Understanding Programming of Fungal Iterative Polyketide Synthases: The Biochemical Basis for Regioselectivity by the Methyltransferase Domain in the Lovastatin Megasynthase

Article abstract of DOI:10.1021/jacs.5b11814

Highly reducing polyketide synthases (HR-PKSs) from fungi synthesize complex natural products using a single set of domains in a highly programmed, iterative fashion. The most enigmatic feature of HR-PKSs is how tailoring domains function selectively during different iterations of chain elongation to afford structural diversity. Using the lovastatin nonaketide synthase LovB as a model system and a variety of acyl substrates, we characterized the substrate specificity of the LovB methyltransferase (MT) domain. We showed that, while the MT domain displays methylation activity toward different β-ketoacyl groups, it is exceptionally selective toward its naturally programmed β-keto-dienyltetraketide substrate with respect to both chain length and functionalization. Accompanying characterization of the ketoreductase (KR) domain displays broader substrate specificity toward different β-ketoacyl groups. Our studies indicate that selective modifications by tailoring domains, such as the MTs, are achieved by higher kinetic efficiency on a particular substrate relative to the rate of transformation by other competing domains.

Full text of DOI:10.1021/jacs.5b11814

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Communication
Understanding Programming of Fungal Iterative Polyketide
Synthases: the Biochemical Basis for Regioselectivity by the
Methyltransferase Domain in the Lovastatin Megasynthase
Ralph A. Cacho, Justin Thuss, Wei Xu, Randy Sanichar,
Zhizeng Gao, Allison Nguyen, John C. Vederas, and Yi Tang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b11814 • Publication Date (Web): 02 Dec 2015
Downloaded from http://pubs.acs.org on December 9, 2015
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Understanding Programming of Fungal Iterative Polyketide Syn-
thases: the Biochemical Basis for Regioselectivity by the Methyl-
transferase Domain in the Lovastatin Megasynthase
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‡
‡
Ralph A. Cacho,1 Justin Thuss,2 Wei Xu,1 Randy Sanichar,2 Zhizeng Gao,2 Allison Nguyen,1 John C. Vederas,2
Yi Tang1
1Department of Chemical and Biomolecular Engineering, Department of Chemistry and Biochemistry, University of California, Los
Angeles, CA 90095, USA; 2 Department of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2
Supporting Information Placeholder
PKSs remain unresolved. Knowledge of how tailoring domains
function will enable both rational manipulation of the megasyn-
thases,8,9 and product prediction from the vast number of HR-PKSs
uncovered through genome sequencing efforts.
ABSTRACT: Highly-reducing polyketide synthases (HR-PKSs)
from fungi synthesize complex natural products using a single set of
domains in a highly programmed, iterative fashion. The most en-
igmatic feature of HR-PKSs is how tailoring domains function se-
lectively during different iterations of chain elongation to afford
structural diversity. Using the lovastatin nonaketide synthase LovB
as a model system and a variety of acyl substrates, we characterized
the substrate specificity of the LovB methyltransferase (MT) do-
main. We showed that while the MT domain displays methylation
activity toward different -ketoacyl groups, it is exceptionally selec-
tive towards its naturally programmed -keto-dienyltetraketide
substrate with respect to both chain length and functionalization.
Accompanying characterization of the ketoreductase (KR) domain
displays broader substrate specificity towards different -ketoacyl
groups. Our studies indicate that selective modifications by tailor-
ing domains, such as the MTs, are achieved by higher kinetic effi-
ciency on a particular substrate relative to the rate of transfor-
mation by other competing domains.
