Identification of a set of genes involved in the formation of the substrate for the incorporation of the unusual "glycolate" chain extension unit in ansamitocin biosynthesis

Article abstract of DOI:10.1021/ja0124764

The unusual "glycolate" extender unit at C-9/C-10 of ansamitocin is not derived from 2-hydroxymalonyl-CoA or 2-methoxymalonyl-CoA, as demonstrated by feeding experiments with the corresponding 1-¹³C-labeled N-acetylcysteamine thioesters but is formed from an acyl carrier protein (ACP)-bound substrate, possibly 2-methoxymalonyl-ACP, elaborated by enzymes encoded by a subcluster of five genes, asm12-17, from the ansamitocin bisosynthetic gene cluster. Copyright

Full text of DOI:10.1021/ja0124764

Published on Web 03/27/2002
Identification of a Set of Genes Involved in the Formation of the Substrate for
the Incorporation of the Unusual “Glycolate” Chain Extension Unit in
Ansamitocin Biosynthesis
Brian J. Carroll, Steven J. Moss, Linquan Bai, Yasuo Kato, Sabine Toelzer, Tin-Wein Yu,* and
Heinz G. Floss*
Department of Chemistry, Box 351700, UniVersity of Washington, Seattle, Washington 98195-1700
Received November 6, 2001
1
2
The ansamitocins, exemplified by ansamitocin P-3 (Figure 1),
are members of the maytansinoids, a family of extraordinarily potent
antitumor agents isolated from higher plants as well as microorgan-
isms.3 The biosynthesis of the ansamitocins in the producing
microorganism, the actinomycete Actinosynnema pretiosum, in-
volves the assembly of a macrocyclic lactam from an aromatic
4
starter unit, 3-amino-5-hydroxybenzoic acid (AHBA), by seven
chain extension steps on a type I modular polyketide synthase
(PKS). Three of the chain extension reactions incorporate propi-
Figure 1. Structures of ansamitocins.
onate, and three incorporate acetate units, that is, two use methyl-
malonyl-CoA and malonyl-CoA, respectively, as substrate and the
third one incorporates an unusual oxygenated two-carbon unit
Scheme 1 . Synthesis of 1-13C-Labeled 2-Hydroxymalonyl- and
2-Methoxymalonyl-N-acetylcysteamine (SNAC) Thioester.
5
sometimes called a “glycolate” unit. Such “glycolate” units are
also found in other macrolactam or macrolide antibiotics, such as
6
7
8
9
geldanamycin, leucomycin, FK520 and FK506, and soraphen.
In most cases, as in ansamitocin, the R-oxygen of this unusual
extender unit is methylated, although there are some exceptions,
1
0
11
such as the aflastatins and zwittermicin. Isotopic tracer
experiments5
-10,12
have shown that these “glycolate” units are not
derived from acetate, but probably arise from carbohydrate me-
tabolism.
Analogy with the other chain extension reactions suggests that
the substrate for the incorporation of a “glycolate” unit should be
2
-hydroxymalonyl-CoA or 2-methoxymalonyl-CoA. To test this
1
3
notion we synthesized C-labeled 2-hydroxy- and 2-methoxy-
malonyl-N-acetylcysteamine (SNAC) thioester as cell-permeable
analogues of the corresponding CoA thioesters acceptable to type
I PKSs in vivo and in vitro (Scheme 1). Feeding of 2-meth-
oxymalonyl-SNAC (>98% 13C, 150 mg/L) to eight 100-mL cultures
of A. pretiosum ATCC 31565 in the presence of the side-chain
precursor isobutyrate gave 9 mg of purified ansamitocin P-3 which
by NMR and mass spectrometry showed no isotope enrichment
13
14
Comparison with the sequences of putative or proven biosynthetic
gene clusters of three other antibiotics containing “glycolate”
extender units9
a,11,16
revealed sets of homologous genes (Figure 2).
over natural abundance. In a second experiment, 2-hydroxymalonyl-
SNAC (89% 13C, 50 mg/L) and 2-methoxymalonyl-SNAC (50 mg/
Notably, each contained a small open reading frame (ORF)
predicted to encode an acyl or peptidyl carrier protein (ACP) which
could serve as an alternate carrier of the hydroxy- or methoxy-
malonate moiety after activation by transfer of the pantetheinyl
moiety of CoA.17 To determine whether this ACP is involved in
ansamitocin formation, we constructed a mutant of A. pretiosum
in which the asm14 gene was inactivated by inserting a 100-bp
DNA fragment into the central coding region. The mutant failed
to produce ansamitocin or accumulate any new compounds. This
demonstrates that asm14 is indeed essential for ansamitocin
formation.
L) were each fed to two 50-mL cultures. ES-MS analysis of the
resulting ansamitocin P-3 again showed no enrichment. We
therefore conclude that the third chain extension step in ansamitocin
biosynthesis does not use the CoA thioester of hydroxymalonate
or methoxymalonate as substrate, although it may involve a different
thioester of one of these acyl groups.
Recently, we have cloned and sequenced a gene cluster from A.
pretiosum which encodes all the biosynthetic machinery necessary
for the formation of ansamitocin.15 A set of five genes, asm13-
17, which appear to form a transcription operon, were considered
The asm13-17 subcluster and a related subcluster, fkbG-K, from
the FK520 biosynthetic gene cluster of Streptomyces hygroscopi-
cus both contain two dehydrogenase genes, one (asm13/fkbK)
homologous to â-hydroxyacyl-CoA dehydrogenases and the other
to be involved in the formation of either the ansamitocin ester side
chain or the substrate for the unusual chain extension step.
1
6
*
To whom correspondence may be sent. E-mail: floss@chem.washington.edu.
4
176 J. AM. CHEM. SOC. 2002, 124, 4176-4177
9
10.1021/ja0124764 CCC: $22.00 © 2002 American Chemical Society

