Characterization of CalE10, the N-oxidase involved in calicheamicin hydroxyaminosugar formation

Full text of DOI:10.1021/ja807557a

Published on Web 12/04/2008
Characterization of CalE10, the N-Oxidase Involved in Calicheamicin
Hydroxyaminosugar Formation
Heather D. Johnson and Jon S. Thorson*
DiVision of Pharmaceutical Sciences and UniVersity of Wisconsin National Drug DiscoVery Group,
UniVersity of WisconsinsMadison, Madison, Wisconsin 53706
Received October 3, 2008; E-mail: jsthorson@pharmacy.wisc.edu
Calicheamicin (CLM) γ1I (Figure 1, 5) is a prominent member
Scheme 1. Proposed Biosynthesis of
of the 10-membered enediyne family.1 Like all enediynes, CLM-
induced oxidative DNA strand scission is enabled by cycloaro-
4-Hydroxyamino-6-deoxy-R-D-glucose Common to CLM (5) and
Esperamicin
matization of the enediyne core to form a highly reactive
diradical species.2 In CLM, this reactive intermediate is posi-
tioned in the DNA minor groove via the aryltetrasaccharide
wherein the unique conformation of the CLM hydroxylamino
glycosidic bond contributes to both DNA specificity and affinity.3
The incredible potency of CLM has been harnessed for clinical
use (Mylotarg),4 and CLM biosynthetic studies have unveiled a
variety of unique features. For example, the recent elucidation
of gene clusters encoding both 9-membered and 10-membered
enediynes revealed a unified, divergent polyketide paradigm for
enediyne core biosynthesis,5 likely originating from a common
polyene precursor.6 Studies on CLM self-resistance also revealed
the first “self-sacrifice” resistance mechanism,7 while CLM
CalO2 displayed a typical P450 Soret peak (418 nm),15 N-His6-
glycosyltransferase-catalyzed “sugar exchange” and “aglycon
exchange” reactions enabled the production of >70 differentially
glycosylated CLM variants.8 Despite the prevalence of deoxy-
and aminosugars in nature,9 only a few naturally occurring
N-oxidized aminosugars, such as the one found in CLM, have
been identified.10 Putative N-oxidase genes for rubranitrose,
kijanose, and the CLM/esperamicin hydroxylaminosugar bio-
synthesis have been put forth, yet the enzymes involved in
aminosugar N-oxidation remain elusive.5b,d,10,11 Herein we
describe the first reported in vitro characterization of an
aminosugar N-oxidase, CalE10, responsible for CLM hydroxy-
laminosugar formation.
A comparison among the gene clusters encoding 10-membered
enediynes5b-d and indolocarbazoles12 presented a genomic basis
from which to propose the biosynthetic pathway for hy-
droxyaminosugar precursor TDP-4-hydroxyamino-6-deoxy-R-D-
glucose (Scheme 1, 4). Specifically, this comparative genomic
analysis (Scheme S1) enabled the elimination from consideration
genes for the biosynthesis of the 10-membered enediyne core
(common to 5, 14, and 16) and the CLM aminopentose (common
to 5, 14, and 18, but not 19). Genes anticipated to be involved
in the biosynthesis of orsellinic acid13 and the terminal rhamnose
precursor9 were also excluded on the basis of the well-established
precedent for these pathways. The remaining genes were
anticipated to be integral to CLM thiosugar or hydroxylamino-
sugar biosynthesis. In conjunction with the well-established
routes to aminosugar biosynthesis,9 and reminiscent of the P450
N-oxidase in ꢀ-lactam biosynthesis (NocL),14 this information
led to the proposed pathway highlighted in Scheme 1 wherein
two P450s (CalO2 and CalE10) were identified as aminosugar
oxidase candidates.
