Construction of the octose 8-phosphate intermediate in lincomycin A biosynthesis: Characterization of the reactions catalyzed by LmbR and LmbN

Article abstract of DOI:10.1021/ja308221z

Lincomycin A is a potent antimicrobial agent noted for its unusual C1 methylmercapto-substituted 8-carbon sugar. Despite its long clinical history for the treatment of Gram-positive infections, the biosynthesis of the C ₈-sugar, methylthiolincosamide (MTL), is poorly understood. Here, we report our studies of the two initial enzymatic steps in the MTL biosynthetic pathway leading to the identification of d-erythro-d-gluco-octose 8-phosphate as a key intermediate. Our experiments demonstrate that this intermediate is formed 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. Subsequent 1,2-isomerization catalyzed by LmbN converts the resulting 2-keto C₈-sugar (octulose 8-phosphate) to octose 8-phosphate. These results provide, for the first time, in vitro evidence for the biosynthetic origin of the C₈ backbone of MTL.

Full text of DOI:10.1021/ja308221z

Communication
pubs.acs.org/JACS
Construction of the Octose 8‑Phosphate Intermediate in Lincomycin
A Biosynthesis: Characterization of the Reactions Catalyzed by LmbR
and LmbN
Eita Sasaki, Chia-I Lin, Ke-Yi Lin, and Hung-wen Liu*
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at
Austin, Austin, Texas 78712, United States
S
* Supporting Information
ABSTRACT: Lincomycin A is a potent antimicrobial
agent noted for its unusual C1 methylmercapto-substituted
8-carbon sugar. Despite its long clinical history for the
treatment of Gram-positive infections, the biosynthesis of
the C8-sugar, methylthiolincosamide (MTL), is poorly
understood. Here, we report our studies of the two initial
enzymatic steps in the MTL biosynthetic pathway leading
to the identification of D-erythro-D-gluco-octose 8-phos-
phate as a key intermediate. Our experiments demonstrate
that this intermediate is formed 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. Subsequent 1,2-isomer-
Figure 1. Examples of natural products containing a C8-sugar scaffold.
ization catalyzed by LmbN converts the resulting 2-keto
C8-sugar (octulose 8-phosphate) to octose 8-phosphate.
These results provide, for the first time, in vitro evidence
for the biosynthetic origin of the C8 backbone of MTL.
established to be derived from D-arabinose 5-phosphate (A5P)
and phospho-enolpyruvate (PEP) in an aldol-like reaction
catalyzed by Kdo8P synthase,8 little is known about how the
other C8 sugar scaffolds are biosynthesized. Herein, we report
the in vitro functional characterization of two enzymes, LmbR
and LmbN, involved in the early stage of MTL (3)
biosynthesis. On the basis of the detailed investigation and
stereochemical analysis of these two consecutive enzymatic
reactions, we identified D-erythro-D-gluco-ocotose 8-phosphate
(29) as a key intermediate in this pathway.
any compounds important for the treatment and study
M
of human disease have their origin in natural products.
These compounds are frequently modified with carbohydrate
appendages that are critical for their biological activities.1 By
exploiting the biosynthetic machinery of these sugars, we can
enhance or vary the biological characteristics of the parent
molecules. To fully realize the therapeutic potential of such
approach, the biosynthetic pathways of these sugars must be
characterized and the underlying chemistry understood at the
mechanistic level.2 Despite recent advance made in deoxy-
hexoses biosynthesis research,3 our knowledge about how
unusual octoses are constructed remains elusive.
Early feeding experiments using 13C-labeled D-glucose
showed that 3 may be assembled through the condensation
of a pentose 5-phosphate (C5) and a C3 unit derived from the
pentose phosphate pathway in a transaldolase-catalyzed
reaction.9 Later, the biosynthetic gene cluster for 1 was isolated
and sequenced,10 and a gene encoding a putative transaldolase,
LmbR, was identified along with several genes homologous to
those found in various NDP-deoxyhexose pathways.3 These
results allowed us to propose a possible biosynthetic pathway
for MTL (3), which consists of three key steps: (i) the
assembly of the C8 scaffold from a C5 acceptor and a C3 donor
in a transaldol reaction catalyzed by LmbR, (ii) the conversion
of the resulting octulose 8-phosphate (10) to an NDP-activated
octopyranose (NDP-octose, 13) through an octose phosphate
intermediate (11), and (iii) modification of the NDP-octose
(13) by enzymes similar to those found in NDP-deoxyhexose
Lincomycin A (1), originally isolated from S. lincolnensis var.
