptose moiety (4) is biosynthesized. In
to catalyze an aldol reaction between a 5'-oxo-4'-
thionucleoside such as 8 and L-threonine (9) to form the
C5'–C6' bond (Scheme 1). The putative AbmH reaction
product 10 may then be coupled with L-serine or a deriva-
particular, the D-configuration (6'R) of the a-amino acid
and the D-xylo-configuration (3'R) of the furanose ring in
the thioheptose core (see 4 and 7) are less commonly found
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Page 2 of 6
tive thereof in the subsequent biosynthetic steps resulting in
production of 11, which is the predicted precursor of 1.
suggested an erythro-configuration at C5'/C6' of 16 (5'S,6'R
or its 5'R,6'S-isomer, see Figure 1A). This assignment is
supported by comparison with the carbamate derivative 19
derived from 1, which has a J5',6' coupling of 9.9 Hz.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To test the proposed role of AbmH, N-His -tagged
6
AbmH was heterologously overexpressed and purified
from Escherichia coli (Figure S2). Since attempts to syn-
thesize 8 as a substrate for AbmH were unsuccessful due to
complications involving the cross-coupling between the 4-
amino and 5'-aldehyde groups, the uracil analogue 12 (ex-
isting as its hydrate form 13) was synthesized as an alterna-
tive substrate (Figure 1A and Supporting Information).
When 12/13 (0.6 mM) was incubated with AbmH (3.2 µM)
and L-threonine (9, 5 mM) in 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) buffer (50 mM, pH
However, compound 8 represented by its synthetically
accessible analogue 12/13 may not be the natural substrate
of AbmH, because it is a D-xylose-based nucleoside, which
is unusual, and several stereoisomers of 16 were also ob-
served during the incubation of 12/13 with AbmH. An al-
ternative candidate is the 3'S-epimer of 8 (i.e., 8ribo, see
Figure 2A), which has a D-ribose configuration. This would
suggest that epimerization of C3' is a necessary step prior
to completion of the thioheptose core of 1. The abm cluster
contains a gene, abmJ, which is annotated as encoding a
radical SAM enzyme. It is well-known that certain radical
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.5), formation of several new compounds was detected by
HPLC (Figure 1B). Based on electrospray ionization-mass
spectroscopy (ESI-MS) and nuclear Overhauser effect
spectroscopy (NOESY), the broad peak with a retention
time at ~9 min was assigned as 15, which is the nonenzy-
matically generated 4'R-epimer of 13 (Figures S28). In
contrast, a product peak at ~8 min displayed a mass con-
sistent with an aldol adduct, such as 16 (calcd m/z for
SAM enzymes are capable of catalyzing epimerization re-
11,12
actions at unactivated centers.
It was thus hypothesized
that AbmH catalyzes the aldol-type reaction of 8ribo with L-
threonine (9) to yield 10ribo prior to AbmJ-catalyzed C3'-
epimerization to produce the thioheptose core in 1 (Figure
2A). To test this hypothesis, abmJ was in-frame deleted in
the producing strain (Figures 2B and S12). The DabmJ
strain produced ferrichrome 5 as shown by HPLC and MS
analysis; however, two new products were also observed.
One had a mass consistent with that of 1 in its iron-chelated
+
+
C
11
H
16
N
3
O S [M+H] 334.0703; obsd 334.0686, see Fig-
7
ure S29). A similar ESI-MS result was also obtained for the
minor peak at 6 min revealing the formation of another
aldol adduct with the same chemical composition. Moreo-
1
+
+
ver, H NMR analysis indicated that the latter peak con-
form (calcd m/z for C37H58FeN12O18S [M+H] 1046.3057;
tained a mixture of two diastereomers of 16 (see Support-
ing Information).
obsd 1046.2985, see Figure S29) and the other had a mass
identical to that of SB-217452 (6, calcd m/z for
+
+
C
16
H
25
N
6
O S [M+H] 477.1398; obsd 477.1382, see Fig-
9
(
A)
AbmH
ure S29). However, neither of these two new products coe-
luted with 1 or 6 by HPLC, implying that they are epimers
of 1 and 6 thereby implicating 22 and 21, respectively.
