Carbocyclic Substrate Analogues Reveal Kanosamine Biosynthesis Begins with the α-Anomer of Glucose 6-Phosphate

Article abstract of DOI:10.1021/acschembio.0c00409

NtdC is an NAD-dependent dehydrogenase that catalyzes the conversion of glucose 6-phosphate (G6P) to 3-oxo-glucose 6-phosphate (3oG6P), the first step in kanosamine biosynthesis in Bacillus subtilis and other closely-related bacteria. The NtdC-catalyzed r

Full text of DOI:10.1021/acschembio.0c00409

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Carbocyclic Substrate Analogues Reveal Kanosamine Biosynthesis
Begins with the α‑Anomer of Glucose 6‑Phosphate
Natasha D. Vetter, Rajendra C. Jagdhane, Brett J. Richter, and David R. J. Palmer*
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ABSTRACT: NtdC is an NAD-dependent dehydrogenase that
catalyzes the conversion of glucose 6-phosphate (G6P) to 3-oxo-
glucose 6-phosphate (3oG6P), the first step in kanosamine
biosynthesis in Bacillus subtilis and other closely-related bacteria.
The NtdC-catalyzed reaction is unusual because 3oG6P undergoes
rapid ring opening, resulting in a 1,3-dicarbonyl compound that is
inherently unstable due to enolate formation. We have reported
the steady-state kinetic behavior of NtdC, but many questions
remain about the nature of this reaction, including whether it is the
α-anomer, β-anomer, or open-chain form that is the substrate for
the enzyme. Here, we report the synthesis of carbocyclic G6P
analogues by two routes, one based upon the Ferrier II
rearrangement to generate the carbocycle and one based upon a Claisen rearrangement. We were able to synthesize both
pseudo-anomers of carbaglucose 6-phosphate (C6P) using the Ferrier approach, and activity assays revealed that the pseudo-α-
anomer is a good substrate for NtdC, while the pseudo-β-anomer and the open-chain analogue, sorbitol 6-phosphate (S6P), are not
substrates. A more efficient synthesis of α-C6P was achieved using the Claisen rearrangement approach, which allowed for a
thorough evaluation of the NtdC-catalyzed oxidation of α-C6P. The requirement for the α-anomer indicates that NtdC and NtdA,
the subsequent enzyme in the pathway, have co-evolved to recognize the α-anomer in order to avoid mutarotation between
enzymatic steps.
anosamine, 3-amino-3-deoxy-D-glucose, is an amino-sugar
known to inhibit the growth of a variety of bacterial and
Because of the rapid mutarotation of reducing sugars, 3-keto
sugars can generate the corresponding 1,3-dicarbonyl in the
acyclic form, and as a result, the C2 proton becomes much
more acidic. Under mildly basic conditions, the 1,3-dicarbonyl
then rapidly enolizes, resulting in a strongly UV-absorbing
species (Figure 1b), and indeed, 3oG6P displays a strong UV
absorbance at 310 nm.8 Because of this rapid enolate
formation, the absorbance due to NADH at 340 nm is masked
by that of the enolate at 310 nm when monitoring the reaction
spectrophotometrically.
We have previously reported the kinetics of NtdC by itself
and with the next enzyme in the pathway, NtdA, which
converts 3oG6P to kanosamine 6-phosphate through a
glutamate-coupled PLP-dependent transamination.16 We have
observed that the equilibrium of both the NtdC reaction and
the NtdC−NtdA-coupled reaction lies heavily toward G6P,
and as a result, we have been unable to isolate the 3oG6P
K
fungal species, in particular, those that lead to plant
pathogenesis.1,2 It is also a precursor to more complex
antibiotics, such as kanamycin, rifamycins, and ansamycins.1,3,4
Biosynthetic pathways of kanosamine have been found in a
variety of bacterial species, such as Bacillus pumilus,5 Bacillus
cereus,6 Amycolatopsis mediterranei,4,7 and Bacillus subtilis. In B.
