A previously unrecognized kanosamine biosynthesis pathway in Bacillus subtilis

Article abstract of DOI:10.1021/ja4010255

The ntd operon in Bacillus subtilis is essential for biosynthesis of 3,3′-neotrehalosadiamine (NTD), an unusual nonreducing disaccharide reported to have antibiotic properties. It has been proposed that the three enzymes encoded within this operon, NtdA, NtdB, and NtdC, constitute a complete set of enzymes required for NTD synthesis, although their functions have never been demonstrated in vitro. We now report that these enzymes catalyze the biosynthesis of kanosamine from glucose-6-phosphate: NtdC is a glucose-6-phosphate 3-dehydrogenase, NtdA is a pyridoxal phosphate-dependent 3-oxo-glucose-6-phosphate:glutamate aminotransferase, and NtdB is a kanosamine-6-phosphate phosphatase. None of these enzymatic reactions have been reported before. This pathway represents an alternate route to the previously reported pathway from Amycolatopsis mediterranei which derives kanosamine from UDP-glucose.

Full text of DOI:10.1021/ja4010255

Communication
pubs.acs.org/JACS
A Previously Unrecognized Kanosamine Biosynthesis Pathway in
Bacillus subtilis
Natasha D. Vetter,† David M. Langill,† Shazia Anjum,†,§ Julie Boisvert-Martel,† Rajendra C. Jagdhane,†
Egiroh Omene,† Hongyan Zheng,†,∥ Karin E. van Straaten,† Isaac Asiamah,‡ Ed S. Krol,‡
David A. R. Sanders,† and David R. J. Palmer*,†
†Department of Chemistry, and ‡College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, SK,
Canada, S7N 5C9
S
* Supporting Information
(PLP)-dependent 3-oxo-glucose-6-phosphate:glutamate amino-
ABSTRACT: The ntd operon in Bacillus subtilis is
essential for biosynthesis of 3,3′-neotrehalosadiamine
(NTD), an unusual nonreducing disaccharide reported
to have antibiotic properties. It has been proposed that the
three enzymes encoded within this operon, NtdA, NtdB,
and NtdC, constitute a complete set of enzymes required
for NTD synthesis, although their functions have never
been demonstrated in vitro. We now report that these
enzymes catalyze the biosynthesis of kanosamine from
glucose-6-phosphate: NtdC is a glucose-6-phosphate 3-
dehydrogenase, NtdA is a pyridoxal phosphate-dependent
3-oxo-glucose-6-phosphate:glutamate aminotransferase,
and NtdB is a kanosamine-6-phosphate phosphatase.
None of these enzymatic reactions have been reported
before. This pathway represents an alternate route to the
previously reported pathway from Amycolatopsis medi-
terranei which derives kanosamine from UDP-glucose.
transferase, and NtdB, a kanosamine-6-phosphate phosphatase.
Kanosamine, or 3-amino-3-deoxy-D-glucose, is known to
have antibiotic1,2 and antifungal3 properties. A kanosamine
biosynthetic pathway starting from UDP-glucose was first
proposed for Bacillus pumilis.4 Later, such a pathway was
characterized in Amycolatopsis mediterreanei,5,6 based on
incubation of cell-free extracts of the bacteria with UDP-
glucose, NAD, and glutamine; an incubation with 6,6′-2H2-
UDP-glucose resulted in 6,6′-2H2-kanosamine in 5% yield.5
Most recently, an operon in Bacillus cereus UW85, named the
kab operon, was proposed to encode enzymes that synthesize
kanosamine.7 This hypothesis was based on the resemblance of
the kab operon to the ntd operon in B. subtilis, although the ntd
operon was so named because it was proposed to synthesize the
disaccharide NTD, which is made up of two kanosamine
residues connected by a 1,1′-α,β linkage. B. cereus UW85 is
known to produce kanosamine and not NTD,8 but the function
of the kab operon has not been experimentally investigated.