OH
OH
S
OH
OH
– H2O
OH
S
L-Thr (9) H
OH
S
OH
S
U
U
U
U
2
N
6'
2
H N
6'
O
HO
4'
5'
5'
H
H
MeCHO
HO
2
C
H
or HO
2
C
H
OH
OH
OH
OH
13
U = uracil)
12
(5'S,6'R)-16
(5'R,6'S)-16
(
(B)
OH
S
OH
S
H
U
2
– H O
H
U
15
HO
AU
13
1
6
O
OH
– AbmH
AbmH
OH
OH
1
5
14
+
(C)
(5'R,6'S)-17: J5',6' = 8.2 Hz
O
time (min)
O
O
O
HN
O
NCONH
NMe
2
O
or
6' 5'
HN
2
O
HN
O
O
NH
HO C
OH
HN
HO
2
C
N
N
OH
S
O
S
6
'
5'
6' 5'
6
'
5'
HO C
O
O
H
2
H
HO
2
C
H
HO OH
OH
(5'S,6'R)-17: J5',6' = 8.2 Hz
18 (5'S,6'S): J5',6' = 5.0 Hz 19 (5'S,6'R): J5',6' = 9.9 Hz
Figure 1. (A) AbmH reaction of 12/13. (B) HPLC-UV
analysis. (C) Phosgene-derivatized AbmH-product 17
(
5'S,6'R or its 5'R,6'S-isomer) and its analogues.
To establish the stereochemistry of the newly formed
stereogenic centers C5'/C6', the major AbmH product was
isolated and derivatized with phosgene to afford a cyclic
10 1
carbamate (see Supporting Information). H NMR analy-
sis of the carbamate (17) showed a value of 8.2 Hz for the
J
5',6' coupling that is significantly different from the report-
ed J5',6' coupling (5.0 Hz) for 18, which has threo-
8a
stereochemistry at C5'/C6' (Figure 1C). This outcome
2
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Journal of the American Chemical Society
(
A)
NH2
NH2
NCONH2
configuration at C3'. Hence, the annotated radical SAM
enzyme AbmJ appears to be necessary for C3' epimeriza-
tion from the S to R stereochemistry during the biosynthesis
of 1. This hypothesis is also supported by the observation
that complementation of the DabmJ mutant with abmJ par-
tially restored production of 1 and 6 (Figure S13). While 1
showed antimicrobial activity against E. coli as previously
N
O
AbmH
L-Thr (
N
O
Me
N
1
2
3
4
5
6
7
8
9
OH
H2N 6' 5'
OH
N
N
AbmJ
OH
S
N
9)
S
O
S
RHN 6' 5'
O
H
HO2
C
H
MeCHO
HO2C
H
3'
HO OH
HO OH
OH
8
ribo
(5'S,6'R)-10ribo
1
(B) EIC m/z = 1046.3
Me
O
Me
O
O
O
Me
FeIII
O
N
N
N
X
2
2
O
1
OH
H
N
1b
N
O
Me
reported, 22 exhibited significantly reduced bacterial
coinjection
wild + ΔabmJ)
O
O
OH
H
N
N
(
6'
S
3'
N
H
N
H
5'
H
growth inhibition based on disc-diffusion bioassays (Figure
S26). These results imply the importance of the proposed
C3'-epimerization for the biological activity of albomycins.