subtilis, kanosamine is synthesized in three steps from glucose
6-phosphate (G6P) catalyzed by the enzymes NtdC, NtdA,
and NtdB, as shown in Figure 1a.8
The first step in this pathway, catalyzed by NtdC (a glucose-
6-phosphate 3-dehydrogenase), converts G6P to 3-dehydro-D-
glucose 6-phosphate, also known as 3-oxo-glucose 6-phosphate
(3oG6P) through an NAD-dependent oxidation. While
dehydrogenases are ubiquitous in nature, this is the only
known enzyme to catalyze this particular reaction on the C3
hydroxyl of a phosphorylated reducing sugar, and in fact, there
are only a handful of reports of 3-keto reducing sugars in the
literature.9−12 There is a notable similarity of this reaction to
the mechanism of glycosyl hydrolase family 4, a group of 6-
phospho glycoside hydrolases that oxidize the C3 hydroxyl of
G6P glycosides with NAD to facilitate hydrolysis of the
glycoside, and the resulting enzyme-bound 3oG6P is reduced
back to G6P by NADH prior to the product release.13−15
Received: May 20, 2020
Accepted: July 16, 2020
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excellent yield. The primary alcohol at C6 was converted to an
iodide under Appel conditions in quantitative yields, and the
elimination of hydrogen iodide with a base produced exo-
methylene derivative 8.
The elimination proved difficult under a variety of
conditions, giving undesired isomerization of the exo-
methylene 8 to the thermodynamically favored endo-alkene
9. Most commonly, DBU has been used in DMF at 70−80
°C,17,19−21 and while these conditions are reported to produce
the desired exo-methylene in good yields, our experience has
shown minimal success. At best, we were able to isolate the
desired product in a 44% yield; however, significant isomer-
ization was still observed. In one instance, we were able to
isolate a significant quantity of a second product, which was
confirmed by NMR and mass spectrometry to be a DBU
nucleophilic adduct (Supporting Information, compound
S27). Using fresh KOtBu in THF,22 we were able to achieve
very good yields quite rapidly under much milder conditions.
Note, however, that this reaction was highly sensitive to
moisture, making it difficult to reproduce.
The Ferrier II carbocyclization of compound 8 was carried
out with palladium chloride in aqueous dioxane, which
proceeded in a 75% yield and produced an inseparable mixture
of pseudo-α and -β diastereomers in a 3:1 ratio, consistent with
a previous report.17 The two isomers could be separated upon
conversion to the silyl ethers 11a and 11b; however,
degradation of the α-isomer was observed, resulting in a
different ratio of products. This degradation was also observed
when we performed this reaction with pure pseudo-α
diastereomer of 10.
Homologation of 11 at C5 using the Tebbe reagent
produced exo-methylene derivative 12 in good yields (Scheme
1). Interestingly, homologation under Wittig conditions
afforded only a 51% yield with the pseudo-α-isomer, resulting
in two elimination by-products being formed, with the loss of
either the C3-OBn (15%) or C1-OTBS (29%); this was not
observed with the pseudo-β isomer. Hydroboration-oxidation
of 12 led predominantly to the undesired ido configuration at
C5 (13) in contrast to results reported by Ko et al.17 Careful
inspection of both the 1H and 2D NMR spectra for 13a, which
matches that obtained by Ko, showed a small coupling
constant (5 Hz) of H4 to H5, suggesting that CH2OH is in the
axial orientation (Figure 2a) and not the equatorial orientation
as previously reported.17 We were able to convert this product
back to the desired gluco configuration (14) in three steps by
oxidation to the corresponding aldehyde, isomerization to the
low-energy isomer under basic conditions,22 and reduction
back to the alcohol. Examination of the NMR spectra of this
product showed a clear, large H4/H5 coupling constant of 11
Hz, indicative of the gluco configuration (Figure 2b).
Phosphorylation with diphenyl phosphoryl chloride afforded
C6P derivatives 15a and 16 in good yields. The TBS
protecting group of the β isomer was easily cleaved upon
acidic workup, while the TBS group of the α-isomer proved
difficult to deprotect under the same conditions. Interestingly,
the use of TBAF to deprotect the TBS group of 15a resulted in
fluorine substitution at phosphorus and not silyl deprotection,
as we had expected (Supporting Information, compound S28).
Final deprotection of both the benzyl and phenyl groups
required two separate hydrogenolysis steps, with the diphenyl
phosphate deprotection proceeding in lower yields. Overall, α-
C6P and β-C6P were obtained in 7 and 1% yields, respectively,
in 14 steps.
Figure 1. (a) Kanosamine biosynthesis in B. subtilis. (b) Enolate
formation of 3oG6P when released into solution. (c) Mutarotation of
G6P and synthetic analogues as mimics of the various forms of G6P.
product. Additionally, we have not been able to synthesize this
compound, likely due to rapid degradation.
