Substrate specificity of the macrolide-glycosylating enzyme pair DesVII/DesVIII: Opportunities, limitations, and mechanistic hypotheses

Article abstract of DOI:10.1002/anie.200503195

(Chemical Equation Presented) Two's Company: DesVII, a glycosyltransferase involved in the biosynthesis of macrolide antibiotics, is unusual in that it requires an additional protein partner, DesVIII, for its full activity. The level of substrate tolerance of the DesVII/DesVIII pair was explored.

Full text of DOI:10.1002/anie.200503195

Communications
Glycosylations
DOI: 10.1002/anie.200503195
Substrate Specificity of the Macrolide-
Glycosylating Enzyme Pair DesVII/DesVIII:
Opportunities, Limitations, and Mechanistic
Hypotheses**
Svetlana A. Borisova, Changsheng Zhang,
Haruko Takahashi, Hua Zhang, Alexander W. Wong,
Jon S. Thorson, and Hung-wen Liu*
Macrolide antibiotics consist of a polyketide macrolactone
with one or more deoxysugar residues attached. Macrolides
target protein translation and are highly effective against
gram-positive bacteria and represent an important class of
compounds in our current antibiotic arsenal to treat patho-
genic bacteria.[1] However, as with most broadly prescribed
antibiotics, the rapid emergence of macrolide-resistant organ-
isms dictates the continuous need for new macrolide ana-
logues to help sustain our battery of effective agents against
the constant onslaught of potentially lethal microbes.[2] The
recent determination of the 3D structure of the 50S ribosomal
subunit complexed with a variety of macrolides clearly reveals
that the sugar substituents of these macrolides contribute to
[*] Dr. S. A. Borisova, Dr. H. Takahashi, Dr. H. Zhang, Dr. A. W. Wong,
Prof. H.-w. Liu
Division of Medicinal Chemistry, College of Pharmacy
and Department of Chemistry and Biochemistry, University of Texas
Austin, TX 78712 (USA)
Fax: (+1)512-471-2746
E-mail: h.w.liu@mail.utexas.edu
Dr. C. Zhang, Prof. J. S. Thorson
Laboratory for Biosynthetic Chemistry and
National Cooperative Drug Discovery Group
Pharmaceutical Sciences Division
School of Pharmacy, University of Wisconsin
Madison, WI 53705 (USA)
[**] This work was supported in part by the National Institutes of Health
(grants GM35906 and GM54346 (H.-w.L.) and AI52218, CA84374
and GM70637) and an NCDDG grant (U19 CA113297) from the
National Cancer Institute (J.S.T.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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binding and sterically infringe upon the polypeptide exit
tunnel—thereby preventing peptide-chain elongation.[3]
Structural studies also show that the C5 disaccharide moieties
of 16-membered macrolides, such as spiramycin and carbo-
mycin, extend further into the exit tunnel toward the adjacent
peptidyl transferase center. Moreover, a direct correlation
between the length of truncated peptides released by a
macrolide-bound ribosome and the length of the saccharide
side chain of the macrolide has been established. Cumula-
tively, these studies clearly demonstrate the essential nature
of interactions between rRNA and deoxy sugars of macrolide
antibiotics and suggest that alterations of these appended
carbohydrates may modulate binding and/or potentially
circumvent certain resistance mechanisms.
biosynthetic loci encoding for aminosugar-bearing metabolite
biosynthesis where they typically reside directly upstream to
the aminosugar glycosyltransferase gene.[15] Thus, DesVIII
was initially implicated as necessary for the incorporation of
aminosugar moieties in these metabolites.[6]
In study reported herein, we extend the established
in vitro DesVII/DesVIII assay to assess the level of aglycone
and sugar substrate tolerance by this catalytic pair. In addition
to providing a set of new macrolides, this work reveals a
notably stringent 6-deoxysugar requirement for DesVII/
DesVIII-catalyzed glycosylation. More importantly, this
work also reveals that the DesVIII “boost” is not limited to
aminosugars and thus vastly contrasts the earlier “aminosugar
carrier” hypothesis.
Given the architectural and synthetic complexity of
macrolides, pathway engineering has emerged as an attractive
means for in vivo analogue production, presenting new
derivatives with variant aglycones and/or sugars.[4] These
in vivo studies have also revealed a relaxed specificity of the
responsible macrolide glycosyltransferases toward both their
sugar and aglycone substrates.[5] In 2004 we successfully
demonstrated the first in vitro activity of a purified macrolide
glycosyltransferase, DesVII,[6] which catalyzes the attachment
of TDP-desosamine (1) to 10-deoxymethynolide (2) or
narbonolide (3) in the biosynthesis of methymycin (6),
neomethymycin (7), pikromycin (8), and narbomycin (5) in
Streptomyces venezuelae (Scheme 1).