The ntd operon, as described by Inaoka et al., is required for
the biosynthesis of NTD,9,10 which is reported to have
antibiotic properties including inhibition of the growth of
Staphylococcus aureus.11 The production of NTD has been
reported for Bacillus pumilus and Bacillus circulans.11,12
Although the operon is normally dormant in B. subtilis such
that no NTD is produced, a mutant strain in which the operon
is activated secretes NTD. Moreover, when Escherichia coli is
transformed with a plasmid bearing the operon, that bacterium
also produces NTD. The authors thus concluded that the ntd
operon “contains a complete set of genes required for NTD
biosynthesis.”9 However, neotrehalose is not known to be
produced by this organism, and the same authors point out that
the addition of neotrehalose or trehalose to cultures did not
result in increased NTD. This strongly suggests that NTD is
synthesized by the linking of kanosamine monosaccharides or
closely related derivatives. One would therefore predict that a
complete set of genes necessary for NTD biosynthesis would
include a nucleotidylyltransferase and/or glycosyltransferase in
order to form the glycosidic bond. The three enzymes encoded
within the ntd operon show no apparent sequence similarity to
such enzymes. Instead, the encoded proteins NtdA, NtdB, and
anosamine is a naturally occurring antibiotic and
K
component of oligosaccharide antibiotics such as
kanamycin (Figure 1). Although this product has been
known for many years, the biosynthetic pathway for kanos-
amine in bacteria has not been demonstrated unequivocally.
Figure 1. Structures of kanosamine and NTD.
Here we show that the enzymes encoded by the ntd operon
from Bacillus subtilis, which were proposed to catalyze the
complete synthesis of 3,3′-neotrehalosadiamine (NTD), in fact
synthesize kanosamine. This is in agreement with a published
proposal for the function of an homologous operon in Bacillus
cereus, but by a different series of reactions than previously
proposed. Here we show that glucose-6-phosphate is converted
in three steps to kanosamine by the action of NtdC, a glucose-
6-phosphate 3-dehydrogenase, NtdA, a pyridoxal phosphate
Received: January 29, 2013
Published: April 11, 2013
© 2013 American Chemical Society
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NtdC are predicted by sequence alignment to be a PLP-
dependent aminotransferase, a member of the HAD super-
family, and an NAD-dependent dehydrogenase, respectively.
The unusual structure, interesting properties, and undeter-
mined biosynthesis prompted us to investigate NTD further.
We recently described the first chemical synthesis of NTD.13
We subcloned each of ntdA,14 ntdB, and ntdC into pET-28b
and expressed them in E. coli BL21-Gold cells. The N-terminal
hexahistidine-tagged proteins were purified by nickel-affinity
chromatography. NtdC is homologous with inositol dehydro-
genase (IDH) and several other NAD-dependent mono-
saccharide dehydrogenases. Although this made UDP-glucose
a less likely candidate substrate, IDH can accommodate inositol
substrates conjugated to aromatic and carbohydrate moieties,15
and therefore recognition of a nucleotidylyl sugar was an
intriguing possibility. However, in our hands NtdC shows no
activity with UDP-glucose using the standard UV assay
monitoring the absorbance at 340 nm due to the appearance
of NADH or NADPH. Glucose is a weak substrate for the
enzyme (as is inositol). The strong resemblance of NtdB to
phosphatases such as sucrose-6-phosphate phosphatase sug-
gested to us that a 6-phospho-sugar might be the correct
substrate for NtdC, and indeed this enzyme showed high
specificity for the NAD-dependent oxidation of glucose 6-
phosphate, and no NADP-dependent activity.
Figure 3. (A) The increase in absorbance over time in the UV−visible
spectrum due to oxidation of glucose-6-phosphate and reduction of
NAD by NtdC at pH 8.0. (B) The reaction in A in the presence of
excess pyruvate and lactate dehydrogenase. The recycling of NADH
eliminates the absorbance at 340 nm, revealing the increase in
absorbance at 310 nm associated with 3-oxo-D-glucose-6-phosphate.