We seek a complete understanding of this unusual enzymatic
function, and unfortunately, we have been unable to generate a
crystal structure of NtdC (with or without G6P). The rapid
mutarotation of G6P in solution means that all forms of the
sugar are easily accessible on the time scale of the enzymatic
reaction (Figure 1c), meaning that we do not know the nature
of the substrate form recognized by the enzyme. Furthermore,
we have observed no activity with any substrates that bear
substitutions at C1, such as methyl or UDP glycosides,
suggesting this position is crucial for substrate binding to the
active site. This prompted us to synthesize carbocyclic
analogues of G6P (α- and β-carbaglucose 6-phosphate,
C6P), which can mimic the cyclic forms of the substrate, as
well as an acyclic analogue (sorbitol 6-phosphate, S6P) to
mimic the open chain form (Figure 1c). Additionally, since
these carbocyclic compounds cannot undergo the same ring
opening as G6P, the stereochemistry at the pseudo-anomeric
position remains fixed, allowing us to determine the anomeric
preference of NtdC.
RESULTS AND DISCUSSION
■
Synthesis of Sorbitol 6-Phosphate. S6P (1) was
synthesized in one step in an 80% yield from G6P using
NaBH4. After purification using anion exchange chromatog-
raphy, 11B NMR showed contaminating boric acid present in
the sample, which was then further removed as trimethyl
borate by stirring with methanol under vacuum.
Synthesis of α-C6P and β-C6P via Ferrier II
Rearrangement. We first chose to synthesize carbocyclic
analogues 2 and 3 using the well-known Ferrier-II carbocyc-
lization reaction and readily accessible glucose precursors,
similar to what Ko et al. reported to generate carbocyclic
glucose 1-phosphate.17 The Ferrier substrate was synthesized
in four steps starting from commercially available methyl 4,6-
O-benzylidene-α-D-glucopyranoside (4, Scheme 1). Benzyla-
tion at C2 and C3 and subsequent regioselective cleavage of
the 4,6-benzylidene18 afforded tri-O-benzyl derivative 6 in an
B
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a
Scheme 1. Synthesis of α-C6P and β-C6P via Ferrier II Rearrangement
a
Numbering of the carbocyclic ring is indicated in red for compounds 10 and 14.
NtdC Activity with Acyclic and Carbocyclic Ana-
logues. Having all three G6P analogues (S6P, α-C6P, and β-
C6P) in hand, we tested their activity with NtdC. α-C6P, while
comparatively slower than G6P (Figure 3a), proved to be a
good substrate with NtdC (Figure 3c), while no reaction was
observed with β-C6P and S6P (Figure 3b and 3d), even at
higher substrate concentrations or varied pH. Under identical
conditions, α-C6P was oxidized at approximately 30% the rate
of G6P, considerably better than the next best substrate of
NtdC, D-glucose (<1%). Additionally, no absorbance at 310
nm was observed, as expected, due to the inability to form the
1,3-dicarbonyl product. Preliminary kinetics at 1 mM NAD
revealed an apparent Km of 0.9 mM for α-C6P, approximately
20-fold higher than G6P (43 μM) (Supporting Information,
Figure S6). Additionally, a significantly lower apparent kcat was
observed for α-C6P (0.8 s−1) compared to G6P (4.1 s−1).
Synthesis of α-C6P via Claisen Rearrangement.
Having discovered that α-C6P is a good substrate for NtdC,
1
Figure 2. (a) H NMR signal of H4 from compound 13a showing
large coupling to H3 and small coupling to H5, indicative of the
CH2OH in the axial position. (b) 1H NMR signal of H4 from
compound 14a showing large coupling to both H3 and H5, indicative
of the CH2OH in the equatorial position. ROBn; R′OTBS.
C
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H4/H5 coupling constant of 11 Hz (Supporting Information).
This was then phosphorylated with tetrabenzyl pyrophosphate
in DMF to give fully benzyl-protected carbaglucal 25 (Scheme
2).
cis-Dihydroxylation of compound 24 was previously
reported to proceed stereoselectively with osmium tetraoxide,
without any added chiral auxiliaries,23,24 and we expected the
same selectivity with 25. Indeed, we observed only one
1
product formed, in 90% yield (Scheme 2). H NMR of 26
showed a large coupling between H3 and H2 (9 Hz) to
confirm that C2−OH is equatorial, and the small coupling
between H2 and H1 (3 Hz) confirmed that C1−OH is axial.
