Interchenar retrotransfer of aureothin intermediates in an iterative polyketide synthase module

Article abstract of DOI:10.1021/ja304454r

The course of the enigmatic iterative use of a polyketide synthase module was deduced from targeted domain inactivation in the aureothin assembly line. Mutational analyses revealed that the N-terminus of AurA is not involved in the iteration process, ruli

Full text of DOI:10.1021/ja304454r

Communication
pubs.acs.org/JACS
Interchenar Retrotransfer of Aureothin Intermediates in an Iterative
Polyketide Synthase Module
Benjamin Busch, Nico Ueberschaar, Yuki Sugimoto, and Christian Hertweck*
Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstrasse 11a, 07745 Jena, Germany, and
Friedrich Schiller University, Jena, Germany
S
* Supporting Information
ABSTRACT: The course of the enigmatic iterative use of
a polyketide synthase module was deduced from targeted
domain inactivation in the aureothin assembly line.
Mutational analyses revealed that the N-terminus of
AurA is not involved in the iteration process, ruling out
an ACP−ACP shuttle. Furthermore, an AurA(KS°,
ACP°)−AurA(AT0) heterodimer proved to be nonfunc-
tional, whereas aureothin production was restored in a
ΔaurA mutant complemented with AurA(KS°)−AurA-
(ACP°). This finding supports a model according to which
the ACP-bound polyketide intermediate is transferred back
to the KS domain on the opposite PKS strand.
odular polyketide synthases are fascinating multifunc-
Mtional biocatalysts involved in the biosynthesis of many
pharmacologically relevant natural products. These giant
enzymes form a molecular processing line to assemble complex
polyketide metabolites from simple acyl and malonyl units.1−3
In general, a distinct set of catalytic domains forming one
module is utilized for one elongation and processing cycle
before the tethered intermediate is transferred to the
downstream module. The number of active domains in the
PKS typically corresponds with the number of required
biotransformation steps, which allows for the prediction of
the basic polyketide skeleton from the PKS architecture and
vice versa. However, there is a growing number of exceptions to
this rule of colinearity.4 Beside the skipping of individual
domains or entire modules, also the irregular reuse of a domain
or one module, referred to as ‘stuttering’, has been reported.5
However, only few modular systems were discovered where the
iterative usage of individual PKS modules is a programmed
event in the biosynthesis.4 First, direct evidence for the iterative
Figure 1. (A) Model aureothin biosynthesis involving an iterative type
I PKS module (AurA); (B) possible mechanisms for intrachenar and
interchenar retrotransfer of the ACP-bound intermediate (Ar = p-
nitrophenyl).
usage of one module was provided by the functional analyses of
the borrelidin (bor)6 and aureothin (aur)7,8 synthases (Figure
1A), which have been shown to catalyze three and two rounds
of Claisen condensations, respectively. Other rare examples of
iterative modules are known from stigmatellin,9 neoaureothin,10
neocarzilin,11 lankacidin,12 and DKxanthene13 PKSs, as well
engineered variants of picromycin14 and erythromycin15 PKSs.
Mechanistically, the iteration process poses a riddle because
modular PKSs form homodimers. Insights into the protein
structures and cross-linking experiments have revealed a double
helical model of modular PKSs where the functional domains of
the homodimer interact with each other.16,17 In analogy to fatty
acid biosynthesis, a polyketide chain is formed through the
interaction of both PKS strands. For the forward direction, the
ketosynthase (KS) domain of chain A transfers the growing
polyketide chain to the acyl carrier protein (ACP) domain of
chain B, and vice versa. The malonyl extender unit is selected
by the acyl transferase (AT) domain that can, in principle, load
the ACPs of both monomers.18 However, the mechanism of
Received: May 18, 2012
Published: July 16, 2012
© 2012 American Chemical Society
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chain transfer in an iteration event is fully unknown. In
principle, one may conceive the following scenarios (Figure
1B): the intermediate is transferred from the ACP domain of
one PKS chain to the (a) ACP domain on the same PKS chain
(‘intrachenar’); (b) ACP domain on the opposite PKS chain
(‘interchenar’); (c) KS domain upstream on the same PKS
chain (‘intrachenar’); or (d) KS domain upstream on the
opposite chain (‘interchenar’). Here, we address this issue by
mutational analysis of the PKS involved in the biosynthesis of
aureothin, an antiproliferative and antifungal metabolite of
Streptomyces thioluteus.7
chains, each lacking a critical PKS domain for chain propagation
(KS, AT, or ACP). Through statistical recombination of two
mutated monomers, three types of AurA dimers may form.