H2N
ΔabmJ
wild type
1 (standard)
O
O
CO2H
HO OH
ferrichrome-Fe(III) (5)
L-Ser
2
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1 (X = NCONH2)
time (min)
The proposed activity of AbmJ also implies that the bio-
synthetic substrate for AbmH is 8ribo rather than 8. There-
fore, to further test this hypothesis, the uracil analogue 26
(existing as its hydrate form 27) was prepared (see Support-
ing Information). Upon incubation of 26/27 and L-
threonine (9) with AbmH, a single aldol product was ob-
served (Figures 2D and S14) in addition to the non-
enzymatically generated 4'R-epimer of 27 (structure not
albomycin isomer (22, 3'S-epimer
)
(C)
O
NCONH2
NOE
OH
enzymatic
digestion H2N
6
NMe
O
enzymatic
digestion
OH
6
NMe
OH
N
N
S
H2N
S
O
2
2
1
1'
3
'
(and
H 3'
H
CO2H
NOE
HO2C
deamination)
HO OH
OH
25
2
3
phosgene
phosgene
O
O
O
NCONH2
NMe
O
O
6
HN
HN
OH
NMe
N
N
1
6'
5'
S
6
'
5'
S
shown). H NMR analysis after phosgene derivatization of
HO2C
O
HO2C
O
H
H
HO OH
OH
the aldol product demonstrated a coupling of J5',6' = 5.1 Hz
indicating an unexpected threo-configuration at C5'/C6'
consistent with 28 (5'S,6'S or its 5'R,6'R-isomer). The for-
mation of the threo-product from 26/27 by AbmH is diffi-
cult to reconcile with the results from the abmJ deletion
experiments, because in the latter case the isolated product
22 has an erythro-configuration at C5'/C6'. Hence, in albo-
mycin biosynthesis, the nascent product of the AbmH reac-
tion (the 28 equivalent) must undergo isomerization to give
24 (5'S,6'R): J5',6' = 9.7 Hz
19 (5'S,6'R): J5',6' = 9.9 Hz
(D)
AbmH
L-Thr (9)
OH
H2N 6' 5'
OH
U
S
U
AbmD H N 6'
U
O
H
S
2
S
5'
MeCHO
HO2C H
K
eq ~ 0.4
HO2C
H
HO OH
HO OH
HO OH
(5'S,6'R)-29
2
6
(5'S,6'S)-28
or
OH
OH
H
2
O
U
S
H N 6' 5
U
HO
2
S
'
H
H
HO2C
HO OH
HO OH
(5'R,6'R)-28
2
7
(
E)
the 5'S,6'R-configuration as seen in 10ribo.
OH
H
AbmH
L-Thr (9) H N 6'
OH
OH
U
U
U
H N 6'
U
– H
2
O
O
2
O
2
O
O
O
5'
5'
HO
+
H
HO2C
H
HO2C
H
Epimerization of amino acids can be catalyzed by PLP-
MeCHO
HO OH
HO OH
HO OH
HO OH
13
dependent enzymes. The gene abmD in the abm gene
31
30
(5'S,6'S)-32
(5'R,6'S)-33
cluster encodes another PLP-dependent enzyme although it
does not show high sequence similarity to known racemas-
es/epimerases. While it has been reported that overexpres-
Figure 2. (A) An alternative proposal of the albomycin
biosynthesis. (B) LC-MS analysis of the metabolites from
the DabmJ strain. Extracted ion chromatogram (EIC) traces
14
sion of abmD increases the level of 1 production, the
catalytic role of the gene product AbmD has not been char-
acterized. To determine whether AbmD can catalyze the
epimerization of the AbmH-product 28 (or its physiological
+
corresponding to [M+H] signals from 1 or 22 are shown.
(C) NOESY analysis and phosgene derivatization of the
digested compounds. (D) Reaction of 26/27 with AbmH
and AbmD to afford 28 and 29. (E) Reaction of 30/31 with
AbmH.
equivalent), N-His -tagged AbmD was heterologously
6
overexpressed and purified from E. coli (Figure S2). The
UV absorption at 413 nm indicated the presence of PLP as
an internal aldimine in AbmD (Figure S3). When the
AbmH product (28, 0.056 mM) was incubated with AbmD
(4.6 µM) in HEPES buffer (50 mM, pH 7.5), a new product
was detected by HPLC (Figures S15 and S16). ESI-MS
analysis indicated that the product has the same mass as 28
(Figure S29). The product structure was characterized to be
29 by NMR and coelution with the synthetic 5'S,6'R-
standard (Figures 2D, S18 and S66-70).
