Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase

Article abstract of DOI:10.1021/ja0753290

6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic

Full text of DOI:10.1021/ja0753290

Published on Web 10/06/2007
Stereospecificity of Ketoreductase Domains of the
6-Deoxyerythronolide B Synthase
Roselyne Castonguay,† Weiguo He,† Alice Y. Chen,§ Chaitan Khosla,‡,§,| and
David E. Cane*,†
Contribution from the Department of Chemistry, Box H, Brown UniVersity, ProVidence, Rhode
Island 02912-9108, and Departments of Chemical Engineering, Chemistry, and Biochemistry,
Stanford UniVersity, Stanford, California 94305
Received July 30, 2007; E-mail: David_Cane@brown.edu
Abstract: 6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible
for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic
erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of
polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To
better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted
system has been developed involving combinations of ketosynthase (KS)-acyl transferase (AT) didomains
with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-
2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH
result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone
4 by either base- or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-MS technique
has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct
comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT]
didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6
gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-δ-lactone (4a).
The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5]-
[ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACP-bound 2-methyl-3-ketoacyl
triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried
only an unepimerized D-2-methyl group. The results confirm the predicted stereospecificity of the individual
KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl
thioester intermediate. The methodology should be applicable to the study of any combination of
heterologous [KS][AT] and [KR] domains.
Modular polyketide synthases (PKSs) are multifunctional
enzymes responsible for the biosynthesis of polyketide metabo-
lites that exhibit a wide range of important biological properties,
including antibacterial, antifungal, anticancer, and immunosup-
pressive activity.1 They catalyze repetitive Claisen-like con-
densations between methylmalonyl or malonyl thioester building
blocks and the growing polyketide acyl thioesters. Using an
assembly line of active sites, each module is responsible for
one cycle of polyketide chain elongation and functional group
modification before passing the resulting intermediate to the
downstream module. Each module contains a set of three core
domains: the â-ketoacyl-ACP synthase (KS), which accepts a
polyketide chain from the appropriate upstream module and
catalyzes the chain-building decarboxylative condensation be-
tween this intermediate and an ACP-bound methylmalonyl or
malonyl chain extension unit; the acyl transferase (AT) domain,
which loads the proper extender unit from the corresponding
methylmalonyl or malonyl-CoA substrate onto the phospho-
pantetheine arm of the acyl-carrier protein (ACP), which in turn
carries the growing polyketide intermediate from domain to
domain. Additional domains that are found in varying combina-
tions in most modules include a ketoreductase (KR), a dehy-
dratase (DH), and an enoyl reductase (ER). Together these
tailoring domains control the final oxidation state of the â-carbon
of the growing polyketide acyl-ACP intermediate. A dedicated
thioesterase (TE) domain, located at the C-terminus of the
furthest downstream module, catalyzes release and concomitant
cyclization of the mature, full-length macrocyclic polyketide.
† Brown University.
‡ Department of Chemical Engineering, Stanford University.
§ Department Chemistry, Stanford University.
| Department of Biochemistry, Stanford University.
The 6-deoxyerythronolide synthase (DEBS) from Saccha-
ropolyspora erythraea, which is by far the most thoroughly
studied modular PKS, is responsible for biosynthesis of
6-deoxyerythronolide (6-dEB, 1), the aglycone precursor of the
broad spectrum antibiotic erythromycin A.2 The macrolide
(1) (a) Walsh, C. T. ChemBioChem 2002, 3, 125-134. Cane, D. E.; Walsh,
C. T.; Khosla, C. Science 1998, 282, 63-68. Walsh, C. T. Science 2004,
303, 1805-1810. (b) For a comprehensive review of PKS and NRPS
biochemistry and molecular biology as of 1997, see the thematic issue of
Chem. ReV. 1997, 97, 2463-2706. (c) Khosla, C.; Gokhale, R. S.; Jacobsen,
J. R.; Cane, D. E. Annu. ReV. Biochem. 1999, 68, 219-253.
9
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J. AM. CHEM. SOC. 2007, 129, 13758-13769
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DEBS Ketoreductase Domains
A R T I C L E S
Figure 1. Modular organization of 6-deoxyerythronolide B synthase (DEBS). The three polypeptides DEBS 1, 2, and 3 are denoted as the yellow rectangles,
and modules, by the solid lines. Each block represents a catalytic domain: KS, ketosynthase; AT, acyltransferase; ACP, acyl-carrier protein; KR, ketoreductase;
DH, dehydratase; ER, enoyl reductase and TE, thioesterase. KR0 represents an inactive KR domain. The phosphopantetheinyl arm of the ACP domain is
represented as a curly line.
6-dEB is assembled by three large polypeptides, denoted
DEBS1, 2, and 3, each consisting of >3000 amino acids, that
catalyze the formation of 6-dEB from a propionyl-CoA starter
unit and six methylmalonyl-CoA extender units (Figure 1).