These are the two functionally inactive “homodimers” lacking
the minimal set of PKS domains, and one potentially active
“heterodimer” bearing the complete set of catalytically active
domains, yet distributed among two subunits (Figure 3).
Initially, we sought to monitor the intermediates generated
by the iterative module on AurA. For this purpose, we cloned
aurA and heterologously expressed it in Streptomyces lividans.
Through high-resolution Orbitrap MS (Exactive) analysis, we
monitored masses that correspond to polyketide intermediates
that are prematurely released occurring after one and two
rounds of elongations. To unequivocally determine their
structures, we synthesized authentic standards of the postulated
aureothin precursors 2 and 3 and compared their retention
time and mass spectra with the observed metabolites. Thus, the
proposed intermediates of AurA could be clearly identified
(Figure 2).
Figure 2. Detection of polyketide intermediates 2 and 3 in an S.
lividans culture heterologously expressing aurA. UV trace (top, 254
nm) and ion extraction chromatograms (Orbitrap, negative mode)
scanning for exact masses of 2 and 3, and comparison with synthetic
reference compounds.
Figure 3. Outline of possible nonfunctional “homodimers” and
potentially functional “heterodimers” of mutated AurA variants (top);
sketch of three-plasmid-system for the reconstitution of the aur
pathway and for mutagenesis of individual AurA subunits (bottom).
To warrant the native PKS function, we decided to employ
the entire aureothin processing line in vivo, exchanging only the
AurA part. An in vivo approach may be limited since one
cannot rule out cross-complementation of functional units.
However, various examples have shown that the function of
PKS components may be altered when taken out of the natural
context.2 As a necessity for conducting mutational analyses of
individual strands of the megasynthase we tested a flexible
multivector host system composed of an integrative plasmid for
For the systematic investigation of the mode of retrotransfer,
we next aimed at combining variants of the iterative aur PKS
module that are deficient in KS, AT, and ACP domains. As a
prerequisite for this, it has been verified earlier in modular PKS
that targeted mutations lead to a complete domain inactivation,
but do not affect the conformation or folding of the
protein.19,20 Thus, it should be viable to reconstitute the
iterative PKS module AurA from two mutated polypeptide
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the entire biosynthesis genes except aurA and two episomal
plasmids for aurA variants. A ΔaurA mutant was successfully
generated by PCR-targeting,10 and in order to rule out polar
effects resulting from gene deletion, the strain was comple-
mented through coexpression of aurA on a free-standing
plasmid (Figure 3).
scenarios (a) and (b) involving ACP−ACP retrotransfers.