CalE10 exhibited two distinct maxima (418 and 386 nm) of equal
intensity indicative of a heme iron mixed spin state. The reduced
CO-bound spectra for both enzymes displayed a typical P450
Soret peak (450 nm) (Figure 1A). Subsequent in vitro assays
employed a series of putative TDP-sugar substrates (Figure 1B,
1, 3, 6-13; 10 mM),8,16 0.5 mg mL-1 P450 (CalO2 or CalE10),
and a standard spinach ferredoxin/reductase system.17 N-His6-
CalE10-catalyzed transformation of 3 afforded two new products
(Figure 1C) with mass and IR consistent with hydroxyaminosugar
4a (Scheme 1, major) and nitrosugar 4b (minor), while amino-
sugar 8 with the same enzyme led to the corresponding
hydroxylamino derivative in trace amounts. Steady state kinetic
analysis of the CalE10-catalyzed oxidation of 3 revealed kinetic
parameters (kcat ) 0.04 ( 0.01 s-1; Km ) 7.6 ( 1.2 µM) similar
to other natural product P450s.17 Consistent with the stringent
aminosugar regiospecificity observed, subsequent ligand-binding
studies revealed a reverse type I difference spectrum17e,18 with
determined Kd values of 9.1 ( 1.1, 17.3 ( 1.8, 165 ( 27, and
>150 µM for 3, 8, 1, and 10, respectively, while TDP or
4-amino-4-deoxy-R-D-Glc-1-phosphate led to no heme perturba-
tion. No apparent sugar nucleotide binding or oxidation was
observed with CalO2, consistent with the ability of CalO2 to
bind substituted aromatic acids (as possible orsellinic acid
surrogates).15
In summary, this study establishes, for the first time, CalE10
as the requisite CLM NDP-aminosugar N-oxidase and confirms
that oxidation occurs at the sugar nucleotide stage prior to
glycosyltransfer. Furthermore, substrate specificity studies re-
vealed CalE10-catalyzed oxidation to be regiospecific with
limited overoxidation in vitro. As the first characterization of
an aminosugar N-oxidase, this study also presents a foundation
for the future study of other N-oxidases involved in hydroxy-
lamino-, nitroso-, and/or nitrosugar formation.
To test the ability of CalO2 and CalE10 to catalyze amino-
sugar N-oxidation, the corresponding enzymes were overpro-
duced in Streptomyces liVidans as N-His6-fusions. While N-His6-
9
17662 J. AM. CHEM. SOC. 2008, 130, 17662–17663
10.1021/ja807557a CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Poncin, M.; Nilakantan, R.; Ellestad, G. A. Science 1989, 244, 697–699.
(d) DeVoss, J. J.; Townsend, C. A.; Ding, W.-D.; Morton, G. O.; Ellestad,
G. A.; Zein, N.; Tabor, A. B.; Schreiber, S. L. J. Am. Chem. Soc. 1990,
112, 9669–9670.
(3) (a) Walker, S.; Murnick, J.; Kahne, D. J. Am. Chem. Soc. 1993, 115, 7954–
7961. (b) Paloma, L. G.; Smith, J. A.; Chazin, W. J.; Nicolaou, K. C. J. Am.
Chem. Soc. 1994, 116, 3697–3708. (c) Ikemoto, N.; Kumar, R. A.; Ling,
T. T.; Ellestad, G. A.; Danishefsky, S. J.; Patel, D. J. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 10506–10510. (d) Kumar, R. A.; Ikemoto, N.; Patel,
D. J. J. Mol. Biol. 1997, 265, 187–201.
(4) For reviews see: (a) Wu, A. M.; Senter, P. D. Nat. Biotechnol. 2005, 23,
1137–1146. (b) Pui, C. H.; Jeha, S. Nat. ReV. Drug DiscoVery 2007, 6,
149–165. (c) Jurcic, J. G. Cytotherapy 2008, 10, 7–12.
(5) (a) C1027: Liu, W.; Christenson, S. D.; Standage, S.; Shen, B. Science
2002, 297, 1170–1173. (b) calicheamicin: Ahlert, J.; Shepard, E.; Lo-
movskaya, 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. (c) dynemicin: Gao, Q.; Thorson, J. S.
FEMS Microbiol. Lett. 2008, 282, 105–114. (d) esperamicin: Ahlert, J.;
Shepard, E. M.; Thorson, J. S. accession number AY267372. (e) ma-
duropeptin: Van Lanen, S. G.; Oh, T.-J.; Liu, W.; Wendt-Pienkowski, E.;
Shen, B. J. Am. Chem. Soc. 2007, 129, 13082–13094. (f) neocarzinostatin:
Liu, W.; Nonaka, K.; Nie, L.; Zhang, J.; Christenson, S. D.; Bae, J.; Van
Lanen, S. G.; Zazopoulos, E.; Farnet, C. M.; Yang, C. F.; Shen, B. Chem.