One of the most powerful tools for the study of the
mechanism and specificity of DEBS-catalyzed reactions has
been the ability to express entire DEBS proteins or individual
DEBS modules, alone or in combination, in heterologous hosts
such as Escherichia coli or Streptomyces coelicolor.3 In Vitro
experiments with purified recombinant modules, most often
carrying an appended TE domain fused to the C-terminus of
the ACP domain, have allowed detailed studies of enzyme
kinetics and mechanism, including determination of substrate
specificity and tolerance.4 For example, DEBS module 3+TE
has been shown to catalyze the conversion of (2S,3R)-2-methyl-
3-hydroxypentanoic acid N-acetylcysteamine thioester (diketide-
SNAC 2) and methylmalonyl-CoA to the triketide ketolactone
3 which is generated by TE-catalyzed release of the correspond-
ing acyclic 2,4-dimethyl-3-keto-5-hydroxypentanoyl thioester
from the ACP (Figure 2A).5 In similar fashion, DEBS module
2+TE, module 5+TE, and module 6+TE, each carrying an
active KR domain, all convert diketide 2 and methylmalonyl-
CoA to the reduced triketide 3-hydroxylactone 4a (Figure 2B).5
Figure 2. (A) Sequence of reactions catalyzed by DEBS module 3 + TE.
Site-directed mutagenesis of specific domains and swapping of
entire domains between different DEBS modules as well as with
heterologous PKS modules have further clarified the role of
individual PKS domains, while allowing the generation of novel,
rationally engineered polyketides.1,2
The KS domain is first acylated by diketide-SNAc 2, and the AT domain
primes the ACP domain with methylmalonyl-CoA. Following the KS-
catalyzed condensation reaction, the TE domain catalyzes the cyclization
and release of the resulting triketide ketolactone 3. (B) Reaction catalyzed
by DEBS module 2 + TE (as well as by DEBS modules 5 + TE and 6 +
TE). After the KS-catalyzed condensation reaction, the KR domain catalyzes
the NADPH-dependent reduction of the 3-ketoacyl-ACP thioester, with
release of the reduced triketide mediated by the TE domain to give the
triketide lactone 4a.
More recently, limited proteolysis of DEBS proteins, in
combination with high-resolution structural studies, has facili-
(2) (a) Khosla, C.; Tang, Y.; Chen, A. Y.; Schnarr, N. A.; Cane, D. E. Annu.
ReV. Biochem. 76, 195-221. (b) Staunton, J.; Wilkinson, B. In Compre-
hensiVe Natural Products Chemistry, Polyketides and Other Secondary
metabolites Including Fatty Acids and Their DeriVatiVes; Sankawa, U., Ed.;
Elsevier: Oxford, 1999; Vol. 1, p 495-532.
tated the dissection and reconstitution of individual DEBS
modules from their constituent domains.2a,6 Thus soluble active
[KS][AT] didomains derived from DEBS modules 3 and 6 in
combination with recombinant ACP domains have both been
(3) Kumar, P.; Khosla, C.; Tang, Y. Methods Enzymol. 2004, 388, 269-293.
(4) Wu, N.; Kudo, F.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc. 2000, 122,
4847-4852.
(5) Gokhale, R. S.; Tsuji, S. Y.; Cane, D. E.; Khosla, C. Science 1999, 284,
482-485.
(6) Kim, C. Y.; Alekseyev, V. Y.; Chen, A. Y.; Tang, Y.; Cane, D. E.; Khosla,
C. Biochemistry 2004, 43, 13892-13898.
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A R T I C L E S
Castonguay et al.
be summarized by a single stereochemical model.12 Since that
time, the Celmer Model has provided a frame of reference for
numerous investigations of the stereochemistry of macrolide
biosynthesis. Although the modular organization of the DEBS
PKS accounts in clear and elegant fashion for the observed
pattern of substitution and oxidation in erythromycin and related
macrolides, the detailed biochemical basis for the complex
stereochemistry of the parent 6-dEB macrolide aglycone remains
to be resolved.
Figure 3. Reactions catalyzed by deconstructed DEBS module 3. After
KS-catalyzed condensation, in the absence of an active KR domain the
triketide ketolactone 3, is released by base- or TE-catalyzed hydrolysis. In
the presence of an active KR domain such as DEBS KR2 and NADPH, the
reduction of the ketone leads to formation of triketide lactone 4.
