Elucidation of the Stereospecificity of C-Methyltransferases from trans-AT Polyketide Synthases

Article abstract of DOI:10.1021/jacs.7b02911

S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial polyketide biosynthesis mediated by trans-AT polyketide synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyltransferases-BaeMT9, DifMT1, and MupMT1-from the bacillaene, difficidin, and mupirocin trans-AT polyketide synthases

Full text of DOI:10.1021/jacs.7b02911

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Communication
Elucidation of the Stereospecificity of C-
Methyltransferases from trans-AT Polyketide Synthases
Xinqiang Xie, Chaitan Khosla, and David E. Cane
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Journal of the American Chemical Society
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Elucidation of the Stereospecificity of C-Methyltransferases
from trans-AT Polyketide Synthases
†
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Xinqiang Xie, Chaitan Khosla, and David E. Cane
†
Department of Chemistry, Brown University, Box H, Providence, Rhode Island 02912-9108, USA
Departments of Chemical Engineering, Chemistry, and Biochemistry, Stanford University, Stanford, California 94305
§
Supporting Information Placeholder
O
OH
ABSTRACT: S-Adenosyl methionine (SAM)-dependent
C-methyltransferases are responsible for the C2-
methylation of 3-ketoacyl-acyl carrier protein (ACP)
intermediates to give the corresponding 2-methy-3-
ketoacyl-ACP products during bacterial polyketide bio-
synthesis mediated by trans-AT polyketide synthases
that lack integrated acyl transferase (AT) domains. A
coupled ketoreductase (KR) assay was used to assign the
stereochemistry of the C-methyltransferase-catalyzed
reaction. Samples of chemoenzymatically-generated 3-
ketopentanoyl-ACP (9) were incubated with SAM and
BonMT2 from module 2 of the bongkrekic acid polyke-
tide synthase. The resulting 2-methyl-3-ketopentanoyl-
ACP (10) was incubated separately with five (2R)- or
(2S)-methyl specific KR domains. Analysis of the de-
rived 2-methyl-3-hydroxypentanoate methyl esters (8)
by chiral GC-MS established that the BonMT2-
catalyzed methylation generated exclusively (2R)-2-
methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results
were also obtained with three additional C-
methyltransferases – BaeMT9, DifMT1, and MupMT1 –
from the bacillaene, difficidin, and mupirocin trans-AT
polyketide synthases
HO
HO
O
3
CH O
BonMT2
CH
3
1
Bongkrekic acid
O
OH
O
O
NH
O
HO
BaeMT9
N
H
CH
3
OH
2
Bacillaene
3 2
OPO H
CH
3
DifMT1
O
O
3
Difficidin
CH
3
O
O
O
OH
O
HO
O
OH
MupMT1
OH
4
mupirocin
Modular polyketide synthases (PKSs) are polyfunc-
tional megaproteins that are responsible for the bacterial
synthesis of an enormous range of physiologically active
natural products, including many important antibiotics,
antitumor agents, and immunosuppressants. Within a
typical PKS, each module is responsible for a separate
round of polyketide chain elongation and modification.
PKS modules fall into two broad functional classes. In
cis-AT polyketide synthases, typified by the extensively
studied 6-deoxyerythronolide B synthase of erythromy-
Figure 1. Representative polyketides produced by trans-AT
polyketide synthases and domain organization of selected
modules harboring C-methyltransferase domains, with the
resultant methyl groups highlighted in red.
