In vitro characterization of LmbK and LmbO: Identification of GDP-d-erythro-α-d-gluco-octose as a key intermediate in lincomycin a biosynthesis

Article abstract of DOI:10.1021/ja412194w

Lincomycin A is a clinically useful antibiotic isolated from Streptomyces lincolnensis. It contains an unusual methylmercapto-substituted octose, methylthiolincosamide (MTL). While it has been demonstrated that the C ₈ backbone of MTL moiety is derived from d-fructose 6-phosphate and d-ribose 5-phosphate via a transaldol reaction catalyzed by LmbR, the subsequent enzymatic transformations leading to the MTL moiety remain elusive. Here, we report the identification of GDP-d-erythro-α-d-gluco-octose (GDP-d-α-d-octose) as a key intermediate in the MTL biosynthetic pathway. Our data show that the octose 1,8-bisphosphate intermediate is first converted to octose 1-phosphate by a phosphatase, LmbK. The subsequent conversion of the octose 1-phosphate to GDP-d-α-d-octose is catalyzed by the octose 1-phosphate guanylyltransferase, LmbO. These results provide significant insight into the lincomycin biosynthetic pathway, because the activated octose likely serves as the acceptor for the installation of the C1 sulfur appendage of MTL.

Full text of DOI:10.1021/ja412194w

Communication
pubs.acs.org/JACS
In Vitro Characterization of LmbK and LmbO: Identification of GDP‑D-
erythro-α‑D-gluco-octose as a Key Intermediate in Lincomycin A
Biosynthesis
Chia-I Lin, Eita Sasaki, Aoshu Zhong, and Hung-wen Liu*
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry, University of Texas at Austin, Austin, Texas
78712, United States
S
* Supporting Information
ABSTRACT: Lincomycin A is a clinically useful antibiotic
isolated from Streptomyces lincolnensis. It contains an
unusual methylmercapto-substituted octose, methylthio-
lincosamide (MTL). While it has been demonstrated that
the C8 backbone of MTL moiety is derived from D-
fructose 6-phosphate and D-ribose 5-phosphate via a
transaldol reaction catalyzed by LmbR, the subsequent
enzymatic transformations leading to the MTL moiety
remain elusive. Here, we report the identification of GDP-
Figure 1. Examples of natural products containing thiooctose moiety.
D-erythro-α-D-gluco-octose (GDP-D-α-D-octose) as a key
intermediate in the MTL biosynthetic pathway. Our data
Scheme 1. Proposed Biosynthetic Pathway for MTL (3)
show that the octose 1,8-bisphosphate intermediate is first
converted to octose 1-phosphate by a phosphatase, LmbK.
The subsequent conversion of the octose 1-phosphate to
GDP-D-α-D-octose is catalyzed by the octose 1-phosphate
guanylyltransferase, LmbO. These results provide signifi-
cant insight into the lincomycin biosynthetic pathway,
because the activated octose likely serves as the acceptor
for the installation of the C1 sulfur appendage of MTL.
arbohydrates are indispensable for all living organisms and
play important roles in determining the biological activities
C
of diverse glycoconjugates, including glycosylated secondary
metabolites.1 Altering the glycosylation pattern of targeted
natural products by manipulating the corresponding sugar
biosynthetic machinery holds promise to enhance or vary the
biological properties of the parent molecules. To achieve this
goal, it is essential to know how unusual sugars are
biosynthesized. Recent research efforts in this area have advanced
our understanding of the formation of deoxyhexoses in nature2
and made possible the glycodiversification of natural products,
taking advantage of substrate promiscuity found for many
glycosyltransferases.3 However, information about the con-
struction, activation, and modification of high-carbon sugars
(>7 C’s) is scarce.4 For example, the biosynthesis of naturally
occurring octoses remains unexplored, except for 3-deoxy-D-
manno-2-octulosonate-8-phosphate (KDO8P), which is derived
from arabinose 5-phophate and phosphoenolpyruvate (PEP).5
Lincomycin A (1), isolated from Streptomyces lincolnensis,6 is a
clinically useful antibiotic against Gram-positive bacteria.7 It