lincolnensis,4 is an octose-containing antimicrobial agent used
for treating Gram-positive bacteria infections. The structure of
1 comprises an N-methyl-4-propyl-L-proline moiety (2) and an
unusual thiooctose, known as methylthiolincosamide (MTL, 3;
Figure 1).5 The lincosamine component, acting as the structural
mimic of the 3′-end of L-Pro-Met-tRNA and deacylated-tRNA,
blocks microbial protein synthesis at the initial phase of the
peptide elongation cycle.6 Only a few natural products that
contain 8-carbon sugar scaffolds have been identified, including
celesticetin (4), octosyl acid A (5), 2-keto-3-deoxy-D-manno-
octulosonate 8-phosphate (Kdo8P, 6), and apramycin (7), in
addition to 1 (Figure 1).7 Except for Kdo8P (6), which is a key
structural component of lipopolysaccharides and has been
Received: August 18, 2012
Published: September 18, 2012
© 2012 American Chemical Society
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biosynthetic pathways to yield MTL (3) as the end product
To verify the proposed transaldolase activity of LmbR, the
recombinant LmbR with a C-terminal His6-tag was overex-
pressed in E. coli and purified to near homogeneity (Figure
S1).16 A transaldolase-catalyzed reaction is known to be
initiated by imine bond formation between an active site lysine
of the enzyme and the 2-keto group of the ketosugar substrate
(e.g., F6P (9a) or S7P (9b), Scheme 2).12,17 To examine
(Scheme 1).11
Scheme 1. Proposed Biosynthetic Pathway for MTL (3)
Scheme 2. Proposed Mechanism for LmbR-Catalyzed
Reaction
whether F6P (9a) is a competent C3 donor for the LmbR-
catalyzed reaction, the purified C-His6-LmbR was incubated
with F6P followed by sodium borohydride treatment. The
recovered LmbR after incubation was subjected to MS analysis.
In addition to the unmodified enzyme (calcd/obsd, 24952 Da),
a set of MS signals corresponding to the reduced forms of the
C-His6-LmbR/F6P conjugate (15: calcd, 25196 Da; obsd,
25193 Da) and C-His6-LmbR/dihydroxyacetone conjugate (16:
calcd, 25026 Da; obsd, 25024 Da) were observed (Figure S2).16
The control reaction without adding F6P showed only the
unmodified enzyme peak. Similar results were noted when S7P
(9b) was used in the incubation (Figure S3).16 These results
indicate that both F6P and S7P are possible substrates of
LmbR, and the reaction proceeds in a similar manner to other
transaldolases involving the formation of an imine adduct (e.g.,
15) followed by Cα−Cβ bond cleavage via a retro-aldol reaction
(e.g., 15→16) to generate the C3 donor unit (16) that reacts
with pentose 5-phosphate (8).
Next, we investigated the possible C5 acceptor substrate for
the LmbR-catalyzed reaction. As discussed, R5P (8a) and X5P
(8b) are the two likely candidates. Thus, the purified C-His6-
LmbR protein was incubated with F6P (9a) and R5P or X5P,
and the reaction mixtures were analyzed using HPLC equipped
with a Corona charged aerosol detector (CAD). When R5P
(8a) was used, a new signal appeared (retention time ∼10 min)
and the substrate signals (retention time for F6P (9a): ∼12
min, for R5P (8a): ∼12 min) decreased in intensity (Figure 2,
trace d). This new product peak, absent in the control reaction
that excluded enzyme (trace g), was isolated and subjected to
ESI-MS. The molecular mass is consistent with that of the
proposed octulose 8-phosphate product (10a; ESI− calcd for
C8H16O11P− [M - H+], 319.0436; obsd, 319.0431). A new
signal (retention time ∼8 min) was detected when X5P (8b)
and F6P (9a) were incubated with the C-His6-LmbR protein
(trace e). This new peak was also isolated and subjected to ESI-
MS. The molecular mass of the observed mass signal agrees
with that of the proposed octulose 8-phosphate product (10b;
ESI− calcd for C8H16O11P− [M - H+], 319.0, obsd, 319.1).
These results clearly indicate that LmbR can accept both R5P
(8a) and X5P (8b) as the C5 substrate. Conveniently, the C8
products derived from each C5 substrate have distinct HPLC
retention times.