CoA or methylmalonyl-CoA, onto the pantetheinyl arm
of a paired acyl carrier protein (ACP) domain within the
same module. The resultant malonyl- or methylmalonyl-
ACP then serves as the substrate for chain elongation by
decarboxylative condensation catalyzed by the paired
ketosynthase (KS) domain so as to generate the respec-
tive 3-ketoacyl-ACP or (2R)-2-methyl-3-ketoacyl-ACP
intermediate. Different degrees of further functional
group modification catalyzed by variable combinations
of ketoreductase (KR), dehydratase (DH), and enoyl-
1
cin biosynthesis, each module harbors a dedicated acyl
transferase (AT) domain that is responsible for loading a
specific chain elongation substrate, most often malonyl-
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Journal of the American Chemical Society
Page 2 of 5
reductase (ER) domains may then occur before transfer
expressed in Escherichia coli as an N-terminal His6-
tagged protein from a codon-optimized synthetic gene
using consensus domain boundaries (Figures S1 and S2,
Table S3). Incubation of BonMT2 with the N-
acetylcysteamine thioester 3-ketopentanoyl-SNAC (5)
and SAM, in the presence of S-adenosyl homocysteine
1
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9
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to the KS domain of the proximal downstream module
for a further round of chain elongation and functional
group modification. By contrast, trans-AT polyketide
synthases lack integrated AT domains, relying instead on
one or more discrete AT proteins to supply the requisite
building blocks, most often malonyl-CoA, to each PKS
(SAH) nucleosidase to relieve potent product inhibition
2
8,10
module. As an alternative to methylmalonyl-CoA, cer-
by SAH,
gave 2-methyl-3-ketopentanoyl-SNAC (6),
tain modules harbor an integrated, S-adenosylmethionine
as established by HPLC-MS (Scheme 1, Figure S3). The
product was further characterized by reduction with
(SAM)-dependent C-methyltransferase (C-MT) that
6,7b,7e
0
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converts the initially generated 3-ketoacyl-ACP inter-
mediate to the corresponding 2-methyl-3-ketoacyl-ACP.
Of 48 trans-AT PKSs for which the resultant natural
product has been assigned, 47 (98%) harbor 1-10 C-
methyltransferases (Tables S1 and S2), as represented by
TylKR1 from module 1 of the tylactone synthase
in
the presence of NADPH to give (2R,3R)-2-methyl-3-
hydroxypentanoyl-SNAC (7a), whose structure and ste-
reochemistry were established by base-catalyzed thio-
ester hydrolysis and treatment with trimethylsilyldiazo-
3
4
4a
bongkrekic acid (1), bacillaene (2), difficidin (3), and
methane (TMS-CHN ). Chiral GC-MS analysis of the
2
5
mupirocin (4) (Figure 1). By contrast, embedded C-
derived methyl esters confirmed predominant formation
of the characteristic reduction product, methyl (2R,3R)-
2-methyl-3-hydroxypentanoate (8a) (Figure S4).
methyltransferases are considerably less common in cis-
AT PKS systems, with only 9 out of 168 (5%) harboring
1
or 2 C-methyltransferases and only gephyronic acid
Scheme 1.
synthase containing as many as 6 (Tables S1 and S2).
The stereochemistry of the various C-methylation re-
actions cannot be inferred from the structure and stereo-
chemistry of the ultimately formed polyketide products,
since the configuration of each of the initially generated
2
-methyl-3-ketoacyl-ACP intermediates is obscured by a
Further biochemical characterization of BonMT2 re-
quired 3-ketopentanoyl-BonACP2 (9), free of potentially
variety of intervening PKS-catalyzed transformations,
including possible epimerization of the unreduced 2-
methyl-3-ketoacyl-ACP intermediate, catalyzed by a
interfering
3-ketopentanoyl-SNAC
(5)
or
3-
11
ketopentanoyl-CoA. To this end, malonyl-BonACP2,
0
6
redox-inactive KR or DH domain, by KR-catalyzed
prepared from recombinant apo-BonACP2 and malonyl-
7
reduction, with or without epimerization, or DH-
12
CoA using the phosphopantetheinyl transferase Sfp,
catalyzed dehydration. Although a number of excised
PKS C-methyltranferases have been expressed as re-
combinant proteins and shown to catalyze the expected
SAM-dependent methylation of 3-ketoacylthioesters, the
stereochemistry of the resulting 2-methyl-3-ketoacyl
was incubated with recombinant BonKS2 which had
been preincubated with propionyl-SNAC (Scheme 2a).
The formation of 9 was established by LC-ESI-MS
analysis of an aliquot after desalting and concentration
by ultrafiltration. After addition of BonMT2, SAM, and
SAH nucleosidase, the incubation was continued for an
additional 4 h. Following removal of BonKS2 and
BonMT2 by ultrafiltration, the mixture was analyzed by
LC-ESI-MS, confirming formation of the predicted 2-
8
,9
thioester products has not been determined.