(C) The reaction in A in the presence of excess L-glutamate and NtdA.
Removal of the intermediate reveals the increasing absorbance due to
NADH at 340 nm. (D) Reaction of kanosamine-6-phosphate and 2-
oxoglutarate in the presence of NtdA, leading to the formation of 3-
oxo-D-glucose-6-phosphate. In all cases, spectra were collected at 5-
min intervals.
This information led us to the hypothesis that the three
enzymes functioned as shown in Figure 2, generating
phosphate and 2-oxoglutarate in the presence of NtdA again
gave rise to the absorbance at 310 nm (Figure 3D), which was
not present when the reaction was performed in an excess of
NtdC and NADH. This demonstrated the reversibility of the
process and established the function of NtdA as shown in
Figure 2. Confirmation of this was provided using reversed-
phase ion-pairing HPLC with evaporative light scattering
detection, with which we could observe the consumption of
kanosamine-6-phosphate and the production of glucose-6-
phosphate in the presence of NtdA, NtdC, 2-oxoglutarate, and
NADH (Supporting Information, Figure S1).
Figure 2. Observed kanosamine pathway.
NtdB phosphatase activity was investigated using several
phosphosugar substrates, including NTD-6-phosphate, NTD-
6′-phosphate, NTD-6,6′-bisphosphate, glucose-6-phosphate,
glucose-1-phosphate, glucosamine-6-phosphate, and kanos-
amine-6-phosphate. The disaccharide analogs were synthesized
by adaptation of our recently reported synthesis of NTD13 as
described in the Supporting Information. NtdB is a very
efficient catalyst of the phosphatase reaction of kanosamine-6-
phosphate (kcat = 32 3 s−1, Km = 101 8 μM), but has no
effect on glucose-6-phosphate, indicating selectivity at the 3-
position. There was a trace amount of activity (<1%) using
glucosamine-6-phosphate, but no other compound tested,
including the synthesized disaccharide phosphates, glucose-6-
phosphate, or para-nitrophenyl phosphate, showed significant
reactivity. Consistent with our results, Huang et al. pointed out
that highly selective members of the HAD superfamily tend to
have specificity constants >105 s−1 M−1.19 This strongly
suggests that NtdB, and by extension, the ntd operon, has
kanosamine rather than NTD. The proposed intermediate 3-
oxo-D-glucose-6-phosphate is the hemiacetal form of a 1,3-
dicarbonyl compound. Such compounds are known to absorb
near 300 nm in basic aqueous solution, due to enolate and/or
enone formation.16−18 Consistent with this, the NtdC-catalyzed
reaction produced the UV spectra shown in Figure 3A. By
recycling the NADH using lactate dehydrogenase and pyruvate,
the absorbance at 340 nm can be removed to reveal the
absorbance due to the product around 310 nm (Figure 3B).
The addition of an excess of L-glutamate and purified NtdA
effectively removes the absorbance at 310 nm, leaving only the
NADH absorbance at 340 nm (Figure 3C). No other amino
acid gave this result, demonstrating the specificity of the NtdA-
catalyzed reaction.
Kanosamine was synthesized chemically by the method of
Guo and Frost5 and converted to kanosamine 6-phosphate
using hexokinase and ATP. A mixture of kanosamine-6-
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evolved for a particular activity, namely the biosynthesis of
kanosamine.
transferase. It is also likely that already identified proteins
encoded within the B. subtilis genome are responsible, but the
catalytic promiscuity of these enzymes is as yet unrevealed.
Jakeman′s observation of promiscuity among bacterial
nucleotidylyltransferases Cps2L and RmlA3,21 which tolerate
a variety of substituents at the 3-position, supports this
hypothesis.
In summary, we have found the ntd operon in B. subtilis to
encode a pathway for kanosamine synthesis. The operon would
be better named the kab operon as proposed for the
homologous operon from B. cereus UW85. The enzymes
appear to be efficient and highly selective. This pathway
represents a distinct strategy for kanosamine synthesis from
that previously described.