interacts with the peptidyltransferase domain of the 50S
ribosomal subunit due to its structural resemblance to the 3′
end of L-Pro-Met-tRNA and deacetylated tRNA. This, in turn,
inhibits the bacterial protein synthesis.8 The structure of
lincomycin A consists of an amino acid, N-methyl-4-propyl-L-
proline (2), and an unusual methylthiolincosamide (MTL, 3)
moiety.9 Similar thiooctoses are also found in antimicrobial
agents, Bu-2545 (4) and celesticetin (5) (see Figure 1).10
Recently, we reported that the C8 backbone of MTL11 is
generated via a transaldol reaction catalyzed by LmbR using D-
fructose 6-phosphate or D-sedoheptulose 7-phosphate as the C3
donor and D-ribose 5-phosphate as the C5 acceptor (Scheme
1).12 Subsequent isomerization catalyzed by LmbN converts the
Received: November 29, 2013
Published: December 31, 2013
© 2013 American Chemical Society
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Journal of the American Chemical Society
Communication
Scheme 2. Biosynthetic Pathways to NDP-Hexose and NDP-
Heptoses
Scheme 3. Synthesis of Octose 1,8-Bisphosphate (9), Octose
1-Phosphate (10), and GDP-D-α-D-octose (11)
a
a
Both 6S- and 6R-isomers of 27 were generated, and the desired 6R-
isomer was isolated using silica gel column chromatography.12
route. However, comparison of the lmb gene cluster with the
NDP-heptose biosynthetic gene clusters from Aneutinibacillus
thermoaerophilus DSM 10155/G+15a and Escherichia coli K-1215b
showed several genes, including lmbN, lmbP, lmbK, and lmbO,
whose translated amino acid sequences have similarities to those
involved in NDP-heptose biosynthesis (Table S2). From this
information, we speculated that MTL biosynthesis might follow a
kinase/phosphatase cascade analogous to that observed in the
NDP-heptose biosynthetic pathway to make NDP-octose.
As depicted in Scheme 1, LmbN catalyzes the C1→C2
isomerization to produce the corresponding octose 8-phosphate
(7→8) in a manner similar to the GmhA-catalyzed isomerization
(16→17).12 The putative kinase, LmbP, might phosphorylate
the C1 hydroxyl group of octose 8-phosphate (8) to afford octose
1,8-bisphosphate (9). The C8 phosphate group of this
bisphosphate intermediate might be hydrolyzed by LmbK
(annotated as phosphatase) to give octose 1-phosphate (10),
which is then converted to the nucleotide-activated octose (11)
by LmbO (annotated as nucleotidylyltransferase) (route A). It is
also conceivable that the reaction proceeds first with
nucleotidylyltransfer by LmbO (9→12), followed by LmbK-
catalyzed C8 dephosphorylation (12→11) (route B). Because
LmbP and LmbO exhibit sequence similarities only to their
counterparts in the GDP-D-α-D-heptose biosynthetic pathways
(Table S2), the C1-activated octose intermediate in lincomycin
biosynthesis is likely a GDP-D-α-D-octose. However, it is well
known that prediction of substrate specificity for enzymes based
solely on sequence alignment can be erroneous; thus the
chemical nature of sugar phosphodinucleotide intermediate in
this pathway must be experimentally verified.2c,d
resulting octulose 8-phosphate (7) to octose 8-phosphate (8).12
The fact that several genes homologous to those found in various
NDP-deoxyhexose pathways13 exist in the lincomycin gene
cluster (designated as lmb) prompted us to hypothesize that an
NDP-activated octopyranose is a possible intermediate in the
MTL biosynthetic pathway.12 However, this hypothesis lacks
literature precedence because NDP-octose has never been
reported to be an biosynthetic intermediate. Thus, investigations
of the existence of this putative NDP-octose intermediate and its
transformation from 8 have been the focus of our research effort.
In this report, we describe the identification of GDP-D-erythro-α-
D-gluco-octose (GDP-D-α-D-octose, 11) as the key intermediate
in the MTL biosynthetic pathway.
In a typical NDP-sugar biosynthetic pathway, the sugar
precursor is first converted to the corresponding sugar 1-
phosphate prior to nucleotidyl transfer to yield the NDP-sugar
product (e.g., 14→15 to make NDP-α-D-glucose, Scheme 2).