Recently, we described a versatile coupled enzyme as-
say utilizing a combination of naturally occurring and
engineered, epimerase-inactive KR domains that are
strictly specific for either (2R)- or (2S)-2-methyl-3-
methyl-3-ketopentanoyl-BonACP2 (10), M 15556, cor-
D
6
b
ketoacyl thioesters. Using this assay, we established
unambiguously that the decarboxylative condensation
catalyzed by the NanKS1 domain of nanchangmycin
synthase generates exclusively a (2R)-2-methyl-3-
ketoacyl-ACP product. In fact, the coupled KR-assay is
entirely general and can be used to assign the stereo-
chemistry of any chemoenzymatically generated 2-
methyl-3-ketoacyl-ACP. We report below the determina-
tion of the stereospecificity of C-methyltransferases
from four different trans-AT PKS systems illustrated in
Figure 1, each of which was shown to catalyze the ex-
clusive formation of the (2R)-2-methyl-3-ketoacyl-ACP
product.
responding to an increase of 14 amu over the substrate
3
-ketopentanoyl-BonACP2 (9), MD 15542 (Table S6,
Figure S8).
Having established that BonMT2 can catalyze the pre-
dicted SAM-dependent, C2-methylation of 3-
ketopentanoyl-BonACP2 (9), we then determined the
stereochemistry of the reaction using the coupled keto-
reductase assay. To this end, BonMT2, in the presence of
SAH nucleosidase, was used to convert a mixture of
SAM and 3-ketopentanoyl-BonACP2 (9), chemoen-
zymatically generated in situ as described above, to the
corresponding 2-methyl-3-ketopentanoyl-BonACP2 (10)
(Scheme 2b). After 15-min incubation at room tempera-
BonMT2, the prospective C-methyltransferase from
module 2 of the bongkrekic acid (1) PKS (Figure 1), was
ture, 800-µL aliquots of the reaction mixture were added
to individual solutions containing excess NADPH and
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Journal of the American Chemical Society
each of the following five ketoreductases: TylKR1,
EryKR6, EryKR1, EryKR6-G324T/L333H, and
In each series of incubations, only the (2R)-2-methyl-3-
ketoacyl-ACP-specific KR domains gave rise to time-
dependent formation of the corresponding reduced
diketide acid methyl esters (8a, 8b, and 8c from
TylKR1, EryKR6, and EryKR1, respectively), while
neither of the two (2S)-2-methyl-3-ketoacyl-ACP-
specific KR domains produced any reduced 2-methyl-3-
hydroxy diketide products. It can therefore confidently
be concluded that BaeMT9, DifMT1, and MupMT1 are
each completely stereospecific for conversion of their 3-
ketoacyl-ACP substrate to the corresponding (2R)-2-
1
2
3
4
5
6
7
8
9
6
b,13
AmpKR2-G355T/Q364H.
Aliquots of each KR in-
cubation mixture were withdrawn after 15, 30, and 60
min, then quenched and hydrolyzed with aq. NaOH. Af-
ter
acidification
the
resulting
2-methyl-3-
hydroxypentanoic acids were converted using TMS-
CHN to the corresponding methyl esters 8 which were
2
analyzed by chiral GC-MS (Figures S9 and S10). Re-
duction with the individual epimerase-inactive, (2R)-2-
methyl-3-ketoacyl-ACP-specific ketoreductases TylKR1
and EryKR6 resulted in time-dependent, diastereospecif-
ic formation of exclusively methyl (2R,3R)-2-methyl-3-
hydroxypentanoate (8a) and methyl (2R,3S)-2-methyl-3-
hydroxypentanoate (8b), respectively, consistent with
1
1
1
1
1
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15
methyl-3-ketoacyl-ACP product.
In bacterial polyketide biosynthesis, nature has
evolved two complementary strategies for the generation
of 2-methyl-3-ketoacyl-ACP intermediates. For canoni-
cal cis-AT PKSs, (2S)-methylmalonyl-CoA serves as the
substrate for each integrated AT domain, resulting in the
formation of a (2R)-2-methyl-3-ketoacyl-ACP interme-
diate by decarboxylative condensation of methylmalo-
nyl-ACP with the electrophilic acyl-ACP primer or par-
the established substrate and product specificity of each
7a,7c
KR domain (Table S7).