Fungal highly-reducing polyketide synthases (HR-PKSs) are
multidomain megasynthases that are involved in the biosynthesis of
diverse polyketide natural products, highlighted by the cholesterol
lowering agent lovastatin and the protein transport inhibitor bre-
feldin A.1,2 HR-PKSs contain a linearly juxtaposed set of domains
that iteratively build the polyketide chain through decarboxylative
condensation and -ketoacyl functionalization. In each HR-PKS, a
single set of domains is repeatedly and permutatively used through
chain elongation cycles to yield the final product. These pro-
grammed tailoring steps are precisely executed by the HR-PKSs to
afford richly functionalized polyketide chains that set up post-PKS
modifications and afford diverse biological activities. For example,
during the synthesis of dihydromonacolin L (DML), the precursor
to lovastatin, the lovastatin nonaketide synthase LovB performs
eight cycles of chain extension and tailoring (Figure 1).3,4 The or-
chestration of different tailoring activities sets up key structural
features in DML, including the decalin core that is proposed to
derive from a triene hexaketide intermediate through Diels-Alder
cyclization;5 and the terminal -hydroxy acid moiety that is im-
portant for inhibition of HMG-CoA reductase.6 Compared to
bacterial counterparts that function in a well-understood assembly-
line like fashion,7 these complex biochemical features of fungal HR-
Figure 1. The programed steps of LovB in the synthesis of dihy-
dromonacolin L (DML). (A) The catalytic steps by LovB; LovB is a
HR-PKS and LovC is the dissociated enoylreductase; and (B) the
tetraketide modification steps shown in detail highlighting the timing
of the MT domain. Domain abbreviations: ketosynthase (KS); malo-
nyl-CoA:ACP acyltransferase (MAT); -methyltransferase (MT), -
ketoreductase (KR), dehydratase (DH),  enoylreductase (ER),
acyl carrier protein (ACP) and NRPS-like Condensation (CON).
The -methylation of -ketoacyl-S-ACP intermediate is a com-
monly observed modification during selected cycles of HR-
PKSs.1,10-12 The reaction is catalyzed by an in-line methyltransferase
(MT) domain using S-adenosylmethionine (SAM) as a cofactor
immediately following ketosynthase (KS)-catalyzed chain elonga-
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tion, and occurs prior to -reductive modifications performed by
between 0.01 and 1 M.4 To allow for quantification and prevent
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ketoreductase (KR), dehydratase (DH) and enoylreductase (ER)
domains. During the eight chain elongation and tailoring iterations
catalyzed by LovB, the MT domain is apparently only active during
the conversion of tetraketide 2-ACP to the on-pathway intermedi-
ate 6-ACP, with the methyl 3-ACP being the product of the MT.
Curiously, no methylation modification occurs on other -
ketoacyl-S-ACP substrates in the other catalytic cycles of LovB
(Figure 1). However, -methylation of the tetraketide is essential
for the remaining steps of the pathway shown in Figure 1A, as the
dissociated ER LovC is unable to recognize the -desmethyl ver-
sion of 5-ACP and the entire catalytic cascade subsequently de-
rails.3,13 The importance of methylation modification on the fideli-
ty of other iterative HR-PKSs has also been observed, in which
bypassing programmed MT function results in production of shunt
products.14 Therefore, the HR-PKSs have clearly evolved to opti-
mize the timing and regioselectivities of the MT domains.
further tailoring reactions of the KR products in the assay, we con-
structed a point mutation H985A in the DH domain of LovB to
yield LovB-DHo (Figure S1).19 LC-MS based product quantifica-
tion was employed for both the methylation (containing SAM) and
ketoreduction (containing NADPH) assays, using standard curves
constructed from the mass signals of synthesized standards.
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We hypothesize that two possible mechanisms of substrate pro-
cessing can account for LovB MT selectivity. First, the HR-PKS
may adopt an assembly-line like model in which each substrate is
passed through the way stations sequentially in the order of MT-
>KR->DH->ER. In the case of LovB, the MT domain only recog-
nizes 2-ACP while excluding all of the other substrates completely.
Alternatively in a kinetically controlled mechanism, we propose
that once formed and released from the KS, the -ketoacyl-S-ACP
substrate can sample all potential modifying domains, including the
MT, KR and KS. The outcome of the tailoring steps is determined
by the relative activities of each domain towards the substrate. The
MT domain is primarily in competition with the KR domain for the
substrate: if a substrate is readily reduced by the KR domain first,
then no methyl transfer will be possible. Conversely, a higher MT
activity relative to the KR will lead to methylation prior to reduc-
tion. To understand the basis for the MT selectivity, individual
rates of the MT and KR domain towards the different -ketoacyl-S-
ACP substrates need to be measured and compared.