Because C5 and C7 of 3 are derived from C2 and C4 of the
C5 acceptor and each has a (R)-configuration, both D-ribose 5-
phosphate (R5P, 8a) and D-xylose 5-phosphate (X5P, 8b)
could serve as the C5 precursor in the transaldolase-catalyzed
reaction. Although the configuration of the 3-OH group of R5P
is opposite to that of X5P, the stereochemical distinction
becomes irrelevant as this stereogenic center is transformed to
the C6 carbon in MTL (3) bearing an amino functionality. The
substitution of a hydroxyl group with an amino moiety at this
position is likely accomplished by a transamination reaction via
the 6-keto intermediate (14). Thus, both R5P and X5P were
considered as possible candidates for the C5 unit. Likewise, the
C3 donor may be D-fructose 6-phosphate (F6P, 9a) or D-
sedoheptulose 7-phosphate (S7P, 9b), both of which are
known C3 precursors for the physiological transaldolase
reaction in the pentose phosphate pathway.12 On the basis of
the general stereochemical course established for most aldolase-
catalyzed reactions, the resulting octulose 8-phosphate product
(10) is expected to inherit the stereochemistry at the C3 and
C4 positions from the corresponding chiral centers of the C3
donor.13 This would yield a C8 sugar intermediate having S and
R configuration at C3 and C4, respectively. However, the
predicted (R)-configuration of the C4 hydroxyl group of 10 is
in contrast to the observed C4-(S)-configuration of the final
product, 3. To account for this discrepancy, the participation of
a putative epimerase (encoded by the lmbM gene) may be
necessary to epimerize the C4 hydroxyl of 12 to afford 13
(Scheme 1, pathway A). Although rare, reactions catalyzed by
aldolases giving the inversed stereochemistry are known.14 It is
thus possible that the LmbR-catalyzed reaction may generate an
octulose 8-phosphate intermediate with the C4-(S)-config-
uration, which can be transformed to 13 (via 11) without going
through 12 (pathway B). In either case, LmbN, which displays
moderate sequence identity with a S7P isomerase, GmhA (31%
identity and 47% similarity to the E. coli protein),15 is proposed
to catalyze the C1−C2 isomerization of the LmbR product
(10) to produce the octose 8-phosphate (11).
Next, we decided to investigate the subsequent isomerization
reaction catalyzed by LmbN using the LmbR reaction products,
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Scheme 3. Synthesis of (4R)- and (4S)-Heptaacetyloctose
Standards
Figure 2. Activity and substrate specificity assays for LmbR and
LmbN. (a) F6P standard; (b) R5P standard; (c) X5P standard; (d)
LmbR with R5P (10 mM) and F6P (10 mM); (e) LmbR with X5P
(10 mM) and F6P (10 mM); (f) LmbR and LmbN with R5P (10
mM) and F6P (10 mM); (g) the control sample with F6P (10 mM)
and R5P (10 mM), but no enzyme; (h) LmbN with F6P (10 mM) and
R5P (10 mM); (i) LmbR and LmbN with X5P (10 mM) and F6P (10
mM).
10a and 10b, as substrates. The recombinant LmbN protein
carrying a C-terminal His6-tag was overexpressed in E. coli
(Figure S1),16 and the purified enzyme was incubated with the
LmbR reaction mixture containing F6P (9a) and either R5P
(8a) or X5P (8b). No change of the HPLC trace for the
reaction with X5P was noted (Figure 2, trace i), but a new peak
with a retention time of ∼9 min was observed for the reaction
using R5P (trace f). This product was absent in a control
reaction in which LmbR was left out (trace h). It is thus clear
that the new product (11a) is derived from the octulose 8-
phosphate (10a), and not from F6P or R5P in the reaction
solution. This new compound was isolated and subjected to
ESI-MS. The molecular mass of the observed MS signal is
consistent with that of the proposed octose 8-phosphate
product (11a; ESI− calcd for C8H16O11P− [M - H+] 319.0436,
obsd, 319.0431). The observed substrate specificity of the
LmbN reaction provides strong evidence that the C5 precursor
of MTL (3) is R5P (8a) instead of X5P (8b), even though both
compounds can be processed by LmbR in vitro. These results
also enable us to assign the (R)-configuration at C6 of the
LmbR product (10a).
