well-characterized 2-DOS containing aminoglycoside antibiotics
(7). The only methyltransferase in the hyg cluster, HygM, may
catalyze the N-methylation of 2-DOS to generate 3-N-methyl-2-
DOS (42), which is then decorated to form the disaccharide
containing a D-gulose and a destomic acid to form hygromycin B
(Fig. 5B). Based on the fact that HygP, GmhB, and HldE can
convert S-7-P to ADP-D-glycero-β-D-altro-heptose, we propose
that the precursor of destomic acid is S-7-P, which is converted
to D-glycero-D-altro-heptose-7-P by HygP. Unlike GmhA and the
isomerase domain of SepB, which catalyze one isomerization
step and control the stereochemistry of only the C2 position,
HygP is a special heptose isomerase that catalyzes consecutive
isomerizations and determines the stereochemistry of both
C2 and C3 positions. D-Glycero-D-altro-heptose-7-P may be
modified by the kinase HygN, the phosphatase HygU, and the
nucleotidyltransferase HygO sequentially to form an NDP-heptose,
which will be further converted to destomic acid by the following
tailoring enzymes (Fig. 5B). The high structure similarities between
destomycins and hygromycin B imply that destomycins are bio-
synthesized in a similar manner as hygromycin B and the heptose of
destomycin B, epi-destomic acid, is also derived from S-7-P.
LPS heptose biosynthesis at its early stage and recruits ADP-sugars
as intermediates. HygP was characterized as an interesting isom-
erase that initiates destomic acid biosynthesis in hygromycin B by
converting S-7-P to D-glycero-D-altro-heptose-7-P through consecu-
tive isomerizations. The findings in this study set the stage for the
generation of novel septacidin derivatives by combinatorial bio-
synthesis. In addition, generation of ADP-D-glycero-β-D-altro-hep-
tose by HygP, GmhB, and HldE enables interesting opportunities
for structural modification of the heptoses in the LPS inner core by
replacing the gmhA gene with hygP in gram-negative bacterium.
Both avenues are currently being actively pursued in our laboratory.
Materials and Methods
Detailed descriptions of materials and methods, including bacterial strains,
construction of mutant strains; isolation and characterization of compounds;
and enzymatic assays and other procedures used are given in SI Appendix.
ACKNOWLEDGMENTS. We thank Dr. Jinwei Ren and Dr. Yaxin Zhu, Institute
of Microbiology, Chinese Academy of Sciences (CAS), for technical support.
The study was supported by Ministry of Science and Technology of China
Grants 2015CB150600 and 2013CB734000 and National Natural Science
Foundation of China Grants 31370095 and 31522001. Y.C. is an awardee
of the “Hundred Talents Program” of CAS. Z.G. is an awardee of the Youth
Innovation Promotion Association of CAS (2017124).
In conclusion, we showed that the special seven-carbon–contain-
ing sugars of septacidin and hygromycin B are both derived from
S-7-P, a heptose in the pentose phosphate pathway. The heptose
biosynthesis in septacidin surprisingly shares the same logic with
1. Thibodeaux CJ, Melançon CE, 3rd, Liu HW (2008) Natural-product sugar biosynthesis
and enzymatic glycodiversification. Angew Chem Int Ed Engl 47:9814–9859.
2. Elshahawi SI, Shaaban KA, Kharel MK, Thorson JS (2015) A comprehensive review of
glycosylated bacterial natural products. Chem Soc Rev 44:7591–7697.
3. Ubukata M, Isono K, Kimura K, Nelson CC, McCloskey JA (1988) The structure of liposidomycin
B, an inhibitor of bacterial peptidoglycan synthesis. J Am Chem Soc 110:4416–4417.
4. Fujita Y, et al. (2011) A-90289 A and B, new inhibitors of bacterial translocase I,
produced by Streptomyces sp. SANK 60405. J Antibiot (Tokyo) 64:495–501.
5. Zeng Y, et al. (2012) Biosynthesis of albomycin δ(2) provides a template for assembling
siderophore and aminoacyl-tRNA synthetase inhibitor conjugates. ACS Chem Biol 7:
1565–1575.