Finally, hydrogenolysis with Pd/C in MeOH/AcOH at 50 psi
of H2 afforded α-C6P in a quantitative yield.
The Claisen rearrangement route offers significant advan-
tages over the Ferrier II route in our experience. Overall, we
were able to synthesize α-C6P on a larger scale, with double
the overall yield (15%) in three fewer steps. Additionally, all
steps were easily purified by column chromatography, with
little or no by-products observed; several steps in this route
allowed for two-step transformations to be conducted without
purification, such as the conversion of diacetate 19 to dibenzyl
21, the tandem oxidation and Wittig olefination to generate 23,
and the Claisen rearrangement and reduction in one-pot to
give carbaglucal 24. The final deprotection required only one
step and proceeded very cleanly such that no further
purification was needed on the final product. The final product
was converted to the monosodium salt by careful titration with
NaOH. In our experience, handling the monosodium salt is far
easier, as it is less hygroscopic than the free acid.
Kinetics of NtdC with α-C6P. Our initial kinetics of NtdC
with α-C6P revealed an apparent Km of 0.9 mM and a kcat of
0.8 s−1. To gain a complete understanding of the kinetics with
this substrate, we obtained a series of saturation curves by
varying the concentration of C6P at various fixed concen-
trations of NAD. The data was fit to a variety of two-substrate
kinetic models, including ping-pong, sequential, and rapid
equilibrium ordered mechanisms (Supporting Information, eq
S1−5, Scheme S1−4). As expected, the best fit obtained was
with a sequential bisubstrate mechanism (eq S1, Figure 5), the
same as previously observed for NtdC with G6P.16 No
cooperativity was observed, and the inclusion of substrate
inhibition terms did not improve the quality of the fit. The
kinetic constants are summarized in Table 1 and are compared
with those reported for G6P.16
Figure 3. Absorbance traces of the NtdC reaction with (a) G6P, (b)
S6P, (c) α-C6P, and (d) β-C6P. Each experiment contained 1 mM
NAD, 1 mM substrate, 100 mM Amp-HCl pH 9.5, and 29 nM NtdC.
Wavelength scans were taken every 10 s for a total reaction time of 5
min.
we wanted to gather a more complete kinetic profile with this
substrate. Upon an examination of our previous synthetic
scheme, we were discouraged by several low-yielding steps,
unwanted isomerizations and by-products, and the loss of
nearly half our overall yield as the unwanted β isomer. This
prompted us to pursue an alternative synthetic route. Several
other carbocyclic sugar analogues have been previously
synthesized using a Claisen rearrangement approach, which
centers on a key thermal [3,3]-sigmatropic rearrangement of a
homologated glycal, as shown in Figure 4.23−26
Figure 4. Thermal Claisen rearrangement to generate carbocyclic
sugars.26
An examination of the calculated kinetic constants revealed a
remarkably high catalytic turnover (kcat of 2.0 s−1), only half
that of the natural substrate, G6P. While the replacement of
the ring oxygen with methylene is approximately isosteric, the
capacity to form polar or hydrogen-bonding interactions is lost,
altering the binding in the active site. This is reflected in the
increased Km of both substrates in the reaction of α-C6P,
indicating that the endocyclic oxygen makes a contribution to
the energy of the ternary complex and its conversion to
products.
Starting from commercially available triacetyl D-glucal, 17,
we were able to deacetylate C6 regioselectively using lipase
from Candida rugosa in good yields (Scheme 2).27 We
envisioned oxidation and homologation of 18 would generate
the desired alkene; however, we were unable to isolate the
desired product from this procedure, likely due to the lability
of the acetyl protecting groups. Instead, the C6−OH of 18 was
silylated with TBSCl in a 90% yield, and the acetyl groups
replaced with benzyl groups in two steps and 64% overall yield.