C O MMU N I C A T I O N S
that asm16 may play a role in activating a precursor molecule,
perhaps loading an acyl compound onto the ACP encoded by asm14
for ansamitocin biosynthesis. It also raises the possibility that
O-methylation catalyzed by Asm17 may occur early in the
elaboration of this unusual chain extension substrate.
Acknowledgment. This work was supported by NIH research
Grant CA 76461 and by a postdoctoral fellowship from the
Deutscher Akademischer Austauschdienst (to S.T.).
Figure 2. Organization of genes putatively involved in the formation of
the novel hydroxylated 2-carbon extender unit in four biosynthetic gene
clusters: asm (AF453501), ansamitocin from A. pretiosum; fkb (AF235504),
FK520 from S. hygroscopicus; sor (U24241), soraphen from Polyangium
cellulosum; zma (AF155831), zwittermicin from Bacillus cereus. The
abbreviations of gene or domain homologues are as follows: BADH,
Supporting Information Available: Experimental procedures for
the synthesis of 2-hydroxymalonyl-SNAC and 2-methoxymalonyl-
SNAC and description of the construction of the asm14 and asm15
mutants of A. pretiosum (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
3
-hydroxyacyl-CoA dehydrogenase; ACP, acyl or peptidyl carrier protein;
ADH, acyl-CoA dehydrogenase; MT, O-methyltransferase; AT, acyltrans-
ferase. Note that the zma cluster has only been partially sequenced.
References
(
(
(
1) Higashide, E.; Asai, M.; Ootsu, K.; Tanida, S.; Kozai, Y.; Hasegawa, T.;
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(
asm15/fkbI) to acyl-CoA dehydrogenases, as well as a methyl-
transferase gene (asm17/fkbG) and an ORF (asm16/fkbH) of
unknown function (Figure 2). This led to the suggestion that these
five genes control the formation of 2-methoxymalonyl-ACP, the
proposed substrate for the “glycolate” extender unit, from glycolytic
intermediates via an ACP-bound glycerate.16 To probe the role of
these genes, we inactivated asm15 in the A. pretiosum genome by
inserting an apramycin resistance gene, aac(3)IV. The mutant did
not produce any ansamitocin P-3, even when supplemented with
isobutyrate, indicating that asm15 is required for formation of the
ansamitocin backbone rather than the ester side chain. Feeding with
3) (a) Komoda, Y.; Kishi, T. In Anticancer Agents Based on Natural Product
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-hydroxymalonyl-SNAC or 2-methoxymalonyl-SNAC did not
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Kaplan, L. DeV. Ind. Microbiol. 1993, 32, 29-45.
restore ansamitocin formation. Analysis of the fermentation of the
asm15 mutant revealed the presence of a small amount of a new
compound, not detected in the wild-type fermentation, which was
shown to have the structure of 10-desmethoxy-ansamitocin P-3
(
9) (a) Schupp. T.; Toupet, C.; Cluzel, B.; Neff, S.; Hill, S.; Beck, J. J.; Ligon,
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10) Ono, M.; Sakuda, S.; Ikeda, H.; Furihata, K.; Nakayama, J.; Suzuki, A.;
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Figure 1).1
8,19
(11) Stohl, E. A.; Milner, J. L.; Handelsman, J. Gene 1999, 237, 403-411.
(
Evidently, this compound results from the, albeit
(
(
12) Bindseil, K. U.; Zeeck, A. Liebigs Ann. Chem. 1994, 305-312.
13) (a) Yue, S.; Duncan, J. S.; Yamamoto, Y.; Hutchinson, C. R. J. Am. Chem.
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Soc. 1987, 109, 1255-1257.
inefficient, incorporation of a malonate unit in the absence of the
substrate for the “glycolate”extender unit. This clearly demonstrates
that asm15, and probably others of the asm13-17 genes, must be
involved in the synthesis of the unusual “glycolate” unit and its
delivery to the PKS. Interestingly, no 10-desmethoxy-ansamitocin
P-3 was detected in the fermentation of the asm14 mutant. This
suggests that the ACP encoded by asm14 is required for the aberrant
