Divergent mechanistic routes for the formation of gem-dimethyl groups in the biosynthesis of complex polyketides

Article abstract of DOI:10.1002/anie.201410124

The gem-dimethyl groups in polyketide-derived natural products add steric bulk, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In

Full text of DOI:10.1002/anie.201410124

Angewandte
Chemie
DOI: 10.1002/anie.201410124
Biosynthesis
Divergent Mechanistic Routes for the Formation of gem-Dimethyl
Groups in the Biosynthesis of Complex Polyketides**
Sean Poust, Ryan M. Phelan, Kai Deng, Leonard Katz, Christopher J. Petzold,* and
Jay D. Keasling*
Abstract: The gem-dimethyl groups in polyketide-derived
natural products add steric bulk and, accordingly, lend
increased stability to medicinal compounds, however, our
ability to rationally incorporate this functional group in
modified natural products is limited. In order to characterize
the mechanism of gem-dimethyl group formation, with a goal
toward engineering of novel compounds containing this
moiety, the gem-dimethyl group producing polyketide synthase
ketide products. Regiospecific methylation of polyketide
products using organic semisynthesis is challenging. To
enable biobased approaches to form new gem-dimethyl-
containing pharmaceutical agents, and to clarify discrepancies
with the current mechanistic understanding, we sought to
determine the reaction mechanism of PKS-based C-methyl-
[2]
ation.
Type I polyketide synthases catalyze Claisen condensa-
tions and tailoring reactions in an assembly line fashion to
elaborate a remarkably diverse collection of secondary
metabolites, many of which have medicinal and industrial
applications. Polyketide chains are extended by sequential
homologation reactions between ketosynthase (KS)-bound
thioesters and a-carboxy building blocks (traditionally
malonyl- or methylmalonyl-acyl carrier protein (ACP)) to
form b-keto-polyketides. Tailoring reactions, such as the
reduction of the b-keto group on the growing polyketide
chain, and the subsequent sulfonation or O-methylation of
the corresponding b-hydroxy group, customarily follow con-
densation. Incorporation of methyl or gem-dimethyl groups
by methyltransferase (MT) containing PKS modules is
(
PKS) modules of yersiniabactin and epothilone were charac-
terized using mass spectrometry. The work demonstrated,
contrary to the canonical understanding of reaction order in
PKSs, that methylation can precede condensation in gem-
dimethyl group producing PKS modules. Experiments showed
that both PKSs are able to use dimethylmalonyl acyl carrier
protein (ACP) as an extender unit. Interestingly, for epothilone
module 8, use of dimethylmalonyl-ACP appeared to be the sole
route to form a gem-dimethylated product, while the yersinia-
bactin PKS could methylate before or after ketosynthase
condensation.
A
pproximately 10% of all approved drugs contain a geminal
[
2,3]
dimethyl group. The introduction of this group into com-
pounds can decrease their rates of chemical and metabolic
thought to follow this biosynthetic paradigm.
The currently accepted view of PKS-based reactions is
that KS-mediated condensation precedes all subsequent
events that take place within the module, including mono-
or dimethylation of the b-ketoacyl-ACP when an MT domain
[1]
degradation, thus improving the efficacy of drugs. Con-
sequently, there is great potential utility for the regiospecific
incorporation of gem-dimethyl groups in engineered poly-
[
2]
is present (Scheme 1B, Route 1). However, another route is
possible in which methylation precedes the condensation
[
*] S. Poust, Prof. Dr. J. D. Keasling
Department of Chemical and Biomolecular Engineering
University of California—Berkeley
Berkeley, CA 94270 (USA)
(
Scheme 1B, Route 2). A pK -based argument supports
a
Route 1, as the pK of a b-ketoacyl-ACP intermediate of
a
Route 1 is about two units lower than the analogous malonyl-
E-mail: keasling@berkeley.edu
[4]
ACP of Route 2 and therefore more easily deprotonated. In
Dr. R. M. Phelan, Dr. K. Deng, Dr. C. J. Petzold,
Prof. Dr. J. D. Keasling
Joint BioEnergy Institute, Lawrence Berkeley National Lab
further support of the proposed Route 1, attack of the enolate
on the upstream acyl-KS should occur more readily with the
less sterically hindered acetyl enolate, than the more sterically
hindered isobutyryl enolate of Route 2. On the other hand, if
methylation precedes condensation as in Route 2, the
observed production of isobutyryl-ACP in the yersiniabactin
PKS (see below) is rationalized by a similar argument that
malonyl-ACP is significantly easier to deprotonate than
5885 Hollis Street, Emeryville, CA 94608 (USA)
Dr. L. Katz, Prof. Dr. J. D. Keasling
Synthetic Biology Engineering Research Center
5885 Hollis Street, Emeryville, CA 94608 (USA)
[
**] The authors would like to thank Isu Yoon for assistance with protein
purification and Satoshi Yuzawa for helpful discussions. This work
was supported by the Joint BioEnergy Institute, which is funded by
the Office of Science, Office of Biological and Environmental
Research of the U.S. Department of Energy (Contract No. DE-AC02-
acetyl-ACP (approximate ten unit difference in pK ) and
a
decarboxylation of dimethylmalonyl-CoA would be facili-
tated by the presence of electron-donating methyl groups.