Our results are in notable contrast to the previously
described biosynthetic pathway for kanosamine. Guo and
Frost5 as well as Arakawa et al.6 have described enzymes from
Amycolatopsis mediterranei which generate kanosamine from
UDP-glucose and shown that kanosamine is subsequently
phosphorylated by a kanosamine kinase. A similar finding has
since been reported in Streptomyces kanamyceticus.20
Kanosamine biosynthesis was previously observed in B.
pumilis. Umezawa et al. observed that kanosamine could be
formed using cell-free extracts from UDP-glucose, ATP, NAD,
Mg2+, and glutamine but also observed kanosamine synthesis
from glucose and ATP (and the absence of UTP). The authors
did not consider that this was due to the formation of glucose-
6-phosphate, but ruled out the formation from glucose by
showing that the addition of 3-oxo-D-glucose did not lead to
kanosamine synthesis. In light of our results, Umezawa′s
observations appear consistent with the pathway of Figure 2,
except that they observed that either glutamine or ammonia
addition was necessary for kanosamine production, unlike the
PLP-dependent glutamate reaction we observe. The use of cell-
free extracts in those experiments rather than purified enzymes
makes a direct comparison to our work difficult. Homologues
of ntdA, ntdB, and ntdC are present in the B. pumilus genome.
Kevany et al. hypothesized that the kab operon from B. cereus
UW85, which is very similar to the ntd operon, encodes a
kanosamine biosynthetic pathway. In keeping with previously
described kanosamine biosynthesis, they proposed that the
pathway began with UDP-glucose. Specifically, KabC (which is
58% identical to NtdC, 75% sequence similarity) was proposed
to be a UDP-glucose 3-dehydrogenase; KabA (58% identical to
NtdA, 78% similar), a glutamine-dependent aminotransferase;
and KabB (57% identical to NtdB, 74% similar), a UDP
glycosyl hydrolase. There are no published experiments on the
Kab enzymes, but given the similarity of the two operons, we
now propose that these authors were correct in that the operon
encodes enzymes that synthesize kanosamine, but incorrect in
the specifics of the biosynthetic steps.
Each of the enzymatic functions reported here belong to
well-established classes of biochemical reactions, but none have
been observed before for these substrates. In fact, there are no
reports of 3-oxo-D-glucose-6-phosphate. Attempts to synthesize
this compound in our laboratory have met with failure. We did
synthesize 3-oxo-D-glucose, and in alkaline aqueous buffer it
absorbs strongly at 310 nm (Supporting Information, Figure
S3). We were also able to detect the presence of a product
consistent with 3-oxo-D-glucose-6-phosphate in the lactate
dehydrogenase-coupled NtdC-catalyzed reaction of glucose-6-
phosphate using quadrupole ion trap mass spectrometry. The
starting material and product fragmented with the loss of
phosphate (m/z 96.97); neutral loss experiments showed a loss
of 162 mass units for glucose-6-phosphate and 160 mass units
for the reaction product, which was consistent with the
precursor ion investigation of the phosphate ion fragment
(Supporting Information, Figures S8−S10). The apparent
lability of 3-oxo-D-glucose-6-phosphate suggests that this
intermediate does not accumulate and might be sequestered
between the active sites of an NtdC-NtdA complex.
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemical syntheses, spectral data, molecular biology, protein
purification, assay conditions, HPLC conditions and chromato-
gram. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
dave.palmer@usask.ca
Present Addresses
§S.A.: Cholistan Institute of Desert Studies, The Islamia
University of Bahawalpur, Pakistan
∥H.Z.: Department of Chemical Engineering and Applied
Chemistry, University of Toronto
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a Saskatchewan Health Research
Foundation Group grant to the Molecular Design Research
Group, an NSERC Undergraduate Summer Research Award to
E.O., and the University of Saskatchewan. Prof. Kozo Ochi
provided ntdABC. We thank Mr. Ken Thoms and the other staff
of the Saskatchewan Structural Sciences Centre.
