Kanosamine biosynthesis: A likely source of the aminoshikimate pathway's nitrogen atom

Article abstract of DOI:10.1021/ja026628m

The biosynthetic source of the nitrogen atom incorporated into the aminoshikimate pathway has remained a question for some time. 3-Amino-3-deoxy-d-fructose 6-phosphate has previously been demonstrated to be a precursor to 4-amino-3,4-dideoxy-d-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid via the inferred intermediacy of 1-deoxy-1-imino-d-erythrose 4-phosphate in Amycolatopsis mediterranei cell-free extract. This investigation examines the possibility that the natural product kanosamine might be a precursor to 3-amino-3-deoxy-d-fructose 6-phosphate. Kanosamine 6-phosphate was synthesized by a chemoenzymatic route and incubated in A. mediterranei cell-free lysate along with d-ribose 5-phosphate and phosphoenolpyruvate. Formation of 4-amino-3,4-dideoxy-d-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid was observed. Subsequent incubation in A. mediterranei cell-free lysate of glutamine and NAD with UDP-glucose resulted in the formation of kanosamine. The bioconversion of UDP-glucose into kanosamine along with the bioconversion of kanosamine 6-phosphate into 4-amino-3,4-dideoxy-d-arabino-heptulosonic acid 7-phosphate and 3-amino-5-hydroxybenzoic acid suggests that kanosamine biosynthesis is the source of the aminoshikimate pathway's nitrogen atom. Copyright

Full text of DOI:10.1021/ja026628m

Published on Web 08/16/2002
Kanosamine Biosynthesis: A Likely Source of the Aminoshikimate Pathway’s
Nitrogen Atom
Jiantao Guo and J. W. Frost*
Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48824
Received April 22, 2002
Scheme 1 a,b
Many biologically active natural products including rifamycin,
mitomycin, and ansamitocin are derived from 3-amino-5-hydroxy-
benzoic acid (AHBA, Scheme 1) by way of the aminoshikimate
pathway.1 Floss and co-workers have demonstrated the role of
4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate2
(aminoDAHP, Scheme 1) in the aminoshikimate pathway and
delineated the loci in the rif biosynthetic gene cluster required for
biosynthesis of 3-amino-5-hydroxybenzoic acid.3 Recently, amino-
DAHP was demonstrated to form in Amycolatopsis mediterranei
cell-free lysate from 3-amino-3-deoxy-D-fructose 6-phosphate
(aminoF6P, Scheme 1) via the inferred intermediacy of 1-deoxy-
1-imino-D-erythrose 4-phosphate (iminoE4P, Scheme 1).4 Biosyn-
thesis of 3-amino-3-deoxy-D-glucose (kanosamine, Scheme 1) is
now examined as a possible source of the aminoshikimate pathway’s
nitrogen atom.
With identification of 3-amino-3-deoxy-D-fructose 6-phosphate
as a precursor to 3-amino-5-hydroxybenzoic acid, attention turned
to delineation of the source of this alkaloid. One natural product
a Conversions (genes): (a) see ref 5c; (b) aminoglucokinase; (c)
that, by combination of an isomerization and phosphorylation, could
glucoisomerase; (d) transketolase (tktA); (e) aminoDAHP synthase (rifH),
DAHP synthase (aroFFBR); (f) aminoshikimate pathway; (g) hydrolysis.
bAbbreviations: AT(D)P, adenosine 5′-tri(di)phosphate; K6P, kanosamine
6-phosphate; aminoF6P, 3-amino-3-deoxy-D-fructose 6-phosphate, R5P,
D-ribose 5-phosphate; S7P, D-sedoheptulose 7-phosphate; iminoE4P, 1-deoxy-
1-imino-D-erythrose 4-phosphate; E4P, D-erythrose 4-phosphate; Pi, inor-
ganic phosphate; aminoDAHP; 4-amino-3,4-dideoxy-D-arabino-heptulosonic
acid 7-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate;
AHBA, 3-amino-5-hydroxybenzoic acid.
be a precursor to 3-amino-3-deoxy-D-fructose 6-phosphate was
kanosamine (Scheme 1). Various microbes generate kanosamine
as a biosynthetic end-product.5 As exemplified by the biosynthesis
of kanamycin,7 kanosamine is also an intermediate en route to other
natural products. The possibility therefore needed to be explored
that A. mediterranei might similarly utilize kanosamine as a
biosynthetic intermediate as well as a vehicle for incorporation of
the nitrogen atom into the aminoshikimate pathway. Working
backward from the intermediacy of 3-amino-3-deoxy-D-fructose
6-phosphate, kanosamine 6-phosphate was first synthesized and
tested as a precursor to aminoDAHP.