There is precedent for the involvement of dimethyl-
malonyl moieties in biochemical reactions. When provided
in vitro to carboxymethylproline synthase (CarB), a member
of the crotonase superfamily, a dimethylmalonyl moiety has
been shown to be a source of a nucleophilic enolate for CÀC
0
0
5CH11231), by the National Science Foundation (award No. EEC-
540879 to the Synthetic Biology Research Center), by the Depart-
ment of Energy, ARPA-E Electrofuels Program (Contract No. DE-
000206-1577), and by the National Science Foundation Graduate
0
Research Fellowship Program (Grant No. DGE 1106400, to SP).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410124.
[
5]
bond-forming reactions, despite its steric bulk. Dimethyl-
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
ipates in the decarboxylative homologation reaction. Epothi-
lone PKS module 8 (EpoM8) forms a similar gem-dimethyl
group based on the final structure of epothilone, but this
module has not been previously studied biochemically. The
AT domain of EpoM8 is bioinformatically predicted to prefer
[11]
methylmalonyl-CoA over malonyl-CoA.
This prediction
was verified biochemically in a competition assay, though
a limited amount of malonyl-ACP was formed when EpoM8
was incubated with malonyl-CoA in the absence of methyl-
malonyl-CoA (Figure SI-2.1 in the Supporting Information).
Thus, the MT domain in the module is predicted to naturally
carry out only a single C methylation. Despite these initial
studies, the exact mechanism and biosynthetic timing that
PKSs employ to produce gem-dimethyl groups remains
unclear.
In order to test if Route 2 is used, we performed in vitro
chain elongation reactions with dimethylmalonyl-ACP, gen-
erated through directed acylation of the apo-ACP domain
using two gem-dimethyl group producing PKS modules:
yersiniabactin and EpoM8. In addition, we sought to under-
stand if the KS domains of these PKSs demonstrated
a preference for the methylation state of the ACP-bound
[
10]
substrate, as reported by Mazur and co-workers. Determi-
nation of these parameters would provide a clearer picture of
which path(s) are operative in the production of this unusual
group. Accordingly, we characterized these gem-dimethyl
group producing PKS clusters using a well-established
tandem mass spectrometry method that can detect inter-
mediates covalently attached to ACP domains in a single-
turnover in vitro assay.
Scheme 1. A) Representative gem-dimethyl group containing com-
pounds. B) Potential mechanistic routes for the methylation and
condensation in gem-dimethyl group producing polyketide synthase
domains. Route 1: KS-catalyzed condensation precedes methylation.
Route 2: Methylation precedes KS condensation. Acetyl-ACP and
isobutyryl-ACP represent potential side products of unproductive
decarboxylation (shown in gray). AT=acyltransferase; KS=ketosyn-
thase; MT=methyltransferase. R represents the acyl chain transferred
from the previous module.
[
12]
The yersiniabactin and EpoM8 PKSs were expressed in
[13]
Escherichia coli strains BLR and BAP1, respectively. These
proteins were purified using Ni-NTA (nickel nitrilotriacetate)
chromatography. The purified yersiniabactin PKS was
approximately 70% holo form, while the EpoM8 was only
the holo form. As both malonyl-CoA and SAM are present in
the expression strains, we analyzed the acylation state of each
PKS immediately after purification to determine if acylation
or methylation occurred during expression. Examination of
the freshly purified yersiniabactin PKS and EpoM8 ACPs
showed isobutyryl-ACP (a decarboxylation product of dime-
thylmalonyl-ACP) on EpoM8 (Figure 1A) and both dime-
thylmalonyl-ACP and isobutyryl-ACP appended to the
yersiniabactin PKS (Figure 1B). This observation shows that
at least the first half of Route 2 is viable for the yersiniabactin
PKS. Interestingly, these results suggest that EpoM8 MT is
able to methylate malonyl-ACP loaded during in vivo expres-
sion in E. coli, even though the preferred substrate for the
EpoM8 AT is methylmalonyl-ACP (which is not natively
present in E. coli). Furthermore, in vitro studies of the
yersiniabactin PKS showed that acetyl-ACP was not methy-
lated in the presence of SAM (Figure SI-2.2), contrary to
previous proposals, which suggested the methylation of
acetyl-ACP accounted for the production of isobutyryl-
malonyl groups are also naturally added to sugar residues in
[
6]
the biosynthesis of cervimycin, and have been proposed as
a source of isobutyryl groups in polyketide synthesis, but the
[7]
latter has not been conclusively demonstrated.