incorporation of a malonate unit in the third chain extension step,
for example, to maintain the PKS in a functionally competent
conformation or to actually deliver the malonate to the AT3 on the
PKS.
The above results indicate that the substrate for the unusual chain
extension reaction incorporating a “glycolate” unit into ansamitocin
and other antibiotics is most likely either 2-hydroxy- or 2-meth-
oxymalonyl-ACP rather than the corresponding CoA thioester. The
asm13-17 operon is probably responsible for the formation of this
substrate from an unidentified intermediate of carbohydrate me-
tabolism. It is noteworthy that the homologues of asm16/fkbH
(
14) Pohl, N. L.; Gokhale, R. S.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc.
1998, 120, 11206-11207.
15) Yu, T.-W.; Bai, L.; Clade, D.; Hoffmann, D.; Toelzer, S.; Trinh, K. Q.;
Xu, J.; Moss, S.; Leistner, E.; Floss, H. G. Proc. Natl. Acad. Sci. U.S.A.
In press.
(
(16) Wu, K.; Chung, L.; Revill, W. P.; Katz, L.; Reeves, C. D. Gene 251,
81-90.
(
17) Lambalot, R. H.; Gehring, A. M.; Flugel, R. S.; Zuber, P.; LaCelle, M.;
Marahiel, M. A.; Khosla, C.; Walsh, C. T. Chem. Biol. 1996, 3, 923-
9
36.
(
18) 10-Desmethoxy-ansamitocin: 1H NMR (499 MHz, CD
3
OD), δ (ppm) 0.90
), 1.23 (3H, d, 6.5 Hz, 2′-CH ), 1.41
), 1.52 (1H, bdd, 13.0 Hz, H-8b), 1.55 (1H, m, H-6), 1.70
3H, bs, 14-CH ), 1.71 (1H, dd, 14.0, 3.0 Hz, H-8a), 2.15 (1H, m, 14.0,
.0 Hz, H-2b), 2.20 (1H, m, 11.0 Hz, H-10b), 2.65 (1H, m, 14.0 Hz,
H-2a), 2.68 (1H, m, 8.5, 4.0 Hz, H-10a), 2.74 (1H, m, H-2′), 2.82 (1H,
bd, 9.5 Hz, H-5), 3.14 (3H, s, NCH ), 3.3 (1H, obsc., H-15b), 3.5 (1H,
obsc., H-15a), 3.98 (3H, s, OCH ), 4.19 (1H, bt, 11.0 Hz, H-7), 4.70 (1H,
dd, 12.0, 2.5 Hz, H-3), 5.67 (1H, ddd, 15.0, 10.5, 4.5 Hz, H-11), 6.18
1H, bd, 10.5 Hz, H-13), 6.40 (1H, dd, 15.0, 11.0 Hz, H-12), 6.97 (1H,
(
(
(
3H, s, 4-CH
3
), 1.22 (3H, d, 2′-CH
3
3
3
3H, bs, 6-CH
3
2
3
3
(
13
s, H-21), 7.14 (1H, s, H-17). C NMR (125.7 MHz, CD
3
OD), δ (ppm)
), 20.9 (2′-CH ),
), 39.5 (c-8), 39.8 (C-6), 47.0 (C-
), 62.1 (C-4), 68.2 (C-5), 76.3 (C-7), 78.1
12.8 (4-CH
3
1
3
), 15.0 (6-CH
3.9 (C-3), 35.1 (C-2′), 36.3 (N-CH
0), 47.5 (C-15), 57.4 (O-CH
3
), 15.9 (14-CH
3
), 18.6 (2′-CH
3
3
3
(
unknown) and asm17/fkbG (methyltransferase) are not present in
3
the putative hydroxy/methoxymalonate subcluster of the soraphen
biosynthetic gene cluster. Instead, a gene, sorC, encoding a three-
domain protein encompassing an acyltransferase, an ACP, and a
methyltransferase, has been identified immediately upstream of sorD
(C-3), 81.2 (C-9), 115.0 (C-21), 120.0 (C-19), 123.1 (C-17), 126.8 (C-
1
1
3), 128.1 (C-11), 132.8 (C-12), 138.6 (C-14), 143. 2 (C-18), 143.3 (C-
6), 155.7 (OCONH), 157.8 (C-20), 171.6 (C-1), 177.9 (C-1′).
(
19) An unusual feature of the ansamitocin structure is the location of the double
bonds at ∆11, 12, and ∆13, 14 instead of ∆10, 11, and ∆12, 14 where
normal PKS processing would place them. This implies that a double
bond shift must occur during the biosynthesis. The structure of the product
from the asm15 mutant indicates that this double bond shift is not
dependent on the presence of the 10-methoxy group
(
Figure 2). Such AT/ACP pairs combined with other genes have
recently been shown to be involved in the activation and modifica-
tion of amino acids,2 suggesting that sorC may be involved in
activating and methylating a precursor of the hydroxy/methoxy-
malonate moiety, which is then converted into methoxymalonyl-
ACP by the action of the sorDEX gene products. This suggests
0,21
(
20) Chen, H.; Thomas, M. G.; O’Connor, S. E.; Hubbard, B. K.; Burkart, M.
D.; Walsh, C. T. Biochemistry 2001, 40, 11651-11659.
(
21) Chen, H.; Walsh, C. T. Chem. Biol. 2001, 8, 301-312.
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J. AM. CHEM. SOC.
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