The KR domains of DEBS modules 1, 2, 5, and 6 all use the
4-si (4-proS) hydride of the NADPH cofactor, identical to the
known stereochemical course for fatty acid synthase KR
domains.13 We have also demonstrated that the stereochemistry
of ketone reduction is an intrinsic property of the PKS KR
domain, with the configuration of the resulting hydroxyl group
independent of either modular context or substrate structure.14
Both Reid15a and Caffrey15b have recently independently
compared the sequences of some 200 modular KR domains
belonging to the superfamily of short-chain dehydrogenases/
reductases (SDRs). Consistent with the known structures of
several members of this family, an active site triad of Ser, Tyr,
and Lys residues is strictly conserved among all the KR domains
analyzed. An interesting correlation was also noted between the
observed formation of D-hydroxy groups and the presence of a
conserved Asp found within a highly conserved LDD motif in
such D-specific reductases. Notably, this conserved Asp is not
present in the KR2, KR5, and KR6 domains responsible for
generating L-3-hydroxyacyl-ACP intermediates that serve as
precursors of the C-3, C-5, and C-11 hydroxyl groups found in
6-dEB (1). Intriguingly, this conserved Asp residue is also found
in KR domains of cryptic stereochemical specificity that are
paired with a dehydratase (DH) domain, such as the KR domains
of DEBS module 4 and of PICS module 2. Although the
stereospecificity of DEBS KR4 remains to be determined, we
have established that PICS KR2 indeed generates a D-hydroxy
group, as predicted by the bioinformatic analysis.16 On the other
hand, attempts to use site-directed mutagenesis to modify the
intrinsic stereospecificity of DEBS KR domains have achieved
only limited success, suggesting that the characteristic Asp and
other conserved residues are not the unique determinants of the
intrinsic stereospecificity of 3-ketoacyl thioester reduction.17
shown to catalyze the formation of triketide ketolactone 3 from
diketide-SNAC 2 and methylmalonyl-CoA (Figure 3).7 The
initially generated ACP-bound acyclic triketide can be released
and lactonized either by base-catalyzed hydrolysis followed by
acidification7,8 or by addition of recombinant DEBS TE or the
closely related PICS TE derived from the picromycin PKS.9
Importantly, addition of a recombinant KR domain, such as
DEBS KR2, to the incubation mixtures results in formation of
the corresponding reduced triketide lactone 4, which can readily
be assayed by TLC-phosphorimaging.8 The stereochemistry of
the 2-methyl and 3-hydroxyl substituents in samples of 4
resulting from combination of different [KS][AT] didomains
with varied [KR] domains has not previously been determined.
Reconstituted PKS systems have several attractive experi-
mental features compared to intact PKS modules. Besides
eliminating problems with incompatible domain boundaries,
which have often interfered with earlier efforts to engineer
catalytically efficient chimeric PKS modules from combinations
of heterologous domains,10 the relative concentrations of the
constituent domains can be independently varied. For example,
it is often advantageous to use the ACP domain, which carries
the extender unit and derived products, in stoichiometric excess
over the catalytic [KS][AT] didomain. It is also possible to vary
the nucleophilic substrate by using Sfp phosphopantetheinyl
transferase11 to load malonyl-CoA analogues directly onto the
ACP,7 thus bypassing the strict substrate specificity of the native
AT domains. In like manner, we have determined the preference
of individual [KS] domains for a variety of [ACP] domains
carrying either the nucleophilic methylmalonyl or electrophilic
polyketide acylthioester substrates.7
The factors controlling the stereochemistry of methyl sub-
stitution in 6-dEB (1) and other polyketides are even less well
understood. Except for the C-6 L-methyl, whose configuration
is the consequence of the ER4-mediated reduction of the
unsaturated 2-methyl pentaketide-ACP4 intermediate, the con-
The macrolide aglycone 6-dEB (1) carries three L-hydroxy
groups, at C-3, C-5, and C-11, and a single D-hydroxy
substituent at C-13. The configuration of the methyl substituents
shows a similar level of complexity, with D-methyl groups at
C-2, C-4, and C-10, and L-methyls at C-6, C-8, and C-12 of
6-dEB (1). More than 40 years ago, Celmer pointed out the
intriguing structural and stereochemical homology among a large
number of 12-, 14-, and 16-membered macrolides, which could
(12) Celmer, W. D. J. Am. Chem. Soc. 1965, 87, 1801-1802.
(13) (a) McPherson, M.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 1998, 120,
3267-3268. (b) Yin, Y.; Gokhale, R.; Khosla, C.; Cane, D. E. Bioorg.
Med. Chem. Lett. 2001, 11, 1477-1479.
(14) Kao, C. M.; McPherson, M.; McDaniel, R. N.; Fu, H.; Cane, D. E.; Khosla,
C. J. Am. Chem. Soc. 1998, 120, 2478-2479.
(15) (a) Reid, R.; Piagentini, M.; Rodriguez, E.; Ashley, G.; Viswanathan, N.;
Carney, J.; Santi, D. V.; Hutchinson, C. R.; McDaniel, R. Biochemistry
2003, 42, 72-79. (b) Caffrey, P. ChemBioChem 2003, 4, 654-657.