Examples of polyketides containing gem-dimethyl groups
include the potential anticancer agents epothilone, pederin,
and bryostatin, and the siderophore yersiniabactin (Sche-
[
8]
me 1A). Currently, the biochemical characterization of
gem-dimethyl group producing PKSs is limited to the
yersiniabactin PKS. Using an in vitro system, Miller and co-
[9]
workers showed that the yersiniabactin PKS used malonyl-
CoA and two S-adenosyl methionine (SAM) molecules to
produce its gem-dimethyl group containing extended product,
3
-(hydroxyphenylthiazolinylthiazolinyl) [HPTT]-b-keto-2,2-
dimethyl-ACP. They also established that the yersiniabactin
AT domain has a 500-fold kinetic preference for malonyl-
CoA over methylmalonyl-CoA. Additionally, Mazur and co-
[10]
workers
observed that the condensation reaction was
dependent on SAM and that isobutyryl-ACP was a major
side product of the reaction; approximately 75% of the ACP
domains of the PKS were occupied by an isobutyryl moiety
following the reaction. The observed dependence of the
[
10]
ACP. These facts strongly suggest that isobutyryl-ACP is
derived from dimethylmalonyl-ACP and implies that Route 2
contributes in the overall formation of a gem-dimethyl group.
To unambiguously determine if the dimethylmalonyl
moiety loaded on the yersiniabactin and EpoM8 PKSs can
[
10]
condensation reaction on SAM could be explained by the
MT domain generating dimethylmalonyl-ACP, which partic-
2
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Figure 2. Dimethylmalonyl-ACP is extended by the EpoM8 and yersi-
niabactin PKS. A) Experiment overview: dimethylmalonyl-ACP was
formed using Sfp, dimethylmalonyl-CoA, and apo-PKS. Then, sub-
strates to form acyl-KS were added to generate extended products
(experimental details in the Supporting Information). Extracted ion
chromatogram (XIC) peak areas for dimethylmalonyl-ACP extension
experiments for B) EpoM8 and C) yersiniabactin PKS. For the yersinia-
bactin case, R represents the acyl chain transferred from the previous
NRPS module: a HPTT moiety. All XIC m/z transitions monitored in
this study are summarized in the Supporting Information.
Figure 1. Analysis of the acylation state of epothilone module 8 PKS
EpoM8 PKS) and yersiniabactin PKS after Ni-NTA purification. Reten-
(
tion times are shown on top of peaks. A) EpoM8 isobutyryl-ACP
chromatogram. B) Yersiniabactin PKS dimethylmalonyl-ACP chromato-
gram and isobutyryl-ACP chromatogram. All XIC m/z transitions
monitored in this study are summarized in the Supporting Informa-
tion. XIC=extracted ion chromatogram; cps=counts per second.
To determine if Route 1 is also operative for each PKS, we
attempted to form the b-keto unmethylated (yersiniabactin
PKS) or monomethylated (EpoM8 PKS) products by incu-
bating malonyl-CoA with acyl-KS, while omitting SAM and
subsequently adding SAM (Figure 3A). We then examined
products before and after SAM addition using LC-MS/MS.
be utilized by the KS domain in an in vitro extension reaction,
we expressed apo-PKS (expressed in E. coli strain BLR, this
resulted in approximately 100% apo form of the EpoM8). We
[5,6]
then synthesized authentic dimethylmalonyl-CoA
loaded the ACP domains with the dimethylmalonyl moiety
and
[
14]
[10]
using the phosphopantetheinyl transferase Sfp. Loading of
dimethylmalonyl-ACP was verified by LC-MS/MS (Fig-
ure SI-2.3). The acyl group to be extended was provided to
the appropriate KS domain by either a diketide-S-N-acetyl-
Contrary to the results of Mazur et al. the yersiniabactin KS
domain was found to use malonyl-ACP as a substrate for
condensation in the absence of SAM, and the MT domain
methylated the resulting b-keto acyl-ACP moiety after SAM
was added (Figure 3B). Small amounts of the unmethylated
and singly methylated yersiniabactin-derived PKS product
were also observed during reactions with the native malonyl-
CoA substrate in the presence of SAM (Figure SI-2.6).