This in vitro study revealed an unusual requirement for
DesVII activity: the need for an additional protein partner,
DesVIII. Recently, a similar requirement for an auxiliary
protein (EryCII) was also established for EryCIII, which
catalyzes the attachment of desosamine (1) to the related
erythronolide core.[7] Interestingly, among the growing
number of in vitro studies of related glycosyltransferases—
including those for nonribosomal peptides,[8] aminocoumar-
ins,[9] enediynes,[10] macrolactams,[11] macrolides,[6,7,12] and
aromatic polyketides[13]—only five, including DesVII and
EryCIII, have been shown to depend on a DesVIII homo-
logue.[14] Although their function remains unclear, the genes
coding for DesVIII homologues are only found within
The recombinant DesVII protein was overproduced in
E. coli as a C-terminal His6-tagged fusion protein and purified
as previously described.[6] The DesVIII protein was produced
in E. coli as a fusion with maltose-binding protein at its
N terminus. The fusion protein was cleaved by treatment with
protease and untagged DesVIII protein was used in the assays
together with DesVII.[16] A number of TDP-sugars were
tested as donor substrates for DesVII/DesVIII with the native
macrolide aglycone 2 (Scheme 2) as the acceptor. These
include TDP derivatives of glucose 9, galactose 10, mannose
11, and a series of amino and acetamido hexoses 12–18. TDP-
sugars deoxygenated at C3 and C4, 19 and 20, as well as TDP-
xylose 21 were also examined. As the natural substrate
desosamine (1) is a 6-deoxyhexose, a variety of 6-deoxy sugars
22–27 and 6-deoxyaminosugars 28–31 were also tested.
Of the sugar nucleotides employed, compound 9 was
commercially available, compounds 1,[17] 26,[18] and 28[17b] were
previously synthesized chemically, compound 30 was pre-
pared by a chemoenzymatic method,[17b] and compounds 23,
25,[16] and 27[19] were obtained through enzymatic synthesis.
The remaining 16 TDP-sugars 10–22, 24, 29, 31 were
generated in situ through an engineered nucleotidylyltrans-
ferase (Ep) catalyzed chemoenzymatic route from the respec-
tive sugar-1-phosphates as previously described.[20] For each
assay, TDP-sugar formation in situ was assessed by HPLC, the
reaction mixture subsequently adjusted to pH 9, and DesVII,
DesVIII, and 2 added. The
progress of the glycosylation
reaction was monitored by
TLC. The products from sam-
ples showing positive TLC
results were subsequently
isolated by preparative TLC
or HPLC, and their identities
established by high-resolu-
tion MS and/or NMR spec-
troscopic analysis.[16]
Of the 23 activated sugars
tested, only seven were
accepted by DesVII/DesVIII
to give products 4 and 32–37
(Scheme 2), of which 35–37
have never been reported.
The in vitro production of
macrolides 32 and 33, previ-
Scheme 1. Final steps of the biosynthesis of macrolide antibiotics methymycin (6), neomethymycin (7),
narbomycin (5), and pikromycin (8) in Streptomyces venezuelae. PikC=cytochrome P450 hydroxylase;
TDP=thymidine diphosphate.
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Scheme 2. Study of sugar nucleotide substrate specificity of DesVII/DesVIII using 10-deoxymethynolide (2) as acceptor.
ously identified shunt metabolites from S. venezuelae disrup-
tion mutants, suggests that reduction of the keto group of
NDP-sugars 23 or 38 likely occurs prior to glycosylation
(Scheme 3) in these engineered mutant strains.[5d,b] This order
of events is further supported by the observation that 23 is not
a substrate of DesVII/DesVIII.
sugars bearing functional groups at C6 (e.g. 9–20) or lacking
C6 (e.g. 21) failed as DesVII/DesVIII substrates, whereas
fairly broad structural variation was tolerated among the 6-
deoxyhexose substrates tested, including both d (e.g. 22, 24)
and l sugars (25). Aminosugars and N-alkyl aminosugars
were found to be substrates (e.g., 1, 28, 30, and 31), but N-acyl
aminosugars were not. The fact that aminosugar 28 is a
substrate whereas N-acetamido sugar 29 is not supports our
previous hypothesis that N-acetyla-
Consistent with the early in vivo studies, a strict require-
ment for 6-deoxysugars was also observed in vitro. TDP-
tion occurs after glycosylation in the
production of 40 and 41 (Sche-
me 3).[5c,f,21]
Furthermore, C3 alkylation and/
or C2deoxygenation appear to be
detrimental (e.g. 27) whereas the
correct anomeric configuration (e.g.