Deprotection of the TBS with TBAF proceeded cleanly, and
subsequent oxidation and Wittig homologation, based on
procedures reported by Gao et al., gave methylene 23 in a 63%
yield over two steps.24 The Claisen rearrangement of 23 was
carried out in diphenyl ether at 210 °C according to reported
procedures,23,25 and immediate reduction with NaBH4
generated carbaglucal analogue 24 in 69% yield. The desired
CONCLUSION
■
The production of antimicrobial compounds such as kanos-
amine by environmental bacteria such as Bacillus spp. is
important in applications such as managing plant diseases.28
The manipulation of kanosamine production and the
application or repurposing of kanosamine biosynthesis
enzymes in synthetic biology demand a comprehensive
1
configuration at C5 was confirmed by H NMR, with a large
D
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Scheme 2. Synthesis of α-C6P via Claisen Rearrangement
reported stereochemistry at C5 is in fact incorrect, and the
previous report describes the synthesis of the carbocyclic
analogue of L-idose.17
Having synthesized all three substrate analogues, we were
able to test their activity with NtdC and our results revealed
that α-C6P is a good substrate for the enzyme, while no
enzyme activity was observed with the other two compounds.
Synthesis of α-C6P was improved upon using a synthetic
scheme centering on a Claisen rearrangement to generate the
desired carbocycle and avoid the formation of the inactive β-
isomer. This approach offered an improved overall yield (15%)
in fewer steps (11 steps), and was amenable to scaling up. We
examined the kinetics of NtdC with α-C6P, which revealed a
relatively high kcat (2.0 0.1) and a Km of 1.9 mM for C6P,
nearly 50 times that of G6P. Additionally, the Km of NAD
increased 10-fold.
Figure 5. Kinetics of NtdC with C6P. (a) Varied [C6P] at fixed
[NAD]: 5.00 mM ( ), 2.50 mM ( ), 1.00 mM ( ), 0.50 mM ( ),
⧫
■
●
▲
○
0.10 mM (×), and 0.05 mM ( ). (b) Varied [NAD] at fixed [C6P]:
⧫
■
●
▲
10.0 mM ( ), 5.00 mM ( ), 2.50 mM ( ), 1.00 mM ( ), 0.50 mM
(×), and 0.10 mM ( ). Reactions were performed in 100 mM Tris-
HCl pH 9, with 29 nM NtdC. Each data point represents duplicate
○
The enzymatic activity with only the α-C6P analogue
indicates that NtdC binds and reacts with the α-glucopyranosyl
form of G6P during catalysis. It also highlights the importance
of the anomeric hydroxyl on substrate binding. Our previous
crystallographic studies have revealed that the next enzyme in
kanosamine biosynthesis, NtdA, binds the α anomer of K6P
exclusively,29 and our current results on NtdC suggests the
same preference. This is consistent with our proposal that
NtdC and NtdA have co-evolved to sequester and process the
ketone product of the NtdC reaction as it is formed.
Production of the wrong anomer would necessitate muta-
rotation via the acyclic 1,3-dicarbonyl form, and enolate
formation could divert the product from the metabolic
pathway (Figure 1b). The sequestration or rapid conversion
of the keto-intermediate may be a general strategy for the
conversion of hydroxyls to amines in enzymatic pathways.16
The relatively high activity of NtdC with α-C6P suggests
that Ntd enzymes could be used to generate other carbocyclic
analogues, such as carbocyclic kanosamine. Looking forward,
the incorporation of carbocyclic kanosamine analogues into
complex natural products using synthetic biology approaches
could yield new and more stable antibiotics.
experiments. Solid lines represent the global fit of the data to eq S1.
Table 1. Kinetic Data of the NtdC Reaction with G6P and
α-C6P
a
G6P
C6P
KNAD
Ksugar
KiNAD
0.040 0.004
0.043 0.004
0.9 0.1
0.48 0.06
1.9 0.2
0.2 0.1
2.0 0.1
(0.41 0.03) × 104
(0.10 0.07) × 104
mM
mM
mM
s−1
M−1 s−1
M−1 s−1
kcat
b
4.1 0.1
k
k
cat/KNAD
cat/Ksugar
(10 1) × 104
(9.6 0.8) × 104
b
a
b
See ref 16. Calculated from a global fitting of the kinetic data to eq
S1, rearranged to treat Vmax/Ka or Vmax/Kb as single parameters.