We synthesized a panel of acyl-S-N-acetylcysteamine (SNAC)
compounds as substrates for the MT and KR assays. The majority
of the -ketoacyl-SNAC compounds (Figure 2) were prepared
using titanium-catalyzed aldol chemistry to synthesize -hydroxy-
carbonyl species that were further functionalized and oxidized to
provide the desired -carbonyl SNAC esters (Supporting Infor-
mation).15,16 Access to shorter, saturated SNAC esters was achieved
using acylated Meldrum’s acid.17,18 The acyl portions of the sub-
strates vary in chain length from diketide (C4) to pentaketide (C10)
as well as functionalization. Compounds 7, 8 and 2 represent the
natural -ketoacyl intermediates in the LovB catalytic cycle, while
compounds 9-11 are model, simplified substrates. We also synthe-
sized the corresponding -methyl--ketoacyl products 12-17 as
standards for quantifying the methylation product amount (Sup-
porting Information). The synthetic strategy outlined above was
expanded to include the -methylated SNAC esters. Rapid keto-
enol interconversion excluded the need for stereoselective methyla-
tion (Supporting information). Furthermore, a number of -
hydroxylacyl-SNAC compounds were synthesized and used as
standards for the ketoreduction assay (Supporting Information).
These standards were conveniently obtained as intermediates in
the synthesis of compounds 2, 7-17. Intact LovB was expressed
and purified from Saccharomyces cerevisiae strain BJ5464-NpgA as
previously described and used in the assays at final concentrations
Figure 2. Full kinetic analysis of LovB MT domain towards different-
ketoacyl-SNAC substrates. 2 is the natural tetraketide substrate based
on DML structure. 8 is the on-pathway triketide substrate of LovB. No
reaction towards diketide 7 or pentaketide 11 was observed.
We first assayed the activity of the MT domain towards the natu-
ral tetraketide 2. Overnight incubation of 2 mM of 2 in the pres-
ence of SAM led to complete consumption of the substrate and the
appearance of 14. Michaelis-Menten saturation kinetics assay gave
a robust kcat of 196 min-1 and KM of 170 M (Figures 2 and S8).
The KM value was surprisingly low considering acyl-SNAC mimic
of the ACP-bound substrates can suffer from significant penalties in
KM due to loss of protein-protein interactions, and are typically in
the millimolar range.11,20,21 Hence the kinetic parameters of 2 sug-
gest that the natural tetraketide can bind exceptionally well to the
active site MT domain of LovB. Having demonstrated the MT
domain activity can be confirmed with 2, we then tested MT cataly-
sis towards -ketoacyl-SNAC substrates of varying chain lengths.
No significant (<1%) methylation can be observed with either the
natural diketide 7 or the model pentaketide substrate 11. The fail-
ure to methylate 7 is in contrast to that of the chaetoviridin HR-
PKS MT domain, which naturally methylates -ketobutyryl-ACP
intermediate as well as 7 in the same assay.22 The MT domain
showed noticeable activity towards converting triketide 8 to 13,
albeit significantly attenuated compared to that towards 2. Kinetic
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analysis showed the MT displays a 2500-fold drop in catalytic effi-
ciency towards 8 compared to 2, which resulted from ~50-fold
attenuation in both the kcat and KM values (Figure S10).
Table 1. Apparent Turnover rate of LovB KR domain.a
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Substrate
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Turnover (min-1)
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2.1±0.02
2.4±0.1
We next assayed the substrate preference of LovB MT towards
more simplified substrates such as the saturated 10 and 9. While
conversion of 10 to 16 was confirmed by using a standard of 16, a
surprising penalty to the catalytic efficiency (0.3% of 2) was ob-
served including a 10-fold decrease in kcat and nearly 40-fold in-
crease in KM (Figure S9). A 7.5-fold drop in catalytic efficiency
compared to 8 was also observed when the  double bond was
saturated in the triketide 9 (Figure S11). Collectively, our methyla-
tion assays with LovB MT domain point to exceptional substrate
specificity towards the natural 3-oxo-oct-4,6-dienyl acyl group.
Changes to chain length and functionalization both resulted in
significant decreases in the methylation rate. The requirement of
correct substrate functionalization further suggests that the MT
domain itself can act as a gatekeeping domain in the programming
of LovB. In the event that other tailoring domains malfunction in
the previous cycles and present an alternative substrate, the MT
domain activities will be significantly attenuated. This would likely
result in enzyme stalling or ketoreduction (bypassing the MT func-
tion) of the substrate, which will eventually result in off-loading of
the polyketide product as previously demonstrated.4
a Substrate concentration at 1 mM, enzyme concentration at 5 M.