EpoM8 was unable to extend methylmalonyl-ACP or
malonyl-ACP in the absence of SAM, but it was able to
form the dimethyated final product for both extender units in
the presence of SAM (Figure 4 and SI-2.7). This suggests that
EpoM8 methylates methylmalonyl-ACP and then uses dime-
thylmalonyl-ACP as the substrate in the condensation
reaction. Additionally, EpoM8 did not form the singly
methylated intermediate (i.e. an intermediate resulting
solely from the extension of methylmalonyl-ACP with no
action by the MT) when full reconstitution reactions were
performed in the presence of methylmalonyl-CoA and SAM.
Together, these findings demonstrate that in vitro, the yersi-
niabactin PKS is able to employ both pathways in Scheme 1,
while EpoM8 is restricted to using solely Route 2.
[
15]
cysteamine substrate, the acyl group of which mimics the
structure of the native intermediate in the case of EpoM8, or
by reconstitution of the upstream nonribosomal peptide
synthetase (NRPS) enzymes in the case of the yersiniabactin
PKS. As shown in Figure 2, both EpoM8 and yersiniabactin
extended dimethylmalonyl-ACP in the absence of SAM to
produce the correct fully extended and methylated product
with retention times matching those observed during fully
reconstituted in vitro reactions (Figure SI-2.4). Additionally,
the side product isobutyryl-ACP was produced from dime-
thylmalonyl-ACP by both enzymes (Figure SI-2.5). A large
amount of isobutyryl-ACP was produced by both enzymes
relative to the extended methylated product, suggesting that
Sfp loading experiments may not fully capture the precisely
orchestrated catalytic cycle of these enzymes. The formation
of acyl-KS also seems to have activated the KS for decarbox-
ylation of dimethylmalonyl-ACP to isobutyryl-ACP (Fig-
ure SI-2.5), potentially indicating a conformational shift in the
enzyme upon KS acylation.
We have clearly demonstrated that the yersiniabactin and
epothilone PKSs are capable of forming dimethylmalonyl-
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
[
16]
electron microscopy.
Future studies will employ cryo
electron microscopy to examine the spatial relationship
between MT with the ACP during the catalytic cycle to
determine the partition between Routes 1 and 2 for the
yersiniabactin PKS. Based on our results, we would suggest
that PKS engineering strategies to incorporate this group
using combinatorial biosynthesis should swap whole modules,
instead of swapping individual MT domains into modules
unaccustomed to a particular order of reactions. Ultimately,
an improved understanding of this remarkable reaction will
allow easier integration of this monomer unit in novel, useful
PKS-based compounds.
Figure 3. Characterization of Route 1 (KS-catalyzed condensation pre-
cedes methylation) for the yersiniabactin PKS. A) Experiment overview:
in part a, SAM was omitted, while malonyl-CoA and acyl-KS were
included to allow the formationof unmethylated extended-ACP. In
part b, SAM was added (experimental details in the Supporting
Information). B) Unmethylated extended-ACP extracted ion chromato-
gram (XIC) peak areas (gray) and methylated extended-ACP peak areas
Received: October 15, 2014
Revised: November 18, 2014
Published online: && &&, &&&&
Keywords: biosynthesis · dimethylmalonyl-ACP · methylation ·
.
polyketides · transferases
(
black). R is a HPTT moiety. All XIC m/z values monitored in this
study are summarized in the Supporting Information.
[
1] J. A. Burkhard, G. Wuitschik, M. Rogers-Evans, K. Mꢀller,
E. M. Carreira, Angew. Chem. Int. Ed. 2010, 49, 9052 – 9067;
Angew. Chem. 2010, 122, 9236 – 9251.
[
[
2] A. T. Keatinge-Clay, Nat. Prod. Rep. 2012, 29, 1050 – 1073.
3] a) M. A. Fischbach, C. T. Walsh, Chem. Rev. 2006, 106, 3468 –
3
4
496; b) C. Hertweck, Angew. Chem. Int. Ed. 2009, 48, 4688 –
716; Angew. Chem. 2009, 121, 4782 – 4811.
[
4] D. A. Evans, “pKa’s of CH bonds in Hydrocarbons and Carbonyl
Compounds ”, can be found under http://evans.rc.fas.harvard.
edu/pdf/evans_pKa_table.pdf, 2005.