Leucine amino peptidase was used to cleave the thionu-
cleoside moiety from the iron-chelating ferrichrome in or-
der to characterize the products isolated from the DabmJ
strain by NMR (Figure 2C). When the resulting thionucleo-
side was derivatized using phosgene, a J5',6' coupling of 9.7
Hz in the corresponding carbamate was noted, indicating an
erythro-configuration at C5'/C6' consistent with 24. More-
1
over, H NMR NOESY analysis of the thionucleoside re-
vealed a correlation between H-6 and H-3' (Figures 2C and
S28). These observations support assignment of the thionu-
cleoside as 23. In contrast, a correlation between H-1' and
H-3' was observed in 25 derived from hydrolysis of 1 (Fig-
ures 2C and S28). Collectively, these results indicate that
the DabmJ metabolite is indeed 22, which has the S-
To verify the position of epimerization (C5' or C6') re-
sulting in 29, the AbmD reaction with 28 was run in D
2
O
(
92% D). Incorporation of a single deuterium into 29 (83%
D) was observed, and no dideuteration was noted as would
be expected for β-epimerization involving hydrogen ex-
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Journal of the American Chemical Society
change at C5' (Figure S19). Likewise, when the reaction tural variation and the biological activity important for the
Page 4 of 6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
18
18
18
was conducted in H
2
O (90% O), no O-labeling was
survival of the producing microorganisms.
found in 29 as would be expected for β-epimerization
involving hydroxyl-exchange at C5' (Figure S19). These
observations rule out b-epimerization (i.e., at C5') as the
process leading to 29 and thus indicate that AbmD-
catalyzed epimerization only occurs at the a-carbon (i.e.,
C6') of the amino acid moiety (Figure S20). Hence, the
AbmD-substrate, which is the AbmH-product, is (5'S,6'S)-
28.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on
the ACS Publications website (http://pubs.acs.org)
Details regarding experimental procedures and spectro-
scopic data (PDF).
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AUTHOR INFORMATION
The demonstrated activities of AbmH and AbmD strong-
ly suggest that 8ribo is the substrate in the AbmH-catalyzed
C5'–C6' formation. However there remains the possibility
that the natural substrates of AbmH and AbmD are actually
Corresponding Author
*h.w.liu@mail.utexas.edu
ORCID
Hung-wen Liu: 0000-0001-8953-4794
4
'-oxynucleosides rather than 4'-thionucleosides such that
the sulfur atom is inserted after the AbmH/AbmD-
catalyzed reactions. To investigate this possibility, com-
pound 30 (existing as its hydrate form 31) was chemically
synthesized and incubated with AbmH under the standard
assay conditions (Figure 2E). Two products were formed in
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by grants from the National Insti-
tutes of Health (GM035906 and 1 S10 OD021508-01 for
NMR) and the Welch Foundation (F-1511). We thank Jessi
Cai for her assistance in the preparation of AbmH.
~
1:1 ratio (Figure S21). One was identified as 32 with a
threo-configuration (5'S,6'S) at C5'/C6' (J5',6' = 5.2 Hz in the
carbamate form) and the other was an erythro-heptose nu-
cleoside (5'R,6'S)-33 (J5',6' = 9.7 Hz in the carbamate form)
based on the results of phosgene derivatization and com-
parison with standards (Supporting Information and Figure
S22). These observations showed that the 4'-oxynucleoside
can also be converted by AbmH, but with much reduced
diastereoselectivity and catalytic efficiency (a 14-fold dif-
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m
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TOC
OH
5'
OH
OH
AbmH
PLP
B
AbmD
PLP
B
B
5'
H
2
N
2
H N
S
S
S
HO
6'
6'
L
-Thr
H
H
H
HO
2
C
2
HO C
OH OH
OH OH
OH OH
B = nucleobase
siderophore
NCONH
2
HO
OH
N
O
Me
H
N
OH
S
5
'
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N
H
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'
AbmJ
radical SAM
O
H
3
'
HO
2
C
OH
Albomycin δ
2
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