In like manner, incubation
with epimerase-active EryKR1 gave the correspond7icn,1g4
methyl (2S,3R)-2-methyl-3-hydroxypentanoate (8c).
By contrast, neither of the epimerase-inactive, (2S)-2-
methyl-3-ketoacyl-ACP-specific, mutant ketoreductases,
EryKR6-G324T/L333H or AmpKR2-G355T/Q364H,
6b,7a,7c,16
tially elaborated polyketide chain.
By contrast,
trans-AT PKSs rely on one or more discrete, primarily
malonyl-CoA-specific AT domains, leading to the gen-
eration of a 3-ketoacyl-ACP intermediate as the product
of the KS-catalyzed chain elongation reaction. Those
trans-AT modules that harbor an integrated SAM-
dependent C-methyltransferase can then generate the
corresponding 2-methyl-3-ketoacyl-ACP. Using the
coupled ketoreductase assay, we have now demonstrated
that all four of the individual C-methyltransferases ana-
lyzed – BonMT2, BaeMT9, DifMT1, MupMT1 – cata-
lyze stereospecific methylation to yield exclusively the
(2R)-2-methyl-3-ketoacyl-ACP product. Furthermore,
multiple sequence alignment of a representative set of 38
PKS C-methyltransferase domains reveals a high level
of sequence conservation and, most importantly, no sig-
nificant sequence segregation according to whether the
eventually formed polyketide displays a trisubstituted Z-
double bond (6 C-MTs, including BonMT2 and
BaeMT9), a trisubstituted E-double bond (12 C-MTs,
including DifMT1), or a 2-methyl-3-hydroxyacyl-ACP-
derived architecture (20 C-MTs, including MupMT1)
(Figure S14), suggesting that the formation of the (2R)-
2-methyl-3-ketoacyl-ACP intermediate is likely to be
general across all PKS systems.
produced
any detectable
methyl
2-methyl-3-
hydroxypentanoate (8), even after 60 min incubation.
These combined results therefore conclusively demon-
strate that the BonMT2-catalyzed C2-methylation of 3-
ketopentanoyl-BonACP2 (9) is completely stereospecif-
ic for formation of (2R)-2-methyl-3-ketopentanoyl-
BonACP2 ((2R)-10).
Scheme 2.
SACP
O
SACP
a
O
CH
3
O
O
BonKS2
SAM, SAHase
BonMT2
SNAC
O
O
9
10
Mal-CoA
SACP
apo-BonACP2
Sfp
2
CO H
SACP
HO
TylKR1
NADPH
O
2
CO Me
b
OH
(2R,3R)-8a
(2R,3R)-11a
SACP
O
SACP
O
SACP
HO
CH
3
EryKR6
O
2
CO Me
SAM, SAHase
O
O
OH
BonMT2
NADPH
9
or BaeMT9
or DifMT1
(2R,3S)-8b
(
2R)-10
(2R,3S)-11b
or MupMT1
SACP
HO
EryKR1
O
2
CO Me
Unlike cis-AT PKSs, the KR domains in trans-AT
PKS modules harboring C-methyltransferases must not
reduce the initially generated 3-ketoacyl-ACP before the
requisite formation of the 2-methyl-3-ketoacyl-ACP. We
surmise that this discrimination results from the relative
values of k /K for processing of the 3-ketoacyl-ACP
OH
NADPH
(2S,3R)-8c
(2S,3R)-11c
AmpKR2-G355T/Q364H
or EryKR6-G324T/L333H
No Reduction
NADPH
cat
m
17
We also carried out the analogous sets of coupled KR
assays with each of the previously characterized C-
methyltransferases from trans-AT PKSs: BaeMT9, from
module 9 of the bacillaene PKS, DifMT1 from module 1
of the difficidin PKS, and MupMT1 from module 1 of8
the mupirocin PKS (Scheme 2b, Figure S10, Table S7).
intermediate by the C-MT and KR domains.