(16) Wu, J.; Zaleski, T. J.; Valenzano, C.; Khosla, C.; Cane, D. E. J. Am. Chem.
Soc. 2005, 127, 17393-17404.
(7) Chen, A. Y.; Schnarr, N. A.; Kim, C. Y.; Cane, D. E.; Khosla, C. J. Am.
Chem. Soc. 2006, 128, 3067-3074.
(8) Chen, A. Y.; Cane, D. E.; Khosla, C. Chem. Biol. 2007, 14, 784-792.
(9) (a) Gokhale, R. S.; Hunziker, D.; Cane, D. E.; Khosla, C. Chem. Biol.
1999, 6, 117-125. (b) Lu, H.; Tsai, S. C.; Khosla, C.; Cane, D. E.
Biochemistry 2002, 41, 12590-12597. (c) Schnarr, N.; Cronin, T.; Khosla,
C.; Cane, D. E. Unpublished observations.
(10) Hans, M.; Hornung, A.; Dziarnowski, A.; Cane, D. E.; Khosla, C. J. Am.
Chem. Soc 2003, 125, 5366-5374.
(11) Walsh, C. T.; Gehring, A. M.; Weinreb, P. H.; Quadri, L. E.; Flugel, R. S.
Curr. Opin. Chem. Biol. 1997, 1, 309-315.
(17) (a) Keatinge-Clay, A. T.; Stroud, R. M. Structure 2006, 14, 737-748. (b)
Baerga-Ortiz, A.; Popovic, B.; Siskos, A. P.; O’Hare, H. M.; Spiteller, D.;
Williams, M. G.; Campillo, N.; Spencer, J. B.; Leadlay, P. F. Chem. Biol.
2006, 13, 277-285. (c) Based on the determination of the crystal structure
of the tylosin KR1 domain and comparison with that of the DEBS KR1
domain, Keatinge-Clay has proposed a binding model to account for the
ketone facial stereospecificity of ketoreductases, as well as their preference
for unepimerized or epimerized 2-methyl-3-ketoacyl-ACP substrates. Cf.
Keatinge-Clay, A. Nat. Chem. Biol. 2007, 14, 898-908.
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DEBS Ketoreductase Domains
A R T I C L E S
Scheme 1. Possible Pathways for the Observed Epimerization
Mediated by DEBS Module 1
figuration of the remaining methyl-bearing carbons must be
fixed either immediately before or following the KS-catalyzed
chain elongation reaction. All six AT domains of DEBS have
been shown to utilize exclusively (2S)-methylmalonyl-CoA,18
with the generation of methyl-bearing centers with D-configu-
ration resulting in net inversion of configuration and retention
of the original C-2 proton of the methylmalonyl unit.19 By
contrast, generation of centers with L-methyl groups (C-8 and
C-12 of 6-dEB) results in the net loss of the original C-2 proton
of the (2S)-methylmalonyl chain extension unit.19a The epimer-
ization itself must occur either immediately preceding or just
following the KS-catalyzed condensation reaction (Scheme 1).
According to pathway A, the KS domain would first epimerize
the (2S)-methylmalonyl-SACP substrate to the corresponding
(2R)-methylmalonyl-SACP, which would then undergo inver-
sion in the course of KS-catalyzed decarboxylative condensation
with the acyl-SACP substrate donated by the proximal upstream
module. In pathway A, control of methyl stereochemistry would
be vested exclusively in the KS domain, with the KR domain
simply reducing the resultant [L-methyl]-2-methyl-3-ketoacyl-
SACP intermediate. Alternatively, in pathway B, the KS domain
first catalyzes the decarboxylative condensation of the (2S)-
methylmalonyl-SACP with inversion of configuration to gener-
ate the [D-methyl]-(2R)-2-methyl-3-ketoacyl-SACP intermedi-
ate.20 For those modules with an associated epimerase activity,
this ketoacyl thioester then would undergo epimerization
followed by diastereoselective reduction mediated by the KR
domain (for example, the KR of DEBS module 1), thereby fixing
the configuration at both C-2 and C-3 of the 2-methyl-3-
hydroxyacyl-SACP chain elongation intermediate.
Figure 4. Reconstituted DEBS modules. Incubation of different combina-
tions of [KS][AT], [ACP], and [KR] domains from different DEBS modules
(color code according to Figure 1) with the appropriate substrates will result
in the formation of triketide lactones whose stereochemistry at C-2 and
C-3 can be determined by GC-MS comparison with authentic standards.
[AT] didomains and recombinant KR domains, based on
rigorous determination of the stereochemistry of the resultant
triketide lactones (Figure 4). The results shed light on both the
timing of epimerization and the diastereoselectivity of the
individual ketoreductase domains. We also report the unexpected
configurational stability of ACP-bound 2-methyl-3-ketoacyl
polyketides.
