A general NMR-based strategy for the in Situ characterization of sugar-nucleotide-dependent biosynthetic pathways

Article abstract of DOI:10.1021/ol501241a

A simple method for the study of sugar-nucleotide-dependent multienzyme cascades is highlighted where the use of selectively ¹³C-labeled sugar nucleotides and inverse ¹³C detection NMR offers fast, direct detection and quantification of reactants and products and circumvents the need for chromatographic separation. The utility of the method has been demonstrated by characterizing four previously uncharacterized sugar nucleotide biosynthetic enzymes involved in calicheamicin biosynthesis.

Full text of DOI:10.1021/ol501241a

Letter
pubs.acs.org/OrgLett
A General NMR-Based Strategy for the in Situ Characterization of
Sugar-Nucleotide-Dependent Biosynthetic Pathways
,†
‡,∥
§
,†
Shanteri Singh,* Pauline Peltier-Pain, Marco Tonelli, and Jon S. Thorson*
†Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, 789 South Limestone Street,
Lexington, Kentucky 40536, United States
‡
Pharmaceutical Sciences Division, University of WisconsinMadison, 777 Highland Avenue, Madison, Wisconsin 53705, United
States
§
National Magnetic Resonance Facility at Madison, University of WisconsinMadison, 433 Babcock Drive, Madison, Wisconsin
5
3706, United States
*
S Supporting Information
ABSTRACT: A simple method for the study of sugar-
nucleotide-dependent multienzyme cascades is highlighted
1
3
where the use of selectively C-labeled sugar nucleotides and
inverse 13C detection NMR offers fast, direct detection and
quantification of reactants and products and circumvents the
need for chromatographic separation. The utility of the
method has been demonstrated by characterizing four
previously uncharacterized sugar nucleotide biosynthetic
enzymes involved in calicheamicin biosynthesis.
5
ugars are critical in cells where they play important roles in
mostly from the natural abundance) still often suffer from a
S
energy metabolism, maintenance of cell integrity, molecular
recognition, and signaling. For many small molecule-based
lack of resolution in the context of complex cofactor/reagent/
product-containing multienzyme reactions. To extend upon the
potential advantages of NMR within this context, herein we
describe an efficient NMR-based method that combines the
sensitivity of inverse detection two-dimensional (2D)-NMR
with the convenient preparation and direct use of NDP-α-D-
1
glycoconjugates, the nature of the sugar dramatically influences
2
the properties of the metabolite to which it is attached,
prompting the development of chemoenzymatic strategies to
manipulate small molecule pharmacology via differential
glycosylation. In nature, many carbohydrate maturation and
attachment processes are mediated via sugar-nucleotide-
dependent multienzyme cascades (Scheme S1 in Supporting
Information).
diverge from a relatively small set of common progenitors and
share a number of conceptual and strategic similarities. Yet, the
biochemical study of the catalysts involved often remains a
challenging endeavor (Scheme S1 in Supporting Information).
The development and implementation of simple, broadly
applicable assay strategies for divergent sugar-nucleotide-
dependent multienzyme transformations is expected to
facilitate such studies.
3
13
[U- C]hexose-based substrates. Distinct from prior multi-
13
4
enzyme preparations of NDP-α-D-[U- C]glucose, an enabling
feature of this work is the single-step synthesis of this key
3
b,c
13
These sugar-nucleotide-dependent pathways
reagent from 2-chloro-4-nitrophenyl-α-D-[U- C]glucose (4 in
Scheme 1) catalyzed by engineered macrolide glycosyltransfer-
6
ase OleD variant TDP16. Cumulatively, this study presents a
user-friendly rapid and general method for sugar nucleotide
characterization in complex mixtures and also provides new
biochemical annotation for a set of four previously uncharac-
terized sugar-nucleotide-dependent enzymes involved in
calicheamicin biosynthesis: a 4,6-dehydratase (CalS3), a 4-
7
aminotransferase (CalS13), a 3,5-epimerase (CalS1), and a 4-
8
ketoreductase (CalS2). Furthermore, the application of the
Over the past two decades several sugar-nucleotide-depend-
6
3
,4
method for rate determination (OleD TDP16 and CalS3) and
ent biosynthetic enzymes have been characterized. In most
cases, these studies have relied upon discontinuous HPLC-
based assays that often (i) require strong ion-pairing agents/
salts incompatible with mass spectrometry; (ii) lack sufficient
resolution to resolve many reactants/products/cofactors
typically found within multienzyme cascades; and (iii) operate
on a time scale unsuitable for short-lived or unstable
intermediates. While NMR offers the advantage of real-time
detection of reactants/products without tedious chromatog-
raphy, the studies to date (which have relied upon direct
potential strategies to address stereospecificity (CalS13 versus
9
WecE ) are briefly highlighted.