In mupirocin, the substructure derived from Mup
module 1 has the (12S,13S)-12-methyl-13-hydroxy con-
figuration (Figure 1), suggesting that MupKR1 converts
the (2R)-2-methyl-3-ketobutyryl-ACP intermediate gen-
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Strittmatter, A. W.; Gottschalk, G.; Sussmuth, R. D.; Borriss, R. J.
erated by MupMT1 to the corresponding (2S,3S)-2-
methyl-3-hydroxybutyryl-ACP product (epimerizing A2-
type KR domain). Interestingly, in sequence alignments
MupKR1 lacks the characteristic A2-type sequence
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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Bacteriol. 2006, 188, 4024-4036. b) Moldenhauer, J.; Chen, X. H.;
Borriss, R.; Piel, J. Angew. Chem. Int. Ed. Engl. 2007, 46, 8195-8197.
c) Butcher, R. A.; Schroeder, F. C.; Fischbach, M. A.; Straight, P. D.;
Kolter, R.; Walsh, C. T.; Clardy, J. Proc. Natl. Acad. Sci. U S A 2007,
104, 1506-1509.
7d,7e
markers
and appears to cluster instead with EryKR6
(5) El-Sayed, A. K.; Hothersall, J.; Cooper, S. M.; Stephens, E.;
Simpson, T. J.; Thomas, C. M. Chem. Biol. 2003, 10, 419-430.
and other non-epimerizing A1-type domains that gener-
ate (2R,3S)-2-methyl-3-hydroxybutyryl-ACP products
(
6) a) Garg, A.; Xie, X.; Keatinge-Clay, A.; Khosla, C.; Cane, D.
(
Figure S15).
E. J. Am. Chem. Soc. 2014, 136, 10190-10193. b) Xie, X.; Garg, A.;
Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2017, 139, 3283-3292.
(7) a) Castonguay, R.; He, W.; Chen, A. Y.; Khosla, C.; Cane, D.
E. J. Am. Chem. Soc. 2007, 129, 13758-13769. b) Castonguay, R.;
Valenzano, C. R.; Chen, A. Y.; Keatinge-Clay, A.; Khosla, C.; Cane,
D. E. J. Am. Chem. Soc. 2008, 130, 11598-11599. c) Valenzano, C.
R.; Lawson, R. J.; Chen, A. Y.; Khosla, C.; Cane, D. E. J. Am. Chem.
Soc. 2009, 131, 18501-18511. d) You, Y. O.; Khosla, C.; Cane, D. E.
J. Am. Chem. Soc. 2013, 135, 7406-7409. e) Keatinge-Clay, A. T.
Chem. Biol. 2007, 14, 898-908.
In bongkrekic acid (1), the configuration of the inter-
mediate 2-methyl-3-hydroxy-4-methylhex-3-enoyl-ACP
generated by BonKR2 is obscured in the final product
by the BonDH2-catalyzed dehydration. To address this
issue, we have recently expressed BonKR2 and estab-
lished that it is a non-epimerizing A1-type KR that ste-
reospecifically converts (2R)-2-methylpentanoyl-ACP to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the
corresponding
(2R,3S)-2-methyl-3-hydroxy-
(8) Stevens, D. C.; Wagner, D. T.; Manion, H. R.; Alexander, B.
K.; Keatinge-Clay, A. T. J. Antibiot. 2016, 69, 567-570.
18
pentanoyl-ACP. Notably, syn dehydration by BonDH2
would be expected to generate the (Z)-3-methyl-2-enoyl-
ACP intermediate from which bongkrekic acid (1) is
directly derived. This and related transformations are
currently under investigation.
(
9) Skiba, M. A.; Sikkema, A. P.; Fiers, W. D.; Gerwick, W. H.;
Sherman, D. H.; Aldrich, C. C.; Smith, J. L. ACS Chem. Biol. 2016,
1, 3319-3327. These authors stated that the acidity of the H-2 proton
1
of the 2-methyl-3-ketoacyl-ACP generated by CurJ precludes che-
moenzymatic assignment of the C2-methyl stereochemistry.