Results
Synthesis of Triketide Ketolactone 3 and Triketide 3-Hy-
droxylactones 4a-4d. The requisite synthetic reference stan-
dards of the triketide ketolactone 3 and the four 2-methyl-3-
hydroxy diastereomers of the reduced triketide lactones 4a-
4d were prepared using a common synthetic scheme based on
the well-established Evans methodology for acyclic diastereo-
selection.14,21 The diastereomeric 2-methyl-3-ketoacyloxazoli-
dinone derivatives 6 and 10 were each prepared as previously
described from the corresponding (4R)- and (4S)-4-benzyl-3-
propyl-2-oxazolidinones via the intermediate 3-ketooxazolidi-
nones 5 and 9, respectively (Schemes 2 and 3). Stereoselective
reduction of 6 and 10 gave the corresponding acyclic syn- or
anti-2-methyl-3-hydroxy derivatives, which were readily con-
verted to the desired triketide lactones 4a-4d by treatment with
lithium hydroperoxide.21c Although hydrolysis of 11 unexpect-
edly gave a mixture of the predicted lactone 4c mixed with
diastereomer 4b, the two compounds could be easily distin-
guished by NMR and were readily resolved by GC-MS
analysis. The configuration of each newly generated stereocenter
A serious obstacle to experimental analysis of these complex
mechanistic and stereochemical issues is that the initially
generated 2-methyl-3-ketoacyl thioesters are expected to be
configurationally labile, undergoing rapid, nonenzyme catalyzed
epimerization if not immediately trapped in situ by reduction
or chain extension. With this in mind, we have used reconstituted
PKS modules to test systematically varied combinations of [KS]-
1
was unambiguously confirmed by detailed H NMR analysis
and comparison where relevant with literature data for the same
compounds prepared by alternative methods.14,22 For the
synthesis of triketide ketolactone 3, attempted direct cyclization
of 3-keto-oxazolidinones 6 or 10 gave low yields of impure
product. The desired 3 could be generated cleanly by Dess-
Martin oxidation of the mixture of triketide 3-hydroxylactones
4b and 4c (Scheme 4). The 3-keto form of 3 is in rapid
equilibrium with the enol tautomer, with the latter being the
(18) Marsden, A. F.; Caffrey, P.; Aparicio, J. F.; Loughran, M. S.; Staunton, J.;
Leadlay, P. F. Science 1994, 263, 378-380. Wiesmann, K. E.; Cortes, J.;
Brown, M. J.; Cutter, A. L.; Staunton, J.; Leadlay, P. F. Chem. Biol. 1995,
2, 583-589.
(19) (a) Weissman, K. J.; Timoney, M.; Bycroft, M.; Grice, P.; Hanefeld, U.;
Staunton, J.; Leadlay, P. F. Biochemistry 1997, 36, 13849-13855. (b) Cane,
D. E.; Liang, T. C.; Taylor, P. B.; Chang, C.; Yang, C. C. J. Am. Chem.
Soc 1986, 108, 4957-4964.
(20) Note on stereochemical conventions: Although strictly speaking it is
incorrect to mix Fischer D/L designations with the more rigorous Cahn,
Ingold, Prelog R/S nomenclature, it is often convenient to retain the D-
and L- labels when discussing a homologous series of polyketides, for which
the specific R or S designation for the same absolute configuration at a
given site might vary in response to the substitution pattern on neighboring
carbons. We have there mixed the two stereochemical conventions in the
interests of greater clarity.
(21) (a) Evans, D. A.; Ennis, M. D.; Le, T.; Mandel, N.; Mandel, G. J. Am.
Chem. Soc. 1984, 106, 1154-1156. (b) Evans, D. A.; Kim, A. S.;
Metternich, R.; Novack, V. J. J. Am. Chem. Soc. 1998, 120, 5921-5942.
(c) Evans, D. A.; Britton, T. C.; Ellman, J. A. Tetrahedron Lett. 1987, 28,
6141-6144. (d) Beck, B. J.; Aldrich, C. C.; Fecik, R. A.; Reynolds, K.
A.; Sherman, D. H. J. Am. Chem. Soc 2003, 125, 12551-12557.
(22) (a) Kao, C. M.; Luo, G.; Katz, L.; Cane, D. E.; Khosla, C. J. Am. Chem.
Soc. 1994, 116, 11612-11613. (b) Jacobsen, J. R.; Cane, D. E.; Khosla,
C. Biochemistry 1998, 37, 4928-4934.
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A R T I C L E S
Castonguay et al.
Scheme 2. Synthesis of Triketide Lactones 4a and 4ba
a (a) (i) Bu2BOTf, i-Pr2NEt, propionaldehyde, CH2Cl2, -78 °C, 60%; (ii) SO3Pyr, Et3N, DMSO, 97%; (b) Sn(OTf)2, Et3N, propionaldehyde, CH2Cl2,
-78 °C, 61%; (c) Na(OAc)3BH, AcOH, 25 °C, 89%; (d) LiOOH, 0 °C, 68%; (e) DIBAL-H, THF, -78 °C, 86%; (f) LiOOH, 0 °C, 51%.