[
5] E. T. Batchelar, R. B. Hamed, C. Ducho, T. D. W. Claridge, M. J.
Edelmann, B. Kessler, C. J. Schofield, Angew. Chem. Int. Ed.
2008, 47, 9322 – 9325; Angew. Chem. 2008, 120, 9462 – 9465.
[
6] T. Bretschneider, G. Zocher, M. Unger, K. Scherlach, T. Stehle,
C. Hertweck, Nat. Chem. Biol. 2012, 8, 154 – 161.
[
7] J. Young, D. C. Stevens, R. Carmichael, J. Tan, S. Rachid, C. N.
Boddy, R. Mꢀller, R. E. Taylor, J. Nat. Prod. 2013, 76, 2269 –
Figure 4. Characterization of Route 1 (KS catalyzed condensation pre-
cedes methylation) for the epothilone PKS. A) Experiment overview:
using methylmalonyl-CoA as an extender unit, SAM was omitted from
the overall reaction (no product formed), or SAM was included,
allowing the complete reaction to take place. B) Unmethylated
extended-ACP extracted ion chromatogram (XIC) peak areas (gray) and
methylated extended-ACP peak areas (black). All XIC m/z values
monitored in this study are summarized in the Supporting Informa-
tion.
2276.
[
8] S. Anand, M. V. R. Prasad, G. Yadav, N. Kumar, J. Shehara,
M. Z. Ansari, D. Mohanty, Nucleic Acids Res. 2010, 38, W487 –
W496.
9] D. A. Miller, L. Luo, N. Hillson, T. A. Keating, C. T. Walsh,
Chem. Biol. 2002, 9, 333 – 344.
[
[
[
[
10] M. T. Mazur, C. T. Walsh, N. L. Kelleher, Biochemistry 2003, 42,
13393 – 13400.
11] B. Julien, S. Shah, R. Ziermann, R. Goldman, L. Katz, C. Khosla,
Gene 2000, 249, 153 – 160.
ACP and isobutyryl-ACP, its decarboxylated derivative, and,
contrary to the traditional understanding of methylation in
type I PKSs, that both enzymes can use dimethylmalonyl-
ACP as an extender unit in acyl chain elongation. For EpoM8,
formation of dimethylmalonyl-ACP through Route 2
appeared to be the only means to form a gem-dimethyl
group (at least in vitro), while the yersiniabactin PKS could
methylate before or after condensation. The increased
decarboxylation of dimethylmalonyl-ACP we observed
upon acylation of the KS suggests that conformational
changes in the enzyme are important during the catalytic
cycle of these gem-dimethyl group producing PKSs, as
recently demonstrated for pikromycin module 5 using cryo
12] P. C. Dorrestein, S. B. Bumpus, C. T. Calderone, S. Garneau-
Tsodikova, Z. D. Aron, P. D. Straight, R. Kolter, C. T. Walsh,
N. L. Kelleher, Biochemistry 2006, 45, 12756 – 12766.
13] B. A. Pfeifer, S. J. Admiraal, H. Gramajo, D. E. Cane, C. Khosla,
Science 2001, 291, 1790 – 1792.
14] L. E. N. Quadri, P. H. Weinreb, M. Lei, M. M. Nakano, P. Zuber,
C. T. Walsh, Biochemistry 1998, 37, 1585 – 1595.
[15] K. K. Sharma, C. N. Boddy, Bioorg. Med. Chem. Lett. 2007, 17,
034 – 3037.
[
[
3
[
16] J. R. Whicher, S. Dutta, D. A. Hansen, W. A. Hale, J. A.
Chemler, A. M. Dosey, A. R. H. Narayan, K. Hakansson,
D. H. Sherman, J. L. Smith, G. Skiniotis, Nature 2014, 510,
560 – 564.
4
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Biosynthesis
Order of events: In order to elucidate the
mechanism of gem-dimethyl group for-
mation in polyketides, the gem-dimethyl
group producing polyketide synthase
(PKS) modules of yersiniabactin and
epothilone were characterized using
mass spectrometry. The study demon-
strated, contrary to the canonical under-
standing of reaction order in PKSs, that
methylation can precede condensation in
PKS modules that produce gem-dimethyl
groups.
S. Poust, R. M. Phelan, K. Deng, L. Katz,
C. J. Petzold,*
J. D. Keasling*
&&& — &&&
Divergent Mechanistic Routes for the
Formation of gem-Dimethyl Groups in the
Biosynthesis of Complex Polyketides
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!