To fully characterize the LmbR and LmbN reactions, the
stereochemistry at C4 of their products, 10a and 11a, must be
determined (Scheme 1). Although a small amount of pure 11a
could be isolated for MS analysis, it was difficult to secure
sufficient amounts for NMR characterization due to the poor
separation of 11a from 10a. Thus, we opted to chemically
synthesize the peracetylated C4-(R)- and C4-(S)-octose
standards (24 and 26, respectively, Scheme 3.16 For
comparative analysis, the LmbR and LmbN products (10a
and 11a, respectively) generated from the incubation with R5P
(8a) and F6P (9a) were first treated with alkaline phosphatase,
and the resulting dephosphorylated sugar compounds were
subjected to peracetylation conditions. The derivatized
enzymatic product mixture and the synthetic standards were
then analyzed using HPLC.
Figure 3. HPLC analysis of acetylated LmbR and LmbN reaction
products. (a) LmbR reaction with R5P (10 mM) and F6P (50 mM)
followed by dephosphorylation and acetylation. (b) LmbR and LmbN
reactions with R5P (10 mM) and F6P (50 mM) followed by
dephosphorylation and acetylation. (c) Control reaction with only
F6P. (d) Synthetic standard 24. (e) Coinjection of the sample derived
from LmbR and LmbN reaction (trace b), and the synthetic standard
24. (f) Synthetic standard 26. (g) Coinjection of the sample derived
from LmbR and LmbN reaction, and the synthetic standard 26.
time ∼17.5 min, trace a) can be attributed to the C-2 anomers
of the octulofuranose heptaacetate (see 28) derived from the
LmbR product 10a. The MS data of the isolated peaks are
consistent with this assignment (ESI+, calcd for C20H27O13+ [M
- AcO−] 475.1, found 475.2). When both LmbR and LmbN
were used, a new peak with a retention time of ∼18 min
emerged (trace b). This peak was isolated and subjected to
high-resolution MS. The results agree with the proposed
heptaacetyloctose product (ESI+, calcd for C22H30O15Na+ [M +
Na]+ 557.1477, found 557.1478). Importantly, the retention
time of this peak matches that of the C4-(R) standard 24 (trace
d). Moreover, this product and 24 coeluted when coinjected
(trace e). In contrast, the C4-(S) isomer (26) eluted with a
longer retention time (trace f) than the peracetylated LmbN
product (see trace g).18 These results unambiguously show that
the C4 position of the LmbR/N reaction products has a (R)-
configuration (Scheme 1, pathway A).
As shown in Figure 3, two sets of signals (retention times
∼13.5 and ∼17.5 min) arise from the LmbR reaction (trace a).
The first set of two peaks matches the signals observed for the
control reaction, which generate pentaacetylated fructose as the
product (trace c). This assignment is supported by MS analysis
of the collected fraction containing these two peaks (ESI+, calcd
In summary, we performed in vitro functional character-
ization of LmbR and LmbN and determined the early
intermediates of the MTL biosynthetic pathway. Our results
demonstrate that the products of LmbR and LmbN reactions
are D-glycero-D-altro-octulose 8-phosphate (27/28) and D-
erythro-D-gluco-ocotose 8-phosphate (29/30), respectively
(Scheme 4). Our experiments also establish that the C8 sugar
backbone of 3 is assembled using R5P (8a) as the C5 acceptor
and F6P or S7P as the C3 donor in a transaldol reaction
catalyzed by LmbR. Interestingly, the C4 stereochemistry of the
for C14H19O9 [M - AcO−] 331.1, found 331.2). The splitting
+
peak pattern likely reflects the formation of two anomeric
isomers (α and β) of the pentaacetylfructose product during
chemical derivatization. The second set of two peaks (retention
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Scheme 4. Reactions Catalyzed by LmbR and LmbN
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ASSOCIATED CONTENT
* Supporting Information
Experimental details, ESI-MS spectra, HPLC trace, and Mosher
ester analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
121.
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AUTHOR INFORMATION
Corresponding Author
h.w.liu@mail.utexas.edu
■
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(16) See Supporting Information for details.
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biosynthetic pathway, catalyzes the C1−C2 isomerization of S7P to
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Interestingly, no isomerized product was observed when S7P was
treated with LmbN (Figure S4).16 This result together with the fact
that LmbN does not process 10b indicates that LmbN has a relatively
strict substrate specificity.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Sei Hyun Choi for useful discussion of the chemical
synthesis. This work was supported in part by the National
Institutes of Health (GM035906) and the Welch Foundation
(F-1511).
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