Scheme 3. Synthesis of Triketide Lactones 4c and 4da
a (a) (i) Bu2BOTf, Et3N, propionaldehyde, CH2Cl2, -78 °C, 54%; (ii) SO3Pyr, Et3N, DMSO, 93%; (b) TiCl4, i-Pr2NEt, propionaldehyde, CH2Cl2, -78
°C, 80%; (c) Zn(BH4)2, CH2Cl2, -78 °C, 99%; (d) LiOOH, 0 °C, 75%; (e) Na(OAc)3BH, AcOH, 25 °C, 29%; (f) LiOOH, 0 °C, 20%.
Scheme 4. Synthesis of Triketide Ketolactone 3a
Table 1. GC-CI-MS Retention Times for Synthetic Triketide
Ketolactone 3 and Triketide Lactones 4a-4d, as Well as the
Corresponding TMS Derivatives 4a-TMS-4d-TMS
retention time
(min:s)
retention time
(min:s)
lactone
TMS-lactone
3
6:09
6:43
6:50
6:33
6:52
4a
4b
4c
4d
4a-TMS
4b-TMS
4c-TMS
4d-TMS
6:55
7:02
6:49
7:17
a (a) Dess-Martin periodinane, CH2Cl2, rt, 38%.
predominant form in water, as previously reported.23 Detailed
synthetic protocols and spectroscopic data can be found in the
Supporting Information.
Scheme 5. Derivatization of Triketide Lactones 4a-4d
Mass Spectrometric Analysis of Triketide Ketolactone 3
and Triketide Lactones 4a-4d. For routine analysis of
enzymatically generated triketide lactones, we wanted a method
that would be simple, sensitive, and robust. Although the
individual diastereomers of lactones 4a-4d can be readily
1
distinguished by H NMR, such analysis normally requires a
minimum of 1-10 µmol of purified material. By contrast, GC-
MS analysis is at least 3-4 orders of magnitude more sensitive
and requires minimal manipulation of the sample prior to
analysis. Indeed, the triketide ketolactone 3 and the four triketide
lactone diastereomers 4a-4d were readily resolved by capillary
GC and detected by chemical ionization (CI)-MS (Table 1).
The sensitivity and resolution were further improved by analysis
of the corresponding trimethylsilyl ether derivatives 4a-TMS-
4d-TMS, which were conveniently generated by treatment of
the concentrated EtOAc extract of the enzymatic incubation
mixture with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)
containing 1% (v/v) TMS-imidazole (Scheme 5, Figure 5, and
Table 1). In this manner, as little as 1 nmol of each TMS-lactone
could readily be detected. For analysis of enzymatic incubation
mixtures, the detection sensitivity was enhanced with concomi-
tant elimination of signals from nonenzymatic side products by
the use of extracted ion monitoring (XIM) of the parent [M +
H]+ (m/z 245). Alternatively, the TMS-lactones 4a-TMS-4d-
TMS could be analyzed by 70 eV electron impact (EI) MS,
monitoring the m/z 171 base peak corresponding to the [M -
TMS]+ species.
Stereochemical Assignment of the Triketide Lactone
Formed by [KS6][AT6], [ACP6], and [KR6]. DEBS module
6 + TE has been shown to catalyze conversion of diketide 2
and methylmalonyl-CoA in the presence of NADPH to triketide
lactone 4a as the exclusive product.5 We have recently reported
that incubation of the reconstituted mixture of recombinant
[KS6][AT6] didomain, [ACP6], and [KR6] with the same
substrates resulted in formation of triketide lactone 4 of
unspecified stereochemistry at C-2 and C-3, as determined by
TLC-phosphorimaging.8 To validate the sensitivity and accuracy
of the mass spectrometric analysis and to establish the stereo-
(23) Luo, G.; Pieper, R.; Rosa, A.; Khosla, C.; Cane, D. E. Bioorg. Med. Chem.
1996, 4, 995-999.