understanding of these enzymes. We have synthesized three
carbohydrate analogues in order to mimic three forms of G6P
in solution to test as substrates with NtdC. An analogue of the
open-chain form, S6P, was synthesized in one step by NaBH4
reduction of G6P. Both carbocyclic analogues, α-C6P and β-
C6P, were synthesized in 14 steps (in 7 and 1% overall yield,
respectively) using a Ferrier II carbocyclization approach to the
carbaglucose core, following a previous report.17 Unfortu-
nately, the hydroboration-oxidation of Ferrier products 12a
and 12b led almost exclusively to the opposite stereochemistry
than reported. While the NMR spectra of our compounds
matched that of their work, we have been able to show that the
ASSOCIATED CONTENT
■
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* Supporting Information
The Supporting Information is available free of charge at
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E
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I., Krol, E. S., Sanders, D. A. R., and Palmer, D. R. J. (2013) A
previously unrecognized kanosamine biosynthesis pathway in Bacillus
subtilis. J. Am. Chem. Soc. 135, 5970−5973.
Full experimental procedures, including synthetic
methods, protein purification, and kinetic methods, as
1
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AUTHOR INFORMATION
Corresponding Author
David R. J. Palmer − Department of Chemistry, University of
Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada;
orcid.org/0000-0001-9300-7460; Email: dave.palmer@
usask.ca
(10) Hayano, K., Tsubouchi, Y., and Fukui, S. (1973) 3-Ketoglucose
reductase of Agrobacterium tumefaciens. J. Bacteriol. 113, 652−657.
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(1998) C-2 and C-3 oxidation of D-Glc, and C-2 oxidation of D-Gal
by pyranose dehydrogenase from Agaricus bisporus. Carbohydr. Res.
310, 151−156.
(12) Kojima, K., Tsugawa, W., and Sode, K. (2001) Cloning and
Expression of Glucose 3-Dehydrogenase from Halomonas sp. α-15 in
Escherichia coli, Biochem. Biochem. Biophys. Res. Commun. 282, 21−27.
(13) Rajan, S. S., Yang, X., Collart, F., Yip, V. L. Y., Withers, S. G.,
Varrot, A., Thompson, J., Davies, G. J., and Anderson, W. F. (2004)
Novel Catalytic Mechanism of Glycoside Hydrolysis Based on the
Structure of an NAD+/Mn2+-Dependent Phospho-α-Glucosidase
from Bacillus subtilis. Structure 12, 1619−1629.
Authors
Natasha D. Vetter − Department of Chemistry, University of
Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada;
orcid.org/0000-0002-0963-8858
Rajendra C. Jagdhane − Department of Chemistry, University
of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
Brett J. Richter − Department of Chemistry, University of
Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
(14) Yip, V. L. Y., and Withers, S. G. (2006) Mechanistic Analysis of
the Unusual Redox-Elimination Sequence Employed by Thermotoga
maritima BglT: A 6-Phospho-β-glucosidase from Glycoside Hydrolase
Family 4. Biochemistry 45, 571−580.
(15) Yip, V. L. Y., Thompson, J., and Withers, S. G. (2007)
Mechanism of GlvA from Bacillus subtilis: A Detailed Kinetic Analysis
of a 6-Phospho-α-glucosidase from Glycoside Hydrolase Family 4.
Biochemistry 46, 9840−9852.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschembio.0c00409
Notes
The authors declare no competing financial interest.
(16) Vetter, N. D., and Palmer, D. R. J. (2017) Simultaneous
Measurement of Glucose-6-phosphate 3-Dehydrogenase (NtdC)
Catalysis and the Nonenzymatic Reaction of Its Product: Kinetics
and Isotope Effects on the First Step in Kanosamine Biosynthesis.
Biochemistry 56, 2001−2009.
ACKNOWLEDGMENTS
■
This work was funded by a NSERC Discovery Grant to
D.R.J.P. and a NSERC PGS award and a University of
Saskatchewan Gerhard Herzberg Memorial Fellowship to
N.D.V. The authors thank K. Brown, P. Zhu, K. Fransishyn,
K. Thoms, and other staff of the Saskatchewan Structural
Sciences Centre for their expertise; D. Sanders for helpful
discussions and shared equipment; and the anonymous
reviewers for their helpful comments on the manuscript.
(17) Ko, K.-S., Zea, C. J., and Pohl, N. L. (2004) Surprising Bacterial
Nucleotidyltransferase Selectivity in the Conversion of Carbaglucose-
1-phosphate. J. Am. Chem. Soc. 126, 13188−13189.
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ABBREVIATIONS
■
C6P, carbaglucose 6-phosphate; 3oG6P, 3-oxo-D-glucose 6-
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