This is intriguing since skipping in a modular PKS has been
shown to involve an ACP−ACP transfer.19 The decrease in
aureothin production (20% of wild type) may be due to
reduced solubility or change in protein folding of the mutated
AurA variant. As negative controls for the domain comple-
mentation experiments, we first individually coexpressed the
aurA(KS0) and aurA(ACP0) genes with the incomplete aur
gene cluster. As expected, the individual domain mutations led
to a complete abrogation of polyketide biosynthesis.21 First, we
explored the possibility of an intrachenar KS−ACP retro-
transfer by coexpressing aurA(KS°, ACP°) and aurA(AT0) in
the ΔaurA mutant. Since aureothin formation could not be
observed in the mutant broth, it appeared that homo- and
heterodimers of these AurA variants were not capable of
producing any polyketide intermediates. Next, to evaluate the
viability of a KS−ACP retrotransfer from one PKS chain to the
other (interchenar, scenario d), we co-expressed the aurA(KS0)
and aurA(ACP0) variants using the ΔaurA mutant. In this case,
aureothin production could be clearly detected. Although the
production rate was substantially lower than in the wild type,
this result is a clear indication for a successful complementation
of the inactivated PKS domains in an AurA(KS°)−AurA-
(ACP°) heterodimer. Aureothin production in this mutant
unequivocally shows that one subset of PKS domains is used
twice during the iteration event, and that the ACP-bound
intermediate is transferred back to the KS located on the
opposite PKS chain. The reduced production rate (15% of wild
type) may be rationalized by less efficient AurA, since only one
subset of domains can be used and the statistical probability of
heterodimer formation.
For the targeted inactivation of the PKS domains, fragments
of aurA were cloned and codons for critical amino acids were
individually exchanged for alanine codons by site-directed
mutagenesis. In the KS1 domain, the conserved cysteine in the
DTACSSS motif was chosen. For the ACP1 domain, we
targeted the conserved serine in the GFDSLR motif that serves
as an anchor for the phosphopantetheinyl residue, and in the
AT1 domain, the conserved serine in the GHSQG motif was
replaced. In this way, we generated four constructs coding for
AurA variants lacking one or two functional domains,
AurA(KS°), AurA(AT°), AurA(ACP°), and AurA(KS°,
ACP°). As to a potential ACP−ACP retrotransfer, we noted
a potential phosphopantetheine binding site upstream of KS1.
Because of the aberrant motif, however, we chose to truncate
the N-terminus of AurA, yielding Aur(N°), to exclude the
potential involvement of this region in the iteration process.
The plasmids harboring the mutated aurA variants were then
introduced into the expression host S. lividans and co-expressed
with the remaining aur biosynthesis genes, and metabolite
production was monitored by HPLC−MS (Figure 4). Since
aureothin biosynthesis was restored in the ΔaurA mutant
complemented with aurA(N°), we could readily exclude
In conclusion, through targeted domain mutagenesis, cross-
complementation experiments, and metabolic profiling, we
have addressed for the first time the course of iterative chain
elongation in a type I polyketide synthase. We found that the
N-terminus with tentative residual ACP function is not involved
in the iteration process. Furthermore, we constructed func-
tionally impaired modules that permit the exclusion of three of
four possible mechanisms for intermediate retro-transfer.
According to our data, the most likely scenario is (d), a
retrotransfer of the biosynthetic intermediate from the ACP
onto the KS domain located on the opposite polypeptide
strand. This finding is not only of significance for under-
standing the mode of action of the large family of related
bacterial multimodular megasynthases, but may also serve as
model for the widely distributed fungal iterative type I PKSs.
Future biochemical studies will help unraveling the exact
mechanism of iteration.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental and analytical procedures, synthesis of reference
compounds, additional HPLC profiles, description of mutant
constructions and heterologous expression, domain analysis of
N-terminal part of AurA. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
Christian.Hertweck@hki-jena.de
Notes
Figure 4. Key results of mutational analyses. HPLC/MS (SIM)
profiles of ΔaurA mutant and complemented mutants.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
Financial support by the DFG is gratefully acknowledged. We
thank Marianne Quaas for invaluable help in cloning and
fermenting.
REFERENCES
■
(1) Meier, J. L.; Burkart, M. D. Chem. Rev. 2009, 38, 2012−2045.
(2) Hertweck, C. Angew. Chem., Int. Ed. 2009, 48, 4688−4716.
(3) Khosla, C.; Kapur, S.; Cane, D. E. Curr. Opin. Chem. Biol. 2009,
13, 135−143.