6. Barnard-Britson S, et al. (2012) Amalgamation of nucleosides and amino acids in
antibiotic biosynthesis: Discovery of an L-threonine:uridine-5′-aldehyde transaldolase.
J Am Chem Soc 134:18514–18517.
7. Busscher GF, Rutjes FPJT, van Delft FL (2005) 2-Deoxystreptamine: Central scaffold of
aminoglycoside antibiotics. Chem Rev 105:775–791.
8. Hong W, Yan L (2012) Identification of gntK, a gene required for the methylation of
purpurosamine C-6′ in gentamicin biosynthesis. J Gen Appl Microbiol 58:349–356.
9. Karki S, Kim JY, Park SH, Kwon HJ (2012) Gene inactivation study on gntK, a putative
C-methyltransferase gene in gentamicin biosynthesis from Micromonospora echino-
spora. J Korean Soc Appl Biol Chem 55:439–442.
10. Kim HJ, et al. (2013) GenK-catalyzed C-6′ methylation in the biosynthesis of genta-
micin: Isolation and characterization of a cobalamin-dependent radical SAM enzyme.
J Am Chem Soc 135:8093–8096.
11. Kuzuyama T, Seki T, Dairi T, Hidaka T, Seto H (1995) Nucleotide sequence of fortimicin
KL1 methyltransferase gene isolated from Micromonospora olivasterospora, and com-
parison of its deduced amino acid sequence with those of methyltransferases involved in
the biosynthesis of bialaphos and fosfomycin. J Antibiot (Tokyo) 48:1191–1193.
12. Price NPJ, et al. (2014) Biosynthesis of 4-aminoheptose 2-epimers, core structural
components of the septacidins and spicamycins. J Antibiot (Tokyo) 67:405–414.
13. Sakai T, et al. (1995) Absolute configuration of spicamycin, an antitumor antibiotic
produced by Streptomyces alanosinicus. J Antibiot (Tokyo) 48:899–900.
22. Kamishohara M, et al. (1993) Structure-antitumor activity relationship of semi-syn-
thetic spicamycin analogues. J Antibiot (Tokyo) 46:1439–1446.
23. Suzuki T, et al. (2002) Total synthesis of spicamycin. J Org Chem 67:2874–2880.
24. Flatt PM, Mahmud T (2007) Biosynthesis of aminocyclitol-aminoglycoside antibiotics
and related compounds. Nat Prod Rep 24:358–392.
25. Brodersen DE, et al. (2000) The structural basis for the action of the antibiotics tetracy-
cline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 103:1143–1154.
26. Kondo S, Iinuma K, Naganawa H, Shimura M, Sekizawa Y (1975) Structural studies on
destomycins A and B. J Antibiot (Tokyo) 28:79–82.
27. Shimura M, Sekizawa Y, Iinuma K, Naganawa H, Kondo S (1975) Destomycin C, a new
member of destomycin family antibiotics. J Antibiot (Tokyo) 28:83–84.
28. Wencewicz TA, Möllmann U, Long TE, Miller MJ (2009) Is drug release necessary for
antimicrobial activity of siderophore-drug conjugates? Syntheses and biological
studies of the naturally occurring salmycin “Trojan Horse” antibiotics and synthetic
desferridanoxamine-antibiotic conjugates. Biometals 22:633–648.
29. Roosenberg JM, 2nd, Miller MJ (2000) Total synthesis of the siderophore danoxamine.
J Org Chem 65:4833–4838.
30. Kneidinger B, et al. (2002) Biosynthesis pathway of ADP-L-glycero-β-D-manno-hep-
tose in Escherichia coli. J Bacteriol 184:363–369.
31. Jiang W, et al. (2015) Cas9-Assisted Targeting of CHromosome segments CATCH en-
ables one-step targeted cloning of large gene clusters. Nat Commun 6:8101.
32. Brooke JS, Valvano MA (1996) Biosynthesis of inner core lipopolysaccharide in enteric
bacteria identification and characterization of a conserved phosphoheptose isomer-
ase. J Biol Chem 271:3608–3614.