While the acyl-SNAC substrates enabled a relative measure of
the domain specificity towards different acyl groups, these remain a
much-simplified model of the actual ACP-bound intermediates
that are in cis with all the tailoring domains. To determine if there
is indeed competitive catalysis between the KR and MT domains
towards the -ketoacyl substrates, we performed a combined
MT/KR assay in which each substrate (2, 8-10) was added to LovB
DHo mutant in the presence of both SAM and NADPH, and the
amounts of each product was compared. The MT-first products
can be both the -methyl--keto (+14 mu) and the -methyl--
hydroxyl (+16 mu) compounds, the latter represent the products of
ketoreduction following methylation. The KR-first products are
the -hydroxyl compounds (+2 mu) of which the MT domain can
no longer methylate.
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We first analyzed the competitive modification of model sub-
strates 9 and 10 since all the products can be quantified using
standards. As shown in Figure 3A and Figures S14-15, when 9 was
used in the assay in the presence of equimolar amounts of SAM and
NADPH, the amount of KR products are significantly more than
the MT products (MT/KR product ratio of 1:4) when quantified
after three hours. This is consistent with the individually deter-
mined kinetic parameters of which the KR is more active towards
triketide 9. Increasing the amount of SAM led to higher amount of
the MT products. Conversely, using 10 led to the reversal of prod-
uct distribution with MT/KR product ratio of 4:1. This is in spite
of the kinetic assays showing comparable kcat/KM for both domains
towards 10. However, the KM of the KR domain towards 10 is very
high as we were not able to reach saturation in the assay. Hence
under assay conditions of 1 mM 10, the binding of the SNAC sub-
strate by the KR is likely substantially weaker compared to the MT.
Having established the substrate scope and kinetic properties of
the MT domain, we next assayed the properties of the KR domain
towards the tri- and tetraketide substrates. Since the KR is func-
tional in every iteration of the HR-PKS, we expect the substrate
specificities towards different -ketoacyl thioesters to be more
relaxed. We used the MS-based quantification of substrate conver-
sion, similar to that used in the MT assay. However, significant
difficulties were encountered when working with the conjugated -
ketoacyl substrates such as 2 and 8, due to i) broadening of the
peak as a result of enolization of the -keto group; ii) retention
time overlap; iii) MS signal overlap due to isotopic abundance of
the substrate and the actual mass of the product; and iv) spontane-
ous dehydration of the -hydroxyl product (see Figure 3B). There-
fore, we used model substrates 9, 15, 10 and 16 to perform the ki-
netics assays. The -methyl compounds 15 and 16 were chosen to
examine the effect of methylation of substrate specificity. Follow-
ing overnight incubation in the presence of NADPH and confirma-
tion of product formation using synthesized standards, we per-
formed time-course analysis using single substrate concentration of
1 mM and enzyme concentration of 5 M to obtain the apparent
turnover rates as shown in Table 1. We also attempted to obtain
saturation kinetics of the KR domain towards the substrates, how-
ever we were not able to reach saturation at solubility limits of the
substrate with the exception of 9 which gave kcat of 34.3 min-1 and
KM of 2 mM (kcat/KM = 18.5 min-1mM-1) (Figure S12). Fitting the
linear region of the kinetics data of 10 yielded a kcat/KM value of 5.4
min-1mM-1 (Figure S13). Although we were not able to obtain full
kinetic data on all of the substrates of interest, one can still con-
clude based on Table 1 that the KR domain does not differentiate
between different substrates significantly (within an order of mag-
nitude). The activity of KR is also not significantly affected by the
presence of the -methyl group, suggesting that KR does not exert
any significant kinetic penalty towards a noncognate substrate.
This further suggests the importance of substrate specificity at the
MT step to determine the first tailoring reaction of the -ketoacyl
substrate.
Figure 3. KR and MT competition assays using model and natural tri-
and tetraketide substrates. (A) Quantification of product distribution
of model substrates 9 and 10. (B) and (C) Product distributions of
natural substrates 8 and 2, respectively.
Shown are the extract ion
chromatograms of different products as indicated.