Scheme 2 a
D-Glucose was selectively protected and the resulting C-3
hydroxyl group oxidized (Scheme 2). Subsequent diastereoselective
reduction provided for an overall net inversion of configuration at
C-3. Activation of the C-3 hydroxyl group as a triflate ester followed
by nucleophilic displacement with NaN3 introduced the requisite
nitrogen atom at C-3. Reduction and subsequent deprotection gave
kanosamine. Hexokinase-catalyzed phosphorylation of kansoamine
employed citric acid as an activator4 and afforded kanosamine
6-phosphate in an overall yield of 43% from starting D-glucose.
Initial attempts at the in vitro bioconversion of kanosamine
6-phosphate into aminoDAHP focused on an assembled system.
Kanosamine 6-phosphate, D-ribose 5-phosphate, and phospho-
enolpyruvate were incubated with yeast phosphoglucose isomerase,
a Reactions: (a) acetone, ZnCl2, H3PO4, 68%; (b) PDC, (CH3CO)2O,
CH2Cl2, reflux, 91%; (c) NaBH4, EtOH/H2O (9:1), 0 °C, 90%; (d) (i)
(CF3SO2)2O, pyridine, CH2Cl2, -20 °C, quantitative, (ii) NaN3, DMF, 50
°C, 92%; (e) LiAlH4, Et2O, 94%; (f) 2 N HCl, 25 °C, quantitative; (g)
ATP, MgCl2, hexokinase, citric acid, pH 8, 90%.
isomerase-generated 3-amino-3-deoxy-D-fructose 6-phosphate and
subsequent hydrolysis of 1-deoxy-1-imino-D-erythrose 4-phosphate
account for the formation of DAHP.4 The bioconversion of
kanosamine 6-phosphate was then repeated upon substitution of
E. coli aroFFBR-encoded DAHP synthase (entry 1, Table 1) with
A. mediterranei rifH-encoded aminoDAHP synthase (entry 2, Table
1). Along with DAHP, formation of aminoDAHP was observed
(entry 2, Table 1).
Escherichia coli tktA-encoded transketolase,6a,b and E. coli aroFFBR
-
encoded DAHP synthase.6b,c Although no aminoDAHP was detected
(entry 1, Table 1), formation of 3-deoxy-D-arabino-heptulosonic
acid 7-phosphate (DAHP) indicated that kanosamine 6-phosphate
was a substrate for isomerase. The action of transketolase on
* Address correspondence to this author. E-mail: frostjw@cem.msu.edu.
9
10642
J. AM. CHEM. SOC. 2002, 124, 10642-10643
10.1021/ja026628m CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Biosynthesis of AminoDAHP from Kanosamine 6-Phosphate and Kanosamine from UDP-Glucose
entry
reaction conditions
productsc (yield,d %)
1
kanosamine 6-phosphate, R5P, PEP; yeast phosphoglucose isomerase (60 units), E. coli TktA
transketolase (9 unitsa), E. coli AroFFBR DAHP synthase (660 unitsb), pH 7.3
kanosamine 6-phosphate, R5P, PEP; yeast phosphoglucose isomerase (60 units), E. coli TktA
transketolase (9 unitsa), A. mediterranei RifH aminoDAHP synthase (64 unitsb), pH 7.3
kanosamine 6-phosphate, R5P, PEP; A. mediterranei cell-free extract
(DAHP synthase activity of 0.2 unitb), pH 7.3
DAHP (39)
2
3
4
5
aminoDAHP (2); DAHP (30)
aminoDAHP (6); DAHP (20);
AHBA (2); Tyr (3); Phe (2)
DAHP (21)
glucose 6-phosphate, R5P, PEP, glutamine, (NH4)2SO4; A. mediterranei cell-free extract
(DAHP synthase activity of 0.2 unitb), pH 7.3
UDP-6,6-[2H2]-glucose, NAD, glutamine; A. mediterranei cell-free extract, pH 6.8
6,6-[2H2]-kanosamine (5)
a Transketolase was assayed according to ref 6a. b AminoDAHP synthase was assayed as DAHP synthase activity according to ref 6a. c See the legend
to Scheme 1 for abbreviations. d Yields are 1H NMR yields of aminoDAHP, DAHP, and AHBA purified to homogeneity and of L-tyrosine and L-phenylalanine
purified to a binary mixture. Response factors and quantification of product concentrations were based on integration relative to 3-(trimethylsilyl)propionate-
2,2,3,3-d4.