Biol. 2005, 12, 293–302. (g) PKSE genome scanning: Zazopoulos, E.;
Huang, K.; Staffa, A.; Liu, W.; Bachmann, B. O.; Nonaka, K.; Ahlert, J.;
Thorson, J. S.; Shen, B. Nat. Biotechnol. 2003, 21, 187–190. (h) PKSE
degenerate PCR: Liu, W.; Ahlert, J.; Gao, Q.; Wendt-Pienkowski, E.; Shen,
B.; Thorson, J. S. Proc. Natl Acad. Sci. U.S.A. 2003, 100, 11959–11963.
(6) Zhang, J.; Van Lanen, S. G.; Ju, J.; Liu, W.; Dorrestein, P. C.; Li, W.;
Kelleher, N. L.; Shen, B. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 1460–
1465.
(7) (a) Biggins, J. B.; Onwueme, K. C.; Thorson, J. S. Science 2003, 301,
1537–1541. (b) Singh, S.; Hager, M. H.; Zhang, C.; Griffith, B. R.; Lee,
M. S.; Hallenga, K.; Markley, J. L.; Thorson, J. S. ACS Chem. Biol. 2006,
1, 451–460.
(8) (a) Zhang, C.; Griffith, B. R.; Fu, Q.; Albermann, C.; Fu, X.; Lee, I.-K.;
Li, L.; Thorson, J. S. Science 2006, 313, 1291–1294. (b) 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.
(9) For reviews see:(a) Nedal, A.; Zotchev, S. B. Appl. Microbiol. Biotechnol.
2004, 64, 7–15. (b) Rupprath, C.; Schumacher, T.; Elling, L. Curr. Med.
Chem. 2005, 12, 1637–1675. (c) Thibodeaux, C. J.; Melanc¸on, C. E.; Liu,
H.-w. Nature 2007, 446, 1008–1016. (d) Salas, J. A.; Me´ndez, C. Trends
Microbiol. 2007, 15, 219–232. (e) Kren, V.; Rezanka, T. FEMS Microbiol.
ReV. 2008, 32, 858–889.
(10) For a review see: Timmons, S. C.; Thorson, J. S. Curr. Opin. Chem. Biol.
2008, 12, 297–305.
(11) (a) Song, J. K; Oh, T. J.; Lee, J. J.; Kim, C. G. Mol. Cells 1997, 7, 674–
681. (b) Lamichhane, J.; Liou, K.; Lee, H. C.; Kim, C.-G.; Sohng, J. K.
Biotechnol. Lett. 2006, 28, 545–553. (c) Zhang, H.; White-Phillip, J. A.;
Melanc¸on, C. E.; Kwon, H.-j.; Yu, W.-l.; Liu, H.-w. J. Am. Chem. Soc.
2007, 129, 14670–14683. (d) Fang, J.; Zhang, Y.; Huang, L.; Jia, X.; Zhang,
Q.; Zhang, X.; Tang, G.; Liu, W. J. Bacteriol. 2008, 190, 6014–6025.
(12) (a) Sa´nchez, C.; Butovich, I. A.; Brana, A. F.; Rohr, J.; Me´ndez, C.; Salas,
J. A. Chem. Biol. 2002, 9, 519–531. (b) Hyun, C.-G.; Bililign, T.; Liao, J.;
Thorson, J. S. ChemBioChem 2003, 4, 114–117. (c) Gao, Q.; Zhang, C.;
Blanchard, S.; Thorson, J. S. Chem. Biol. 2006, 13, 733–743. (d) Sa´nchez,
C.; Me´ndez, C.; Salas, J. A. Nat. Prod. Rep. 2006, 23, 1007–1045.
(13) (a) Gaisser, S.; Trefzer, A.; Stockert, S.; Kirschning, A.; Bechthold, A. J.
Bacteriol. 1997, 179, 6271–6278. (b) Jia, X. Y.; Tian, Z. H.; Shao, L.; Qu,
X. D.; Zhao, Q. F.; Tang, J.; Tang, G. L.; Liu, W. Chem. Biol. 2006, 13,
575–585. (c) Shao, L.; Qu, X. D.; Jia, X. Y.; Zhao, Q. F.; Tian, Z. H.;
Wang, M.; Tang, G. L.; Liu, W. Biochem. Biophys. Res. Commun. 2006,
345, 133–139.