25 versus 26) is essential for DesVII/
DesVIII activity. Most importantly,
parallel assays in the absence of
DesVIII led to a dramatic decrease
in DesVII activity (ranging from a
Scheme 3. Biosynthetic pathway to TDP-desosamine (1) from TDP-glucose (9). Also shown are macrolide
products isolated from the fermentation broths of various gene-disruption mutants of S. venezuelae. Kdes=
S. venezuelae mutants in which the respective des genes are inactivated.
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more than 30-fold decrease in yield to no product at all),
including those assays with substrates that lack amino
functionality (e.g. 24 and 25).[16] Clearly, the DesVIII aug-
mentation effect is not limited to aminosugar substrates only.
Given the well-characterized in vivo aglycone tolerance of
DesVII/DesVIII,[22] we next set out to explore this potential
in vitro, particularly in the context of non-natural sugar
donors. Consistent with earlier observations, we found
DesVII/DesVIII to be capable of adding desosamine to the
natural acceptors 10-deoxymethynolide (2) and narbonolide
(3) to give 4 and 5, respectively. Interestingly, the hydroxy-
lated analogues of 2, methynolide (42) and neomethynolide
(43), can also be glycosylated to yield 6 and 7, respectively.
Remarkably, all six confirmed non-natural in vitro sugar
substrates 22, 24, 25, 28, 30, and 31 from the studies described
above, were also successful DesVII/DesVIII substrates with
aglycones 2, 3, 42, and 43 (Scheme 4). In the context of the
previous hypothesis that PikC-catalyzed aglycone hydroxyl-
ation (i.e. the conversions of 4 into 6/7 and 5 into 8) is a
postglycosylation event,[23] these results demonstrate a toler-
ance of DesVII/DesVIII towards the hydroxylation pattern of
the aglycone and imply some flexibility in the glycosylation/
hydroxylation sequence of biosynthetic steps. Many of the
macrolides first identified through pathway engineering
(32,[5d] 34, 50–52,[5e] 45–46,[14a] 61–62,[5b] 63–66[24]) were also
identified in this study.
Interestingly, we found 1, 22, 24, 25, 28, 30, and 31 to be
substrates with the 16-membered tylactone (44) as well to give
48, 68, 67, 53, 59, 49, and 60, respectively (Scheme 4). Of these
products, 48 and 49 had been previously identified in vi-
Scheme 4. Structures of macrolides identified in this study.
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vo,[25,14a] while others have not been reported. Also, although
[1] a) H. Takashima, Curr. Top. Med. Chem. 2003, 3, 991; b) M.
Gaynor, A. S. Mankin, Front. Med. Chem. 2005, 2, 2 1.
[2] a) A. Coates, Y. Hu, C. Page, Nat. Rev. Drug Discovery 2002, 1,
895; b) T. C. Henninger, Expert Opin. Ther. Pat. 2003, 13, 787.
[3] a) F. Schlünzen, R. Zarivach, J. Harms, A. Bashan, A. Tocilj, R.
Albrecht, A. Yonath, F. Franceschi, Nature 2001, 413, 814;
b) J. L. Hansen, J. A. Ippolito, N. Ban, P. Nissen, P. B. Moore,
T. A. Steitz, Mol. Cell 2002, 10, 117; c) F. Schlünzen, J. M. Harms,
F. Franceschi, H. A. S. Hansen, H. Bartels, R. Zarivach, A.
Yonath, Structure 2003, 11, 329; d) R. Berisio, F. Schlünzen, J.
Harms, A. Bashan, T. Auerbach, D. Baram, A. Yonath, Nat.
Struct. Biol. 2003, 10, 366; e) R. Berisio, J. Harms, F. Schluenzen,
R. Zarivach, H. A. S. Hansen, P. Fucini, A. Yonath, J. Bacteriol.
2003, 185, 4276; f) D. Tu, G. Blaha, P. B. Moore, T. A. Steitz, Cell
2005, 121, 257.