For this study, genes encoding a representative set of
previously uncharacterized calicheamicin aryltetrasaccharide
biosynthetic enzymes (annotated based on BLAST results as
TDP-glucose-4,6-dehydratase CalS3, TDP-glucose-3,5-epimer-
ase CalS1, TDP-glucose-4-ketoreductase, CalS2, and TDP-
Received: April 30, 2014
1
13
13
31
detection of H or C and/or indirect detection of C and P
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ol501241a | Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
1
3
Scheme 1. Synthesis of 2-Chloro-4-nitrophenyl-β-D-[U- C]glucose and Proposed Biosynthetic Pathway of TDP-4-
a
Hydroxyamino-6-deoxy-glucose and TDP-3-Methoxy-rhamnose en Route to Calicheamicin
a
(
a) Ac O pyridine; (b) HBr, AcOH/DCM, 0 °C, 2 h; (c) 2-chloro-4-nitrophenol, TBABr, DCM/NaOH (1:1), rt, 18 h; NaOMe, MeOH, rt, 12 h;
2
13 13 13 13
D-[U- C]glucose, 1; penta-O-acetyl-D-[U- C]glucose, 2; 1-bromo-tetra-O-acetyl-α-D-[U- C]glucose, 3; 2-chloro-4-nitrophenyl-β-D-[U- C]-
glucose, 4; TDP-α-D-[U- C]glucose, 5; TDP-4-keto-6-deoxy-α-D-[U- C]glucose, 6; TDP-4-keto-β-L-[U- C]rhamnose, 7; TDP-β-L-[U- C]-
rhamnose, 8; TDP-3-methoxy-β-L-[U- C]rhamnose, 9; TDP-4-amino-6-deoxy-α-D-[U- C]glucose, 10; TDP-4-hydroxyamino-6-deoxy-α-D-
13
13
13
13
1
3
13
13
[
U- C]glucose, 11.
8
glucose-4-aminotransferase CalS13; Scheme 1) were amplified
from Micromonospora echinospora genomic template DNA using
the polymerase chain reaction (PCR) and subsequently cloned
into a pET28a vector. The resulting constructs were used to
transform the Escherichia coli BL21(DE3) cells to provide
overproduction strains of the corresponding target enzymes as
We used 4 as the initiating substrate, bypassing the native
thymidylyltransferase-catalyzed (CalS7) formation of 4. To
8
confirm the putative steps/enzymes responsible for the
formation of 10 in the calicheamicin pathway, the reaction
was initiated by the addition of 0.5 μM of TDP16 to a reaction
mixture containing 4 and 0.1 equiv excess of TDP with respect
to 4 (HSQC black resonances, Figure 1A) to drive the
equilibrium toward the desired 13C-labeled sugar nucleotide 5
(HSQC green resonances, Figure 1A). Upon reaction
N-terminal His -fusion proteins to afford rapid purification via
Ni-NTA affinity chromatography (see Supporting Informa-
6
tion).
+
An enabling feature of this work is the single-step OleD
TDP16-catalyzed formation of 5 from 4 (Scheme 1), generated
in three simple steps (30% overall yield) from 1. The in situ
NMR reactions were conducted in a 5 mm NMR tube at 25 °C
using standard assay conditions (see Supporting Information)
completion based upon NMR, 0.2 mM NAD and 11.5 μM
of CalS3 were added to the reaction mixture and the formation
of 6 (HSQC blue resonances, Figure 1A) was subsequently
monitored. Following the completion of the CalS3 reaction,
200 μM PLP, 10 mM L-glutamic acid, and 14 μM of CalS13
were added to the reaction and monitored for the appearance
of signals consistent with the formation of 10 (HSQC purple
resonances reflecting a mixture of 6 and 10, Figure 1A).