For the biosynthesis of NDP-deoxyhexoses, generation of the
hexose-1-phosphate precursor is achieved either by direct C-1
phosphorylation or via C-6 phosphorylation followed by a
mutase-catalyzed 6→1 phosphoryl migration (13→14, Scheme
2).2 A different scenario was observed in the biosynthesis of
NDP-heptoses (Scheme 2). Studies of the formation of ADP-D-
glycero-β-D-manno-heptose (ADP-D-β-D-heptose, 20) and GDP-
D-glycero-α-D-manno-heptose (GDP-D-α-D-heptose, 23) revealed
a kinase/phosphatase cascade to produce heptose 1-phosphate in
three steps: (1) isomerization of D-sedoheptulose 7-phosphate to
D-glycero-D-manno-heptose 7-phosphate (16→17); (2) anomeric
phosphorylation of heptose 7-phosphate to heptose 1,7-
bisphosphate by a kinase (17→18 and 17→21); (3) conversion
of heptose 1,7-bisphosphate to heptose 1-phosphate via a
phosphatase-catalyzed hydrolysis (18→19 and 21→22).14
Sequence analysis of the lmb gene cluster from S. lincolnensis
ATCC 25466 found no mutase homologue required for
installation of the C1 phosphoryl group via a CN→C1 migration
To determine whether the proposed pathways are valid, the
lmbP, lmbK, and lmbO genes were amplified from the genomic
DNA of S. lincolnensis ATCC 25466 using polymerase chain
reaction and were individually cloned into pET28 vectors. The
recombinant LmbP, LmbK, and LmbO with N-terminal His6 tags
were overexpressed in E. coli. LmbK could be purified to near
homogeneity as a soluble protein (Figure S1), but both LmbP
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Journal of the American Chemical Society
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Figure 4. Activity assays for LmbK and refolded LmbO: (a) refolded
LmbO with α-glucose 1-phosphate and GTP; (b) refolded LmbO with
β-glucose 1-phosphate and GTP; (c) LmbK and refolded LmbO with α-
glucose 1,6-bisphosphate and GTP; (d) co-injection of the sample from
LmbO reaction with α-glucose 1-phosphate (trace a) and the GDP-D-α-
glucose standard; and (e) GDP-D-α-glucose standard.
Figure 2. Activity assays for LmbK and refolded LmbO. All reactions
contain the synthetic bisphosphate 9, LmbK, and refolded LmbO. (a)
CTP, (b) TTP, (c) UTP, (d) ATP, and (e) GTP.
LmbO (route A). To test this hypothesis and verify which
nucleotide is utilized in the lincomycin biosynthetic pathway, the
synthetic bisphosphate 9 was incubated with LmbK and the
refolded LmbO in the presence of ATP, CTP, GTP, TTP, or
UTP (see Supporting Information section S6 for details). The
resulting mixtures were analyzed by HPLC (Figure 2). Although
no new peak was detected in the reaction mixture using ATP,
CTP, TTP, or UTP, a sample derived from the incubation
mixture with GTP showed a new peak with a retention time of
9.45 min (Figure 2, trace e). The same results were obtained
when the synthetic 10 was incubated with the refolded LmbO
(Figure S4). This new product peak was collected and subjected
to ESI-MS. The recorded molecular mass (calcd for
Figure 3. Activity assays for the refolded LmbO: (a) synthetic GDP-
octose (11) standard; (b) LmbK and refolded LmbO with bisphosphate
9 and GTP; (c) refolded LmbO with bisphosphate 9 and GTP; and (d)
refolded LmbO with the synthetic octose 1-phosphate (10) and GTP.
−
C18H28N5O18P2 [M−H+], 664.0910; obsd, 664.0922) is
and LmbO were expressed only as inclusion bodies. The
denatured LmbO could be refolded (protocol detailed in the
Supporting Information), but attempts to obtain the soluble
form of LmbP using a similar approach were not successful. To
circumvent this obstacle, we synthesized the putative substrate
for LmbK (compound 9). The synthetic scheme is shown in
Scheme 3.
consistent with the proposed product, GDP-D-α-D-octose (11),
which was also chemically prepared from 10, as shown in Scheme
3. As expected, the isolated product from the LmbK/LmbO
reaction displayed a HPLC retention time identical to that of the
synthetic standard (Figure 3, traces a and b).