[4] a) C. MØndez, J. A. Salas, Trends Biotechnol. 2001, 19, 449; b) E.
Rodriguez, R. McDaniel, Curr. Opin. Microbiol. 2001, 4, 52 6;
c) J. S. Thorson, T. J. Hosted, Jr., J. Jiang, J. B. Biggins, J. Ahlert,
Curr. Org. Chem. 2001, 5, 139; d) W. R. Strohl, Metab. Eng. 2001,
3, 4; e) U. Rix, C. Fischer, L. L. Remsing, J. Rohr, Nat. Prod. Rep.
2002, 19, 542; f) X. He, H.-w. Liu, Annu. Rev. Biochem. 2002, 71,
701; g) A. P. Salas, L. Zhu, C. Sµnchez, A. F. Braæa, J. Rohr, C.
MØndez, J. A. Salas, Mol. Microbiol. 2005, 58, 17.
[5] a) L. Zhao, D. H. Sherman, H.-w. Liu, J. Am. Chem. Soc. 1998,
120, 10256; b) L. Zhao, N. L. S. Que, Y. Xue, D. H. Sherman, H.-
w. Liu, J. Am. Chem. Soc. 1998, 120, 12159; c) L. Zhao, J. Ahlert,
Y. Xue, J. S. Thorson, D. H. Sherman, H.-w. Liu, J. Am. Chem.
Soc. 1999, 121, 9881; d) S. A. Borisova, L. Zhao, D. H. Sherman,
H.-w. Liu, Org. Lett. 1999, 1, 133; e) H. Yamase, L. Zhao, H.-w.
Liu, J. Am. Chem. Soc. 2000, 122, 12397; f) L. Zhao, S. Borisova,
S.-M. Yeung, H.-w. Liu, J. Am. Chem. Soc. 2001, 123, 7909.
[6] S. A. Borisova, L. Zhao, C. E. Melançon III, C.-L. Kao, H.-w.
Liu, J. Am. Chem. Soc. 2004, 126, 6534.
44 contains two potential glycosylation sites, the DesVII/
DesVIII system was regioselective for C5. Most importantly,
parallel assays lacking DesVIII again led to a dramatic
reduction in the yield of the glycosylated products. Such an
ability to utilize a non-natural aglycone and a non-natural
sugar in the coupling reaction is unusual for enzyme catalysis,
but appears to be more common among glycosyltransferases
involved in the biosynthesis of secondary metabolites.[4,5] The
demonstrated aglycone as well as sugar promiscuity of this
system presents an opportunity for macrolide glycodiversifi-
cation.
In summary, the in vitro DesVII/DesVIII characterization
described herein not only demonstrates its potential for
macrolactone glycodiversification,[26] but also further expands
our understanding of the DesVII/DesVIII glycosylation
machinery. Based on these studies, DesVII/DesVIII exhibits
a stringent 6-deoxysugar requirement but an otherwise
relaxed substrate specificity toward both TDP-sugar donor
and aglycone acceptor. The catalytic capabilities of DesVII/
DesVIII, as indicated by the formation of 19 previously
unreported macrolides in this limited exploration, hold high
promise in biosynthetic application to construct new macro-
lide derivatives. More importantly, these results reveal that
DesVIII equally augments DesVII activity toward both
aminosugars and non-aminosugars. The lack of preferential
aminosugar enhancement contrasts the previously proposed
“aminosugar-carrier” role for this intriguing protein. DesVIII
has also been speculated to play a role in the enhancement of
the aglycone–glycosyltransferase interaction considering the
high hydrophobic nature of the aglycones 2 and 3. However,
given the fact that the DesVIII augmentation effect holds for
both hydrophobic (such as 2 and 3) and more-hydrophilic
aglycones (such as 42 and 43), a specific “aglycone-carrier”
role for DesVIII may also be called into question. Since
DesVII shows good sequence homology to members of the
glycosyltransferase GT-B superfamily, which contain two
distinct domains for harboring the aglycone and TDP-sugar
substrate, it is more likely that binding of DesVIII to DesVII
may optimize a DesVII conformational change bringing these
two domains to close proximity during coupling, or may
simply facilitate the proper folding of DesVII and thereby
improve its catalytic efficiency. In fact, our preliminary
experiments showed that DesVIII is only required for initial
activation of DesVII and the activated DesVII can catalyze
the glycosyl transfer alone. Interestingly, a similar observation
was recently reported for activation of glycosyltransferase
EryCIII by DesVIII homologues EryCII and AknT.[7] The
authors proposed that auxiliary proteins induce a one-time
conformational change of glycosyltransferase to its active
form. Experiments are in progress to investigate further the
catalytic properties of DesVII and DesVIII.