In the TDP-methoxy-rhamnose (9, Scheme 1) pathway of
calicheamicin, CalS11 has been established as the TDP-
rhamnose (8) C3-O-methyltransferase (O-MT) and is the
only natural product sugar O-MT studied thus far that acts on
the sugar nucleotide substrate prior to a subsequent
6
in 25 mM sodium phosphate buffer containing 10−20% D O,
2
pH 7.5. For all reactions, the NMR probe was tuned and
shimmed with an NMR tube containing the entire reaction
mixture in the absence of enzyme and reactions were
subsequently initiated via the addition of enzyme, before the
1
13
acquisition of a series of 2D- H− C HSQC (referred just as
HSQC henceforth) spectra. Reaction progress was monitored
by the appearance of new product-based resonances and
consequent decrease in the intensity of substrate signals in the
HSQC spectra over time. For rate measurements, a total of
10
glycosyltransfer reaction. While the biosynthesis of 8 has
12
been extensively studied in a range of systems, the formation
of 8 in the context of calicheamicin has not been characterized.
To characterize the enzymes corresponding to the calicheami-
cin TDP-methoxy-rhamnose pathway, 6 (HSQC blue reso-
nances, Figure 1B) was generated as described in the previous
paragraph and 9.5 μM of CalS1 (the putative 3,5-epimerase)
was added to the reaction mixture. Interestingly, no change in
the HSQC spectra was observed in the presence of CalS1,
suggesting the reaction equilibrium to favor reactants or, as
4
0−80 HSQC spectra were collected, depending on the speed
of the reaction. At the completion or near completion of each
1
13
enzyme reaction, 2D H− C planes of HCCH-TOCSY and/or
HCCH-COSY (see Supporting Information) were collected to
assist with the resonance assignment. The product formation of
all the reactions was also subsequently confirmed via HRMS
analysis (see Supporting Information).
While the culminating CalE10-catalyzed N-oxidation of
aminosugar nucleotide 10 en route to calicheamicin (Scheme
) has been characterized, the putative steps/enzymes
responsible for the formation of 10 remained uncharacterized.
13
proposed by Melo and Glaser, the 3,5-epimerase and 4-keto
reductase to require the formation of a functional complex.
Consistent with this, the addition of 6.2 μM of CalS2 and 5
1
1
1
B
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Organic Letters
Letter
Figure 2. Progress of TDP16 (square) and CalS3 (circle) reactions.
Colors correlate to the reactants/products in Scheme 1.
from 6, the calicheamicin CalS13 is anticipated to provide a C4
(
(
(
S) configuration while the E-coli WecE is known to provide C4
9
R). Comparison of the H4′ chemical shift values of CalS13
2.76 ppm) and WecE (3.44 ppm) products (circled in Figure
3
) with previously reported data are consistent with the
1
13
Figure 1. (A) Overlay of 2D- H− C-HSQC spectra of TDP16, CalS3
1
13
and CalS13 reactions. (B) Overlay of 2D- H− C-HSQC spectra of 6
HSQC with blue signals), CalS1, CalS2, and CalS11 reactions. Colors
(
correlate to the products in Scheme 1.
mM NADPH after ∼3 h led to the appearance of new signals
indicative of formation of 8 (HSQC light blue resonances
reflecting a mixture of 6 and 8, Figure 1B). Importantly, the
corresponding control lacking CalS1 failed to provide 8.
1
13
Figure 3. Overlay of 2D- H− C-HSQC spectra of CalS13 (red) and
WecE (black) reactions. The C4′−H4′ correlations corresponding to
CalS13 and WecE products are circled.
1
3
Subsequent addition of 2 mM [U- C]CH -S-adenosyl-L-
3
1
0
methionine and 9 μM CalS11 to the CalS2 reaction resulted
in the formation of 9 (HSQC red resonances, Figure 1B).
To assess the utility of this method for rate determination, a
series of HSQC spectra for two model reactions (TDP16 and
CalS3) were recorded in triplicate over several hours. Based
upon a calibration curve obtained from integrating the
anomeric signals in the HSQC spectra (using NMRPipe
9
expected stereospecificities of amino transfer, suggesting this
method to offer access to stereochemical information if the
chemical shift information on at least one stereoisomer is
available. While the method presented suggests broad utility,
delineation of reaction stereospecificity for unknown molecules
1
1
remains dependent upon the ability to obtain H− H coupling
1
4
software ) of standards 4 and 5 (Figure S2 in Supporting
Information), the concentration of the corresponding reaction
product/substrate in the spectra was plotted against reaction
time and the corresponding rate, as reflected by the slope,
determined by linear regression. Interestingly, the rate of
constants.