Incubation of 3-amino-3-deoxy-D-fructose 6-phosphate with cell-
free lysate of A. mediterranei (ATCC 21789) has previously been
reported to give higher yields of aminoDAHP than incubations
employing the assembled bioconversion system.4 Accordingly,
reaction of kanosamine 6-phosphate with D-ribose 5-phosphate and
phosphoenolpyruvate in A. mediterranei cell-free lysate led to higher
yields of aminoDAHP and formation of 3-amino-5-hydroxybenzoic
acid (entry 3, Table 1). As a control experiment, D-glucose
6-phosphate, D-ribose 5-phosphate, and phosphoenolpyruvate were
incubated in A. mediterranei cell-free lysate with glutamine and
(NH4)2SO4 as possible sources of nitrogen (entry 4, Table 1). No
aminoDAHP formation was observed.
Streptomyces kanamyceticus (kanamycin),7 A. mediterranei (rifa-
mycin),3c Streptomyces laVendulae (mitomycin),8 and Actino-
synnema pretiosum (ansamitocin).9
Acknowledgment. Professor Heinz G. Floss provided rifH.
Research was supported by a contract from F. Hoffmann-La Roche
Ltd. and a grant from the National Institutes of Health.
Supporting Information Available: Synthesis of kanosamine
6-phosphate and its conversion to aminoDAHP; synthesis of UDP-
6,6-[2H2]-glucose and its conversion to 6,6-[2H2]-kanosamine (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
Reaction of kanosamine with ATP, D-ribose 5-phosphate, and
phosphoenolpyruvate in A. mediterranei cell-free lysate did not
produce quantifiable levels of either aminoDAHP or DAHP. As a
consequence, attention turned to biosynthesis of kanosamine in A.
mediterranei. Kanosamine biosynthesis was first observed and
studied in Bacillus pumilus (formerly Bacillus aminoglucosidicus)
in the 1960s.5a-c Incubation of UDP-[U-14C]-glucose in B. pumilus
cell lysate with NAD and glutamine led to the formation of [U-14C]-
kanosamine.5c Likewise, incubation of UDP-6,6-[2H2]-glucose with
NAD and glutamine in A. mediterranei cell-free lysate led to the
formation of 6,6-[2H2]-kanosamine (entry 5, Table 1). Based on
analysis by electrospray mass spectrometry, UDP-6,6-[2H2]-glucose
having an M + 2 ion with relative intensity 98.08% yielded
kanosamine having an M + 2 ion with 97.83% relative intensity.
Incubation of UDP-glucose with NAD, glutamine, ATP, D-ribose
5-phosphate, and phosphosphoenolpyruvate in A. mediterranei cell-
free lysate did not produce quantifiable concentrations of amino-
DAHP. Low levels of aminoglucokinase (b, Scheme 1) in lysed A.
mediterranei cells may explain the formation of kanosamine from
UDP-glucose as well as the lack of quantifiable aminoDAHP
formation from UDP-glucose and kanosamine.
Kanosamine biosynthesis has been directly implicated as the
source of the aminoshikimate pathway’s nitrogen atom. This follows
from the observed bioconversions of kanosamine 6-phosphate into
aminoDAHP and 3-amino-5-hydroxybenzoic acid (entries 2 and
3, Table 1), along with the observed bioconversion of UDP-glucose
into kanosamine (entry 5, Table 1). As a consequence, elaboration
of the source of the aminoshikimate pathway’s nitrogen atom must
now include elaboration of the biosynthesis of kanosamine. In turn,
biosynthesis of kanosmine emerges as a pathway possibly shared
by microbes (biosyntheses) as diverse as Bacillus spp. (kanosamine),5
References
(1) Floss, H. G. Nat. Prod. Rep. 1997, 14, 433.