REFERENCES
■
(1) Iwai, Y.; Tanaka, H.; Oiwa, R.; Shimizu, S.; Omura, S. Biochim.
Biophys. Acta 1977, 498, 223.
(2) Muhizi, T.; Grelier, S.; Coma, V. J. Agric. Food Chem. 2009, 57,
8770.
(3) Janiak, A. M.; Milewski, S. Medical Mycology 2001, 39, 401.
(4) Umezawa, S.; Shibahar., S.; Omoto, S.; Takeuchi, T.; Umezawa,
H. J. Antibiot. 1968, 21, 485.
(5) Guo, J. T.; Frost, J. W. J. Am. Chem. Soc. 2002, 124, 10642.
(6) Arakawa, K.; Muller, R.; Mahmud, T.; Yu, T. W.; Floss, H. G. J.
Am. Chem. Soc. 2002, 124, 10644.
(7) Kevany, B. M.; Rasko, D. A.; Thomas, M. G. Appl. Environ.
Microbiol. 2009, 75, 1144.
(8) Milner, J. L.; SiloSuh, L.; Lee, J. C.; He, H. Y.; Clardy, J.;
Handelsman, J. Appl. Environ. Microbiol. 1996, 62, 3061.
(9) Inaoka, T.; Takahashi, K.; Yada, H.; Yoshida, M.; Ochi, K. J. Biol.
Chem. 2004, 279, 3885.
(10) Inaoka, T.; Ochi, K. J. Bacteriol. 2007, 189, 65.
(11) Tsuno, T.; Ikeda, C.; Numata, K.; Tomita, K.; Konishi, M.;
Kawaguchi, H. J. Antibiot. 1986, 39, 1001.
An important implication of these results is that the
biosynthesis of NTD must require additional enzymatic
activities. Such activities are likely to include a kanosamine
phosphoisomerase, a nucleotidylyltransferase, and a glycosyl-
(12) Numata, K. I.; Satoh, F.; Hatori, M.; Miyaki, T.; Kawaguchi, H.
J. Antibiot. 1986, 39, 1346.
5972
dx.doi.org/10.1021/ja4010255 | J. Am. Chem. Soc. 2013, 135, 5970−5973

Journal of the American Chemical Society
Communication
(13) Anjum, S.; Vetter, N. D.; Rubin, J. E.; Palmer, D. R. J.
Tetrahedron 2013, 69, 816.
(14) van Straaten, K. E.; Langill, D. M.; Palmer, D. R. J.; Sanders, D.
A. R. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2009, 65,
426.
(15) Daniellou, R.; Phenix, C. P.; Tam, P. H.; Laliberte, M. C.;
Palmer, D. R. J. Org. Biomol. Chem. 2005, 3, 401.
(16) Barycki, J. J.; O’Brien, L. K.; Strauss, A. W.; Banaszak, L. J. J.
Biol. Chem. 2000, 275, 27186.
(17) Iglesias, E. New J. Chem. 2002, 26, 1352.
(18) Barash, I.; Pupkin, G.; Netzer, D.; Kashman, Y. Plant Physiol.
1982, 69, 23.
(19) Huang, H.; Patskovsky, Y.; Toro, R.; Farelli, J. D.; Pandya, C.;
Almo, S. C.; Allen, K. N.; Dunaway-Mariano, D. Biochemistry 2011, 50,
8937.
(20) Park, J. W.; Park, S. R.; Nepal, K. K.; Han, A. R.; Ban, Y. H.;
Yoo, Y. J.; Kim, E. J.; Kim, E. M.; Kim, D.; Sohng, J. K.; Yoon, Y. J.
Nat. Chem. Biol. 2011, 7, 843.
(21) Huestis, M. P.; Aish, G. A.; Hui, J. P. M.; Soo, E. C.; Jakeman, D.
L. Org. Biomol. Chem. 2008, 6, 477.
5973
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