The above results clearly indicate that the refolded LmbO is
catalytically active and suggest a pathway in which C8
dephosphorylation by LmbK preceeds prior to nucleotidylyl-
transfer catalyzed by LmbO (Scheme 1, route A). However, a
reversal of the sequence of these two reactions is also conceivable
(route B). To check this possibility, 9 was incubated with the
refolded LmbO in the presence of GTP. As shown in Figure 3,
trace c, no new peak in the range of GDP-activated sugars is
observed. In contrast, formation of 11 is clearly visible (Figure 3,
trace d) when the synthetic 10 was incubated with the refolded
LmbO and GTP. Evidently, C8 dephosphorylation is a
prerequisite for the subsequent nucleotidyl activation. These
results unequivocally establish that 11 is indeed an intermediate
in MTL biosynthesis and its formation follows route A, not route
B.
In heptose biosynthesis, the bisphosphate products (e.g., 18
and 21) generated in the kinase reactions have defined anomeric
configurations that correlate with the anomeric specificities of the
subsequent nucleotidylyltransferase (e.g., HldE and HddC)-
catalyzed transformations (e.g., 19→20 and 22→23, Scheme 2).
An analogous anomeric specificity also appears to be conserved
in LmbK- and LmbO-catalyzed reactions because their products
are all α-anomers. To investigate whether LmbO tolerates
flexibility in its anomeric specificity, the refolded LmbO was
The synthetic bisphosphate 9 was first incubated with LmbK,
and the reaction mixtures were analyzed by HPLC using a
Corona charged aerosol detector (CAD) and a Dionex CarboPac
PA-1 anion exchange column. A new peak was detected (Figure
S2). This new product was collected and characterized by NMR
spectroscopy. The 1H−31P heteronuclear multiple quantum
correlation (HMQC) spectrum of the substrate 9 displayed two
31P signals, one at δ 2.53 coupled to the two C-8 proton signals at
δ 3.86 and 3.83, and the other at δ 0.49 coupled to the anomeric
proton at δ 5.35 (Figure S3). In contrast, the NMR spectrum of
the isolated product displayed a single 31P signal at δ 3.17
coupled to the anomeric proton at δ 5.34. Loss of the C8
phosphoryl group from 9 during the LmbK-catalyzed reaction is
thus evident. To gain support for the structural assignment of the
isolated product, an octose 1-phosphate (10) standard was also
synthesized (Scheme 3). The LmbK product was found to
coelute with the synthetic 10 on HPLC, and the spectral
characteristics of the enzymatic product are in good agreement
with the synthetic standard. These results fully established the
identity of the LmbK product as 10 starting from 9.
After formation of 10, the next reaction step is likely nucleotide
activation catalyzed by the putative nucleotidylyltransferase,
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Journal of the American Chemical Society
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incubated separately with GTP and each of the two anomers of
glucose 1-phosphate, used as substrate analogues. Since
formation of GDP-α-glucose was found only in the sample of
α-glucose 1-phosphate (Figure 4, traces a and b), LmbO shows
stringent α-anomeric stereospecificity for both octose and hexose
substrates. Interestingly, although LmbO is capable of processing
six-carbon sugar 1-phosphate, LmbK could not hydrolyze α-D-
glucose 1,6-bisphosphate (Figure 4, trace c). A similar
observation was reported for heptose 1,7-bisphosphate phos-
phatase GmhB, which could not recognize α-D-glucose 1,6-
bisphosphate as a substrate.15 Apparently, the location of the
phosphoryl group being removed on the substrate is essential for
these phosphatases.
Together these results provide significant insight into the
lincomycin biosynthetic pathway, part of which is reminiscent of
the NDP-heptose pathway. Transformation of octose 8-
phosphate (8) to GDP-D-α-D-octose (11) is shown to involve
kinase and phosphatase reactions as intermediary steps. Our data
also reveal that the dephosphorylation step, catalyzed by LmbK,
is critical for the nucleotide activation reaction. However, direct
demonstration of the predicted kinase activity of LmbP was
unsuccessful due to the difficulty in refolding insoluble LmbP.
Nevertheless, the involvement of a kinase-catalyzed step and the
α-stereospecificity of LmbP reaction are supported by the
effective reconstitution of the biosynthesis of GDP-D-α-D-octose
using the synthetic bisphosphate 9 as substrate.
supported by grants from the National Institutes of Health
(GM035906) and the Welch Foundation (F-1511).
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details, ESI-MS, and HPLC traces. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
h.w.liu@mail.utexas.edu
■
(18) Palaniappan, N.; Ayers, S.; Gupta, S.; Habib, E.-S.; Reynolds, K. A.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Drs. Hak Joong Kim and Sei Hyun Choi for their
helpful discussion of the chemical synthesis. This work was
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