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13762 J. AM. CHEM. SOC. VOL. 129, NO. 44, 2007

DEBS Ketoreductase Domains
A R T I C L E S
Table 2. Identification of Triketide Lactone Isolated after
Incubation of [KS][AT], [ACP], and [KR] Domains from Different
DEBS Modules
[KS][AT]
didomain
[ACP]
[KR]
triketide
lactone
domain
domain
[KS6][AT6]
[KS6][AT6]
[KS3][AT3]
[KS3][AT3]
[ACP6]
[ACP6]
[ACP3]
[ACP3]
[KR6]
[KR2]
[KR6]
[KR2]
4a
4a
4a
4a
using the [KS][AT] didomains from DEBS modules 3 or DEBS
module 6 in combination with either DEBS [KR2] or [KR6],
both of which had been shown to generate the triketide lactone
product 4, with reduction of the 3-ketotriketide intermediate by
KR2 being more efficient than KR6-catalyzed reduction.8 In
each case, we used an ACP domain derived from the same
module as the [KS][AT] didomain, since we had previously
established that each KS domain has a significant preference
for its cognate ACP domain.7 Notably, the KS3 domain is from
DEBS module 3 whose normal tetraketide product eventually
carries an epimerized L-methyl group, corresponding to the C-8
methyl of 6-dEB (1). KS6 on the other hand generates a product
with a D-methyl group, as found at C-2 of 6-dEB (1) produced
by the complete DEBS, or C-2 of 4a, produced by DEBS
module 6 + TE. As summarized in Table 2, all four combina-
tions of [KS][AT] and [KR] domains gave exclusively triketide
lactone isomer 4a. In each case, the [KS][AT] didomain was
preincubated with diketide-SNAC 2 for 1 h, followed by
addition of the [ACP] and [KR] proteins along with the
remaining substrates, methylmalonyl-CoA and NADPH. Ad-
dition of the [ACP] and [KR] domains together or with an
interval of 45 min between introduction of [ACP] and addition
of the [KR] domain all gave triketide lactone 4a of unchanged
stereochemical purity. The 45 min delay did result in a small
increase in the proportion of unreduced triketide ketolactone 3
in the product mixture due to competing buffer-catalyzed release
of the ACP-bound acyclic 3-ketotriketide, as previously reported.
The use of DEBS TE to catalyze the enzymatic release of the
triketide lactone in place of the usual base-catalyzed hydrolysis/
acid cyclization protocol also had no effect on the observed
stereochemical purity of the resultant triketide lactone product
4a, thereby ruling out any influence of the base treatment on
the configurational integrity of the product.
Stereochemistry of the Triketide Lactone Formed by the
Hybrid Module [KS3][AT3][KR5][ACP5][TE]. We have
previously reported the construction of a chimeric module
engineered by fusing the [KS3][AT3] chain elongation didomain
to the reductive [KR5][ACP5] didomain, along with an ap-
pended DEBS TE domain.7 Incubation of this [KS3][AT3][KR5]-
[ACP5][TE] with diketide 2 and methylmalonyl-CoA in the
presence of NADPH led to a mixture of triketide ketolactone 3
and the reduced triketide lactone 4 of unspecified configuration.
GC-CI-MS analysis has now established that this chimeric
module produces exclusively triketide lactone 4a, with no trace
of diastereomers 4b-4d (Scheme 6). The formation of triketide
lactone 4a by the hybrid module is completely consistent with
the results obtained with the reconstituted modules, as well as
the exclusive formation of 4a by the native DEBS module 5 +
TE itself.5
Figure 5. GC-CI-MS analysis of the four TMS-derivatized diastereomers
of (4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-δ-lactones (4a-
TMS-4d-TMS).
Figure 6. GC-CI-MS (XIC at m/z 245) of the derivatized triketide lactone
4a-TMS formed by incubation of [KS6][AT6], [ACP6], and [KR6] with
diketide-SNAC 2, methylmalonyl-CoA, and NADPH.
chemistry of the resulting triketide lactone, the recombinant
[KS6][AT6], [ACP6], and [KR6] fragments from DEBS module
6 were incubated for 1.5 h with diketide-SNAC 2, methylma-
lonyl-CoA, and NADPH, followed by base-catalyzed hydrolysis
of the ACP-bound product and acidification. Following treat-
ment of the crude EtOAc extract with BSTFA containing 1%
(v/v) TMS-imidazole, GC-CI-MS analysis with XIM detection
of the m/z 245 components revealed the presence of a single
peak, retention time 6:55 min, corresponding to TMS-lactone
4a-TMS (Figure 6). GC-CI-MS analysis of the underivatized
extract also confirmed the formation of the single triketide
lactone stereoisomer 4a, retention time 6:43 min, accompanied
as previously reported by the unreduced triketide ketolactone
3. Preincubation of [KS6][AT6] with diketide-SNAC 2 for 1 h
to allow complete acylation of the active Cys of the KS,8
followed by addition of [ACP6] and [KR6] along with methyl-
malonyl-CoA and NADPH, gave identical results. Decreasing
the concentration of [KR6] had no effect on the stereochemical
purity of the product nor, most interestingly, did delaying the
addition of [KR6] and NADPH by 20 or 45 min following the
addition of [ACP6] and methylmalonyl-CoA. In all cases, the
exclusive triketide lactone product formed was diastereomer 4a,
under conditions that could easily have detected as little as
2-3% of any of the other three diastereomers, 4b-4d.