The competition assays were then performed using the natural
substrates 2 and 8 and analyzed by selected ion monitoring as
shown in Figures 3B and 3C. When LovB DHo was added to 8 in
the presence of both SAM and NADPH, we observed a 10:1 ratio
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of KR to MT-catalyzed products consistent with the natural pro-
AUTHOR INFORMATION
1
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8
gramming rules of LovB. Most of the KR products were found to
contain the m/z 215 ion and split into two major peaks. The earlier
peak at TR~4 min is the -hydroxyl compound (parent m/z 233
also observed) and has undergone dehydration during ionization.
A standard of the -hydroxyl compound gave an identical ioniza-
tion pattern. The second peak at TR~6 min is the actual dehydrated
dienyl-SNAC, which forms readily in aqueous solution. When the
natural tetraketide 2 was used in the competition assay, only the
methylated product 14 was observed. Selected ion monitoring
revealed that no reduced products can be found in the assay, there-
by confirming the much higher catalytic efficiency of the MT do-
main towards 2 compared to that of KR. Interestingly, no further
-ketoreduction of 14 can be detected in the assay. Directly using
14 in a KR-only assay also did not yield any ketoreduced products.
This observation is unexpected as the acyl portion of 14 is the natu-
ral substrate of KR in the predicted programmed steps of LovB
(Figure 1). Although the exact reason for this result is unresolved,
one possible explanation may be recognition of the acyl portion of
14 (in the -keto form) requires interactions with the ACP as ob-
served in other PKS systems by NMR studies .23
Corresponding Author
yitang@ucla.edu, john.vederas@ualberta.ca
Author Contributions
‡These authors contributed equally.
ACKNOWLEDGMENT
These investigations were supported by the by NIH (1R01GM085128
and 1DP1GM106413) YT; and the Natural Sciences & Engineering
Research Council of Canada (NSERC) and by the Canada Research
Chair in Bioorganic & Medicinal Chemistry to JCV.
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Our assays using both natural and model substrates provide an
explanation for the programmed methylation step observed in the
iterative cycles of LovB. We suggest the MT and KR domains
compete for each of the -ketoacyl substrates released by the KS
domain, and the relative rates determine the outcome of the imme-
diate tailoring domain choice. The MT domain of LovB has been
precisely tuned to be highly selective for the natural tetraketide 2
and to outcompete the KR at this particular step only. Both chain
length and functional variation in the acyl substrate can lead to
substantial penalties in catalytic efficiency for the MT domain. In
contrast, the KR domain appears to be less substrate dependent in
terms of catalytic efficiency. As a reflection of the competition
between MT and KR, a 30-fold drop in the catalytic efficiency of
MT towards 10 (as compared to 2) can lead to ~20% of the sub-
strate being ketoreduced without being first methylated. As the
correct methyl substitution is essential for recognition in some (but
not all) downstream steps,4 this may pose a significant barrier to
some precursor-directed biosyntheses of polyketides using HR-
PKSs. Particular structural variations in precursors can derail the
programmed steps of the domains and lead to production of shunt
products instead. However, it is clear from previous work that late
steps catalyzed by LovB can proceed without methylation to make
a des-methyl dihydromonacolin L.4
Our findings with the LovB MT domain poses intriguing ques-
tions as to how substrate specificity is achieved at the molecular
level, how other MT domains in HRPKSs have alternative substrate
specificities and the possible influence of the HRPKS quaternary
structure in the intrinsic biosynthetic programming rules of these
megasynthases. For example, in the fusarielin HRPKS,24 the MT
domain is functional on the di-, tri- and pentaketide intermediates,
while inactive on the tetraketide. This is a complete reversal of
specificity compared to LovB, and structural comparisons between
the two MT domains will provide insights into their differences.
ASSOCIATED CONTENT
Supporting Information
Experimental details and synthetic procedures. This material is availa-
ble free of charge via the Internet at http://pubs.acs.org.
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(24) Sørensen, J. L.; Hansen, F. T.; Sondergaard, T. E.; Staerk, D.;
Lee, T. V.; Wimmer, R.; Klitgaard, L. G.; Purup, S.; Giese, H.;
Frandsen, R. J. N. Environ. Microb. 2012, 14, 1159.
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Figure 1
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Figure 3
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