(
10) Hendricks, C. L.; Ross, J. R.; Pichersky, E.; Noel, J. P.; Zhou,
Z. S. Anal. Biochem. 2004, 326, 100-105.
ASSOCIATED CONTENT
(11) The use of the ACP-conjugated substrate is essential since the
rate of epimerization of the 2-methyl-3-ketoacyl-ACP has been shown
Supporting Information.
-1
to be <0.003 min , compared to a kepim for the considerably more
Experimental methods, including C-MT design and expres-
sion, coupled KR assays, kinetic assays, and GC-MS and
ESI-MS analysis. This material is available free of charge
at http://pubs.acs.org.
-1
labile -SNAC derivative of ~0.15 min (refs 7a and 7c).
(
12) Quadri, L. E.; Weinreb, P. H.; Lei, M.; Nakano, M. M.; Zuber,
P.; Walsh, C. T. Biochemistry 1998, 37, 1585-1595.
(13) Zheng, J.; Piasecki, S. K.; Keatinge-Clay, A. T. ACS Chem.
Biol. 2013, 8, 1964-1971.
AUTHOR INFORMATION
Corresponding Author
(14) Garg, A.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2013,
1
35, 16324-16327.
(15) Although competing reduction of non-methylated 3-
ketopentanoyl-ACP would not have affected the validity of the as-
signment of the stereochemistry of the C2-methylation using the cou-
pled KR assay, the corresponding 3-hydroxypentanoyl-ACP was
notably absent from the assay products, as analyzed by chiral GC-MS
of the derived methyl esters. Control kinetic assays and stereochemi-
cal analysis showed that while each of the KR domains used in the
assay was capable of reducing the unmethylated 3-ketopentanoyl-
David E. Cane, David_Cane@brown.edu
Notes
No competing financial interests have been declared.
ACKNOWLEDGMENT
This work was supported by grants from the U. S. National
Institutes of Health, GM022172 to D.E.C. and GM087934
to C.K. We thank Prof. Adrian Keatinge-Clay and Drew T.
Wagner of the University of Texas, Austin for a generous
gift of the expression plasmids for BaeMT9, DifMT1, and
MupMT1.
SNAC substrate, the relative kcat/K
ketopentanoyl-SNAC by TylKR1, EryKR6, or EryKR1 was typically
-5 times greater than that for reduction of 3-ketopentanoyl-SNAC,
with the relative kcat/K values for the (2S)-methyl-specific EryKR6-
m
for reduction of (±)-2-methyl-3-
3
m
G324T/L333H and AmpKR2-G355T/Q364H being 55 and 2, respec-
tively (Tables S4, S5, and S8, Figures S5-S7, S11-S13). Both TylKR1
and AmpKR2-G355T/Q364H reduced 3-ketopentanoyl-SNAC exclu-
sively to (3R)-3-hydroxypentanoyl-SNAC, while reduction by either
EryKR6 or EryKR6-G324T/L333H gave essentially racemic 3-
hydroxypentanoyl-SNAC. A further control showed that the 3-
ketopentanoyl-ACP intermediate was generated continuously over the
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(1) a) Cane, D. E. J. Biol. Chem. 2010, 285, 27517-27523. b)
Hopwood, D. A. Methods in Enzymology. Complex enzymes in mi-
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marins and carbohydrates; Academic Press: United States, 2009; Vol.
1
-h time course of the ketoreductase assay.
16) Marsden, A. F.; Caffrey, P.; Aparicio, J. F.; Loughran, M. S.;
(
Staunton, J.; Leadlay, P. F. Science 1994, 263, 378-380.
(17) The kinetics and relative substrate specificity of the competi-
tion between C-MT and KR domains have been analyzed in detail for
the iterative fungal PKS lovastatin synthase: Cacho, R. A.; Thuss, J.;
Xu, W.; Sanichar, R.; Gao, Z.; Nguyen, A.; Vederas, J. C.; Tang, Y. J.
Am. Chem. Soc. 2015, 137, 15688-15691.
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(2) a) Cheng, Y. Q.; Tang, G. L.; Shen, B. Proc. Natl. Acad. Sci. U
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(
(18) X. Xie, unpublished observation.
Hertweck, C. Chem. Biol. 2012, 19, 1164-1174.
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