[7] Y. Yuan, H. S. Chung, C. Leimkuhler, C. T. Walsh, D. Kahne, S.
Walker, J. Am. Chem. Soc. 2005, 127, 14128.
[8] a) H. C. Losey, M. W. Peczuh, Z. Chen, U. S. Eggert, S. D. Dong,
I. Pelczer, D. Kahne, C. T. Walsh, Biochemistry 2001, 40, 4745;
b) H. C. Losey, J. Jiang, J. B. Biggins, M. Oberthür, X. Y. Ye,
S. D. Dong, D. Kahne, J. S. Thorson, C. T. Walsh, Chem. Biol.
2002, 9, 1305; c) X. Fu, C. Albermann, J. Jiang, J. Liao, C. Zhang,
J. S. Thorson, Nat. Biotechnol. 2003, 21, 1467; d) T. L. Li, F.
Huang, S. F. Haydock, T. Mironenko, P. F. Leadlay, J. B. Spencer,
Chem. Biol. 2004, 11, 107; e) W. Lu, M. Oberthür, C. Leimkuh-
ler, J. Tao, D. Kahne, C. T. Walsh, Proc. Natl. Acad. Sci. USA
2004, 101, 4390; f) X. Fu, C. Albermann, C. Zhang, J. S. Thorson,
Org. Lett. 2005, 7, 1513.
[9] a) C. Albermann, A. Soriano, J. Jiang, H. Vollmer, J. B. Biggins,
W. A. Barton, J. Lesniak, D. B. Nikolov, J. S. Thorson, Org. Lett.
2003, 5, 933; b) C. L. Freel Meyers, M. Oberthür, J. W. Ander-
son, D. Kahne, C. T. Walsh, Biochemistry 2003, 42, 4179.
[10] C. Zhang, J. S. Thorson, unpublished results.
[11] a) A. Minami, R. Uchida, T. Eguchi, K. Kakinuma, J. Am. Chem.
Soc. 2005, 127, 6148; b) A. Minami, K. Kakinuma, T. Eguchi,
Tetrahedron Lett. 2005, 46, 6187.
[12] a) L. M. Quirꢀs, J. A. Salas, J. Biol. Chem. 1995, 270, 18234;
b) L. M. Quirꢀs, R. J. Carbajo, A. F. Braæa, J. A. Salas, J. Biol.
Chem. 2000, 275, 11713; c) H. Y. Lee, H. S. Chung, C. Hang, C.
Khosla, C. T. Walsh, D. Kahne, S. Walker, J. Am. Chem. Soc.
2004, 126, 9924; d) M. Yang, M. R. Proctor, D. N. Bolam, J. C.
Errey, R. A. Field, H. J. Gilbert, B. G. Davis, J. Am. Chem. Soc.
2005, 127, 9336.
Received: September 8, 2005
Revised: January 18, 2006
Published online: March 15, 2006
[13] W. Lu, C. Leimkuhler, M. Oberthür, D. Kahne, C. T. Walsh,
Biochemistry 2004, 43, 4548.
Keywords: antibiotics · biosynthesis · carbohydrates ·
glycosylation · macrolides
[14] a) C. E. Melançon III, H. Takahashi, H.-w. Liu, J. Am. Chem.
Soc. 2004, 126, 16726; b) W. Lu, C. Leimkuhler, G. J. J. Gatto,
.
2752
www.angewandte.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Angewandte
Chemie
R. G. Kruger, M. Oberthur, D. Kahne, C. T. Walsh, Chem. Biol.
2005, 12, 527.
[15] Examples of desVIII homologues include: a) doxorubicin: S. L.
Otten, X. Liu, J. Ferguson, C. R. Hutchinson, J. Bacteriol. 1995,
177, 6688; b) tylosin: A. R. Gandecha, S. L. Large, E. Cundliffe,
Gene 1997, 184, 197; c) erythromycin: R. G. Summers, S.
Donadio, M. J. Staver, E. Wendt-Pienkowski, C. R. Hutchinson,
L. Katz, Microbiology 1997, 143, 3251; d) oleandomycin: C.
Olano, A. M. Rodriguez, J. M. Michel, C. MØndez, M. C. Raynal,
J. A. Salas, Mol. Gen. Genet. 1998, 259, 299; e) megalomicin: Y.
Volchegursky, Z. Hu, L. Katz, R. McDaniel, Mol. Microbiol.