33. McArthur F, Andersson CE, Loutet S, Mowbray SL, Valvano MA (2005) Functional
analysis of the glycero-manno-heptose 7-phosphate kinase domain from the bi-
functional HldE protein, which is involved in ADP-L-glycero-D-manno-heptose bio-
synthesis. J Bacteriol 187:5292–5300.
34. Wang L, et al. (2010) Divergence of biochemical function in the HAD superfamily: D-glyc-
ero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB). Biochemistry 49:1072–1081.
35. Ding L, Seto BL, Ahmed SA, Coleman WG, Jr (1994) Purification and properties of the
Escherichia coli K-12 NAD-dependent nucleotide diphosphosugar epimerase, ADP-L-
glycero-D-mannoheptose 6-epimerase. J Biol Chem 269:24384–24390.
36. Kneidinger B, Graninger M, Puchberger M, Kosma P, Messner P (2001) Biosynthesis of
nucleotide-activated D-glycero-D-manno-heptose. J Biol Chem 276:20935–20944.
37. Osborn AR, Kean KM, Karplus PA, Mahmud T (2017) The sedoheptulose 7-phosphate
cyclases and their emerging roles in biology and ecology. Nat Prod Rep 34:945–956.
38. Mahmud T, et al. (1999) Biosynthetic studies on the α-glucosidase inhibitor acarbose
in Actinoplanes sp.: 2-epi-5-epi-valiolone is the direct precursor of the valienamine
moiety. J Am Chem Soc 121:6973–6983.
14. Igarashi Y, et al. (2005) Anicemycin,
a new inhibitor of anchorage-independent
growth of tumor cells from Streptomyces sp. TP-A0648. J Antibiot (Tokyo) 58:322–326.
15. Dutcher JD, Vonsaltza MH, Pansy FE (1963) Septacidin, a new antitumor and anti-
fungal antibiotic produced by Streptomyces fimbriatus. Antimicrob Agents
Chemother (Bethesda) 161:83–88.
16. Acton EM, Ryan KJ, Luetzow AE (1977) Antitumor septacidin analogues. J Med Chem
20:1362–1371.
17. Sukkurwala AQ, et al. (2014) Screening of novel immunogenic cell death inducers
within the NCI mechanistic diversity set. OncoImmunology 3:e28473.
18. Hayakawa Y, et al. (1985) Spicamycin, a new differentiation inducer of mouse myeloid leuke-
mia cells (Ml) and human promyelocytic leukemia cells (HL-60). Agric Biol Chem 49:2685–2691.
19. Kamishohara M, et al. (1994) Antitumor activity of a spicamycin derivative, KRN5500,
and its active metabolite in tumor cells. Oncol Res 6:383–390.
20. Gadgeel SM, et al. (2003) A phase I clinical trial of spicamycin derivative KRN5500 (NSC
650426) using a phase I accelerated titration “2B” design. Invest New Drugs 21:63–74.
21. Weinstein SM, et al. (2012) A spicamycin derivative (KRN5500) provides neuropathic
pain relief in patients with advanced cancer: A placebo-controlled, proof-of-concept
trial. J Pain Symptom Manage 43:679–693.
39. Dong H, Mahmud T, Tornus I, Lee S, Floss HG (2001) Biosynthesis of the validamycins:
Identification of intermediates in the biosynthesis of validamycin A by Streptomyces
hygroscopicus var. limoneus. J Am Chem Soc 123:2733–2742.
40. Czyzyk DJ, Liu C, Taylor EA (2011) Lipopolysaccharide biosynthesis without the lipids: Rec-
ognition promiscuity of Escherichia coli heptosyltransferase I. Biochemistry 50:10570–10572.
41. Komaki H, Hosoyama A, Kimura A, Ichikawa N, Igarashi Y (2017) Draft genome sequence
of an anicemycin producer, Streptomyces sp. TP-A0648. Genome Announc 5:e01468-16.
42. Walker JB (2002) Enzymatic synthesis of aminoglycoside antibiotics: Novel ad-
enosylmethionine:2-deoxystreptamine N-methyltransferase activities in hygromycin
B- and spectinomycin-producing Streptomyces spp. and uses of the methylated
products. Appl Environ Microbiol 68:2404–2410.
6 of 6
|
Tang et al.