Figure 1. (a) Oxidized spectrum of CalE10 and difference spectra of
reduced CO-bound species (inset). (b) Putative sugar nucleotide substrates
used in this study. (c) HPLC analyses of assays with 3 as the substrate: (i)
no P450 (control); (ii) CalO2; (iii) CalE10. A trace amount of 1 remains
from chemoenzymatic synthesis of 3. See Supporting Information for
experimental details.
(14) Kelly, W. L.; Townsend, C. A. J. Am. Chem. Soc. 2002, 124, 8186–8187.
Acknowledgment. We thank the School of Pharmacy Analytical
Instrumentation Center for analytical support and Professor Hung-
wen (Ben) Liu for graciously providing overexpression constructs
for rfbB and desI. This work was supported by the NIH (Grants
CA84374 and U19 CA113297). H.D.J. is UW Chemical Biology
Interface Trainee (T32 GM008505).
(15) McCoy, J. G.; Johnson, H. D.; Singh, S.; Bingman, C. A.; Lei, I.-K.;
Thorson, J. S.; Phillips, G. N., Jr. Proteins, in press.
(16) (a) Jiang, J.; Biggins, J. B.; Thorson, J. S. J. Am. Chem. Soc. 2000, 122,
6803–6804. (b) Jiang, J.; Biggins, J. B.; Thorson, J. S. Angew. Chem., Intl.
Ed. 2001, 40, 1502–1505. (c) Albermann, C.; Jiang, J.; Thorson, J. S.
ChemBioChem 2003, 4, 443–446. (d) Fu, X.; Albermann, C.; Jiang, J.;
Liao, J.; Zhang, C.; Thorson, J. S. Nat. Biotechnol. 2003, 21, 1467–1469.
(17) (a) Lambalot, R. H.; Cane, D. E.; Aparicio, J. J.; Katz, L. Biochemistry
1995, 34, 1858–1866. (b) Xue, Y.; Wilson, D.; Zhao, L.; Liu, H.-w.;
Sherman, D. H. Chem. Biol. 1998, 5, 661–667. (c) Walczak, R. J.; Hines,
J. V.; Strohl, W. R.; Priestley, N. D. Org. Lett. 2001, 3, 2277–2279. (d)
Davydov, D. R.; Botchkareva, A. E.; Kumar, S.; He, Y. Q.; Halpert, J. R.
Biochemistry 2004, 43, 6475–6485. (e) Ogura, H; Nishida, C. R.; Hoch,
U. R.; Perera, R.; Dawson, J. H.; Ortiz de Montellano, P. R. Biochemistry
2004, 43, 14712–14721. (f) Lawson, R. J.; Leys, D.; Sutcliffe, M. J.; Kemp,
C. A.; Cheesman, M. R.; Smith, S. J.; Clarkson, J.; Smith, W. E.; Haq, I.;
Perkins, J. B.; Munro, A. W. Biochemistry 2004, 43, 12410–12426. (g)
Mendes, M. V.; Anton, N.; Martin, J. F.; Aparicio, J. A. Biochem. J. 2005,
386, 57–62.
Supporting Information Available: Assay procedures and spec-
troscopic data. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) For reviews see: (a) Thorson, J. S.; Sievers, E. L.; Ahlert, J.; Shephard, E.;
Whitwam, R. E.; Onwueme, K. C.; Ruppen, M. Curr. Pharm. Des. 2000,
6, 1841–1879. (b) Galm, U.; Hager, M. H.; Van Lanen, S. G.; Ju, J.;
Thorson, J. S.; Shen, B. Chem. ReV. 2005, 105, 739–758. (c) Van Lanen,
S. G.; Shen, B. Curr. Topics Med. Chem. 2008, 8, 448–459.
(2) (a) Zein, N.; Sinha, A. M.; McGahren, W. J.; Ellestad, G. A. Science 1988,
240, 1198–1201. (b) Zein, N.; McGahren, W. J.; Morton, G. O.; Ashcroft,
J.; Ellestad, G. A. J. Am. Chem. Soc. 1989, 111, 6888–6890. (c) Zein, N.;
(18) (a) Kahn, R. A.; Bak, S.; Svendsen, I.; Halkier, B. A.; Moller, B. L. Plant
Physiol. 1997, 115, 1661–1670. (b) Kahn, R. A.; Fahrendorf, T.; Halkier,
B. A.; Moller, B. L. Arch. Biochem. Biophys. 1999, 363, 9–18. (c) Bak,
S.; Feyereisen, R. Plant Physiol. 2001, 127, 108–118.
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