2000, 37, 752; f) aclarubicin: K. Raty, T. Kunnari, J. Hakala, P.
Mantsala, K. Ylihonko, Mol. Gen. Genet. 2000, 264, 164;
g) nogalamycin: S. Torkkell, T. Kunnari, K. Palmu, P. Mantsala,
J. Hakala, K. Ylihonko, Mol. Genet. Genomics 2001, 266, 276;
h) b-rhodomycin: Y. Miyamoto, O. Johdo, Y. Nagamatsu, A.
Yoshimoto, FEMS Microbiol. Lett. 2002, 206, 163; i) mycinami-
cin: Y. Anzai, N. Saito, M. Tanaka, K. Kinoshita, Y. Koyama, F.
Kato, FEMS Microbiol. Lett. 2003, 218, 135.
[16] See Supporting Information for details.
[17] a) C.-w. Chang, L. Zhao, H. Yamase, H.-w. Liu, Angew. Chem.
2000, 112, 2244; Angew. Chem. Int. Ed. 2000, 39, 2160; b) H.
Chen, H. Yamase, K. Murakami, C.-w. Chang, L. Zhao, Z. Zhao,
H.-w. Liu, Biochemistry 2002, 41, 9165.
[18] Y. Zhao, J. S. Thorson, J. Org. Chem. 1998, 63, 7568.
[19] H. Takahashi, Y.-n. Liu, H. Chen, H.-w. Liu, J. Am. Chem. Soc.
2005, 127, 9340.
[20] a) J. Jiang, J. B. Biggins, J. S. Thorson, J. Am. Chem. Soc. 2000,
122, 6803; b) J. Jiang, J. B. Biggins, J. S. Thorson, Angew. Chem.
2001, 113, 1550; Angew. Chem. Int. Ed. 2001, 40, 1502; c) W. A.
Barton, J. Lesniak, J. B. Biggins, P. D. Jeffrey, J. Jiang, K. R.
Rajashankar, J. S. Thorson, D. B. Nikolov, Nat. Struct. Biol. 2001,
8, 545; d) W. A. Barton, J. B. Biggins, J. Jiang, J. S. Thorson, D. B.
Nikolov, Proc. Natl. Acad. Sci. USA 2002, 99, 13397; e) J. Jiang,
C. Albermann, J. S. Thorson, ChemBioChem 2003, 4, 443.
[21] The N-acetyl-substituted 31 is a poor Ep substrate and thus was
not available in sufficient quantities for the in vitro studies.
[22] a) L. Tang, R. McDaniel, Chem. Biol. 2001, 8, 547; b) Y. J. Yoon,
B. J. Beck, B. S. Kim, H.-Y. Kang, K. A. Reynolds, D. H.
Sherman, Chem. Biol. 2002, 9, 203.
[23] a) M. C. Betlach, J. T. Kealey, G. W. Ashley, R. McDaniel,
Biochemistry 1998, 37, 14937; b) D. E. Cane, E. I. Graziani, J.
Am. Chem. Soc. 1998, 120, 2682; c) E. I. Graziani, D. E. Cane,
M. C. Betlach, J. T. Kealey, R. McDaniel, Bioorg. Med. Chem.
Lett. 1998, 8, 3117; d) Y. Xue, D. Wilson, L. Zhao, H.-w. Liu,
D. H. Sherman, Chem. Biol. 1998, 5, 661.
[24] J. S. J. Hong, S. H. Park, C. Y. Choi, J. K. Sohng, Y. J. Yoon,
FEMS Microbiol. Lett. 2004, 238, 391.
[25] S. Gaisser, J. Reather, G. Wirtz, L. Kellenberger, J. Staunton,
P. F. Leadlay, Mol. Microbiol. 2000, 36, 391.
[26] a) J. Yang, D. Hoffmeister, L. Liu, X. Fu, J. S. Thorson, Bioorg.
Med. Chem. 2004, 12, 1577; b) J. M. Langenhan, B. R. Griffith,
J. S. Thorson, J. Nat. Prod. 2005, 68, 1696; c) B. R. Griffith, J. M.
Langenhan, J. S. Thorson, Curr. Opin. Biotechnol. 2005, 16, 622.
Angew. Chem. Int. Ed. 2006, 45, 2748 –2753
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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