(2) (a) Kim, C.-G.; Kirschning, A.; Bergon, P.; Ahn, Y.; Wang, J. J.; Shibuya,
M.; Floss, H. G. J. Am. Chem. Soc. 1992, 114, 4941. (b) Kirschning, A.;
Bergon, P. Wang, J.-J.; Breazeale, S.; Floss, H. G. Carbohydr. Res. 1994,
256, 245. (c) Kim, C.-G.; Kirschning, A.; Bergon, P.; Zhou, P.; Su, E.;
Sauerbrei, B.; Ning, S.; Ahn, Y.; Breuer, M.; Leistner, E.; Floss, H. G. J.
Am. Chem. Soc. 1996, 118, 7486.
(3) (a) Kim, C.-G.; Yu, T.-W.; Fryhle, C. B.; Handa, S.; Floss, H. G. J. Biol.
Chem. 1998, 273, 6030. (b) Eads, J. C.; Beeby, M.; Scapin, G.; Yu, T.
W.; Floss, H. G. Biochemistry 1999, 38, 9840. (c) August, P. R.; Tang,
L.; Yoon, Y. J.; Ning, S.; Mu¨ller, R.; Yu, T.-W.; Taylor, M.; Hoffmann,
D.; Kim, C.-G.; Zhang, X.; Hutchinson, C. R.; Floss, H. G. Chem. Biol.
1998, 5, 69. (d) Yu, T.-W.; Mu¨ller, R.; Mu¨ller, M.; Zhang, X.; Draeger,
G.; Kim, C.-G.; Leistner, E.; Floss, H. G. J. Biol. Chem. 2001, 276, 12546.
(4) (4)Guo, J.; Frost, J. W. J. Am. Chem. Soc. 2002, 124, 528.
(5) (a) Umezawa, S.; Umino, K.; Shibahara, S.; Hamada, M.; Omoto, S. J.
Antibiot. 1967, 20, 355. (b) Umezawa, S.; Umino, K.; Shibahara, S.;
Omoto, S. Bull. Chem. Soc. Jpn. 1967, 40, 2419. (c) Umezawa, S.;
Shibahara, S.; Omoto, S.; Takeuchi. T.; Umezawa, H. J. Antibiot. 1968,
21, 485. (d) Dolak, L. A.; Castle, T. M.; Dietz, A.; Laborde, A. L. J.
Antibiot. 1980, 33, 900. (e) Tsuno, T.; Ikeda, C.; Numata, K.; Tomita,
K.; Konishi, M.; Kawaguchi, H. J. Antibiot. 1986, 39, 1001. (f) Fusetani,
N.; Ejima, D.; Matsunaga, S.; Hashimoto, K.; Itagaki, K.; Akagi, Y.; Taga,
N.; Suzuki, K. Experientia 1987, 43, 464. (g) Milner, J. L.; Silo-Suh, L.;
Lee, J. C.; He, H.; Clardy, J.; Handelsman, J. Appl. EnViron. Microbiol.
1996, 62, 3061.
(6) (a) Draths, K. M.; Pompliano, D. L.; Conley, D. L.; Frost, J. W.; Berry,
A.; Disbrow, G. L.; Staversky, R. J.; Lievense, J. C. J. Am. Chem. Soc.
1992, 114, 3956. (b) Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R.
M.; Frost, J. W. Biotechnol. Bioeng. 1999, 64, 61. (c) Weaver, L. M.;
Herrmann, K. M. J. Bacteriol. 1990, 172, 6581.
(7) Umezawa, H.; Ueda, M.; Maeda, K.; Yagishita, K.; Kondo, S.; Okami,
Y.; Utahara, R.; Osato, Y.; Nitta, K.; Takeuchi, T. J. Antibiot. 1957, 10,
181.
(8) Mao, Y.; Varoglu, M.; Sherman, D. H. J. Bacteriol. 1999, 181, 2199.
(9) (a) Higashide, E.; Asai, M.; Ootsu, K.; Tanida, S.; Kozai, Y.; Hasegawa,
T.; Kishi, T.; Sugino, Y.; Yoneda, M. Nature 1977, 270, 721. (b) Carrroll,
B. J.; Moss, S. J.; Bai, L.; Kato, Y.; Toelzer, S.; Yu, T.-W.; Floss, H. G.
J. Am. Chem. Soc. 2002, 124, 4176. (c) Yu, T.-W.; Bai, L.; Clade, D.;
Hoffmann, D.; Tolzer, S.; Trinh, K. Q.; Xu, J.; Moss, S. J.; Leistner, E.;
Floss, H. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7968.
JA026628M
9
J. AM. CHEM. SOC. VOL. 124, NO. 36, 2002 10643