(4) Moss, S. J.; Martin, C. J.; Wilkinson, B. Nat. Prod. Rep. 2004, 21,
575−593.
(5) Wilkinson, B.; Foster, G.; Rudd, B. A. M.; Taylor, N. L.; Blackaby,
A. P.; Sidebottom, P. J.; Cooper, D. J.; Dawson, M. J.; Buss, A. D.;
Gaisser, S.; Bohm, I. U.; Rowe, C. J.; Cortes
Staunton, J. Chem. Biol. 2000, 7, 111−117.
́
, J.; Leadlay, P. F.;
(6) Olano, C.; Moss, S. J.; Brana, A. F.; Sheridan, R.; Math, V.;
Weston, A. J.; Mendez, C.; Leadlay, P. F.; Wilkinson, B.; Sala, F. Mol.
Microbiol. 2004, 52, 1745−1756.
(7) He, J.; Hertweck, C. Chem. Biol. 2003, 10, 1225−1232.
(8) He, J.; Hertweck, C. ChemBioChem 2005, 6, 908−912.
(9) Gaitatzis, N.; Silakowski, B.; Kunze, B.; Nordsiek, G.; Bloker, H.;
̈
Hofle, G.; Muller, R. J. Biol. Chem. 2002, 277, 13082−13090.
̈
̈
(10) Traitcheva, N.; Jenke-Kodama, H.; He, J.; Dittmann, E.;
Hertweck, C. ChemBioChem 2007, 8, 1841−1849.
(11) Otsuka, M.; Ichinose, K.; Fujii, I.; Ebizuka, Y. Antimicrob. Agents
Chemother. 2004, 48, 3468−3476.
(12) Tatsuno, S.; Arakawa, K.; Kinashi, H. J. Antibiot. 2007, 60, 700−
708.
(13) Meiser, P.; Weissman, K. J.; Bode, H. B.; Krug, D.; Dikschat, J.;
Sandmann, A.; Muller, R. Chem. Biol. 2008, 15, 771−781.
̈
(14) Beck, B. J.; Aldrich, C. C.; Fecik, R. A.; Reynolds, K. A.;
Sherman, D. H. J. Am. Chem. Soc. 2003, 125, 4682−4683.
(15) Kapur, S.; Lowry, B.; Yuzawa, S.; Kenthirapalan, S.; Chen, A. Y.;
Cane, D. E.; Khosla, C. Proc. Nat. Acad. Sci. U.S.A. 2012, 109, 4110−
4115.
(16) Staunton, J.; Caffrey, P.; Aparicio, J. F.; Roberts, G. A.; Bethell,
S. S.; Leadlay, P. F. Nat. Struct. Biol. 1996, 3, 188−192.
(17) Staunton, J.; Weissman, K. J. Nat. Prod. Rep. 2001, 18, 380−416.
(18) Gokhale, R. S.; Lau, J.; Cane, D. E.; Khosla, C. Biochemistry
1998, 37, 2524−2528.
(19) Thomas, I.; Martin, C. J.; Wilkinson, C. J.; Staunton, J.; Leadlay,
P. F. Chem. Biol. 2002, 9, 781−787.
(20) Beck, B. J.; Yoon, Y. J.; Reynolds, K. A.; Sherman, D. H. Chem.
Biol. 2002, 9, 575−583.
(21) When coexpressing aurA(AT0), in some cases traces of
aureothin could be detected by HPLC−MS (SIM). This surprising
non-zero aureothin production could be rationalized by an alternative
ACP-loading, possibly by the AT of the fatty acid synthase complex;
see: Wesener, S. R.; Potharla, V. Y.; Cheng, Y. Q. Appl. Environ.
Microbiol. 2011, 77, 1501−1507. KS°/AT° or AT°/ACP°
heterodimers could not produce unambiguous results because of
various possible avenues for intermediate transfer.
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