In summary, we have established a simple in situ NMR-based
method to follow sugar-nucleotide-dependent multienzyme
cascades in real time. The method is highly sensitive, affording
simultaneous detection of all suitably labeled reactants,
intermediates, and products available in quantities of ≥25 μg
of compound and can be applied to measure kinetic
parameters. As such, the method circumvents a major liability
of prior methods, namely the difficulty of detecting short-lived
intermediates/products. In addition, the commercial availability
−1
TDP16 measured by this method (15.2 ± 1.2 min , Figure 2)
was consistent with previously reported values (25.6 ± 0.5
−1
6
min ) while the rate determined for CalS3 (0.84 ± 0.03
−1
min , Figure 2) is similar to that of other TDP-Glc 4,6-
dehydratases from secondary metabolite producing strains
−1
15
13
(
ranging from 1.5 to 16.4 min ).
of additional [U- C]sugar precursors along with the recent
To explore the stereospecificity of CalS13, two model
transaminase reactions were examined which differ solely by
their stereospecificity of amino transfer. Specifically, starting
engineering of OleD variants to accommodate ADP, CDP,
GDP, TDP, or UDP and a range of novel sugars suggests the
potential for broad applicability.
6,16
C
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Organic Letters
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ASSOCIATED CONTENT
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This includes general methods, overproduction and purification
of enzymes, enzyme reactions, NMR and HRMS data. This
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AUTHOR INFORMATION
Corresponding Authors
■
*
*
(
6) Gantt, R. W.; Peltier-Pain, P.; Cournoyer, W. J.; Thorson, J. S.
Nat. Chem. Biol. 2011, 7, 685−691.
7) Zhao, L.; Ahlert, J.; Xue, Y.; Thorson, J. S.; Sherman, D. H.; Liu,
E-mail: jsthorson@uky.edu.
E-mail: ssingh555@uky.edu.
Present Address
∥Glycom A/S, Denmark.
Notes
(
H. W. J. Am. Chem. Soc. 1999, 121, 9881−9882.
(8) (a) Ahlert, J.; Shepard, E.; Lomovskaya, N.; Zazopoulos, E.;
Staffa, A.; Bachmann, B. O.; Huang, K.; Fonstein, L.; Czisny, A.;
Whitwam, R. E.; Farnet, C. M.; Thorson, J. S. Science 2002, 297,
1173−1176. (b) Zhang, C.; Griffith, B. R.; Fu, Q.; Albermann, C.; Fu,
X.; Lee, I. K.; Li, L.; Thorson, J. S. Science 2006, 313, 1291−1294.
(c) Zhang, C.; Bitto, E.; Goff, R. D.; Singh, S.; Bingman, C. A.;
Griffith, B. R.; Albermann, C.; Phillips, G. N., Jr.; Thorson, J. S. Chem.
Biol. 2008, 15, 842−853.
The authors declare the following competing financial
interest(s): The authors report competing interests. J.S.T. is a
co-founder of Centrose (Madison, WI).
ACKNOWLEDGMENTS
■
(
9) (a) Zhao, L.; Yeung, B. S.; Liu, H. W. J. Am. Chem. Soc. 2001,
23, 7909−7910. (b) Hwang, B. Y.; Lee, H. J.; Yang, Y. H.; Joo, H. S.;
Kim, B. G. Chem. Biol. 2004, 11, 915−925.
10) Singh, S.; Chang, A.; Helmich, K. E.; Bingman, C. A.; Wrobel, R.
This work was supported by NIH RO1 CA84374 (JST), NIH
R37 AI52218 (JST), NCATS (UL1TR000117) and made use
of the UW NMR FAM [supported in part by the NIH
P41RR02301, P41GM10399, RR02781, RR08438), University
of Wisconsin, NSF (DMB-8415048, OIA-9977486, BIR-
1
(
(
L.; Beebe, E. T.; Makino, S.; Aceti, D. J.; Dyer, K.; Hura, G. L.;
Sunkara, M.; Morris, A. J.; Phillips, G. N., Jr.; Thorson, J. S. ACS Chem.
Biol. 2013, 8, 1632−1639.
9
214394), DOE, and USDA].
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7662−17663. (b) Timmons, S. C.; Thorson, J. S. Curr. Opin. Chem.
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