Incubation of Heterologous Combinations of [KS][AT],
[ACP], and [KR] Domains. Having established the sensitivity
and accuracy of the GC-CI-MS analysis of the diastereomers
of triketide lactone 4, we carried out a series of incubations
Reduction of the ACP-Bound 3-Ketotriketide by Sodium
Borohydride. The formation of triketide lactone 4a carrying
9
J. AM. CHEM. SOC. VOL. 129, NO. 44, 2007 13763

A R T I C L E S
Castonguay et al.
Scheme 6. Stereochemistry of Triketide Lactone Formation
Catalyzed by Hybrid Module [KS3][AT3][KR5][ACP5][TE]
an L-hydroxy group at C-3 as a result of incubations with either
KR2 or KR6 is consistent with the known stereochemistry of
3-ketoacyl thioester reduction that is catalyzed by each ketore-
ductase domain.5 Furthermore, the D-methyl stereochemistry of
4a is consistent with the established stereospecificity of DEBS
modules 2 and 6. On the other hand, the exclusive formation
of lactone 4a with an unepimerized D-2-methyl group is
unexpected for the KS3 domain of DEBS module 3. These
results also do not distinguish whether either KS3 or KS6
generates exclusively the D-2-methyl-3-ketoacyl triketide prod-
uct, or whether the KR domain selectively reduces only the D-2-
methyl diastereomer that may be present in an epimeric mixture
of chain-elongation intermediates. Notably, allowing the initially
generated ACP-bound 3-ketoacyl triketide to incubate for an
additional 45 min prior to introduction of ketoreductase had no
detectable effect on the diastereomeric purity of the resulting
triketide lactone 4a. We therefore sought to trap the ACP-bound
2-methyl-3-ketoacyl triketide by in situ reduction with NaBH4,
in order to establish directly the configuration of the C-2 methyl
group.
Figure 7. Trapping of ACP-bound 2,4-dimethyl-3-keto-5-hydroxytriketide
by NaBH4 reduction.
The R-methyl group of a â-ketoacyl thioester or oxoester
normally undergoes rapid epimerization even at neutral pH.
Indeed, we observed that room temperature incubation of the
model â-ketoester, ethyl 2-methylacetoacetate, in 100 mM
sodium phosphate in D2O, pD 7.2, showed a t1/2 for proton
exchange of 4.7 min, as monitored directly by 1H NMR. Were
the ACP-bound 2-methyl-3-ketoacyl thioesters to behave simi-
larly, an interval of 20 to 45 min before addition of the KR
domain to the incubation mixture would correspond to 4-9
proton-exchange half-lives, resulting in effectively complete
epimerization of the 2-methyl substituent.
Incubation of [KS3][AT3] with diketide 2 for 1 h, followed
by the addition of [ACP3] and methylmalonyl-CoA and
incubation for a further 45 min, with termination by basic
hydrolysis led to the formation of triketide ketolactone 3, as
expected. Reductive quenching of a parallel enzymatic reaction
after 45 min of incubation by addition of an 80-fold excess of
NaBH4, followed by base hydrolysis (Figure 7), gave an ∼1:
1.6 mixture of triketide lactones 4a and 4b, as established by
the standard GC-CI-MS analysis of the corresponding TMS-
derivatives (Figure 8). Neither 4c nor 4d could be detected in
the product mixture. The acyclic ketone had thus been reduced
nonstereospecifically by NaBH4, as expected, but the methyl
group was clearly only in the D configuration. When NaBH4
was used to reductively quench an incubation mixture containing
[KS6][AT6] and [ACP6], a 1.3:1 mixture of 4a and 4b was
obtained, establishing that both KS3 and KS6 generate the
identical (2R,4R,5R)-2,4-dimethyl-3-keto-5-hydroxypentanoyl-
ACP product from diketide 2 and methylmalonyl-CoA.
To validate the reliability of the NaBH4 quenching protocol,
a series of control experiments was carried out (Table 3). To
rule out the possibility that the observed quenching products
result from NaBH4 reduction of free triketide ketolactone 3 that
may have been released from the ACP by buffer-catalyzed
Figure 8. GC-CI-MS (XIC at m/z 245) of TMS-derivatized triketide
lactones 4a-TMS and 4b-TMS isolated from incubation of [KS3][AT3]
and [ACP3] with diketide-SNAC 2 and methylmalonyl-CoA, followed by
quenching with NaBH4 and base hydrolysis.
Table 3. NaBH4 Quenching of ACP6-Bound 3-Ketoacyl Triketide
triketide lactones
treatment
(ratio)
NaBH4 (15 min)
4a, 4b
(1.3:1)
3 plus NaBH4 in MeOH
4a, 4b, 4c, 4d
(1:16:6.5:7)
4a, 4b, 4c, 4d
(1:5.5:29:5.8)
4a, 4b
3 plus NaBH4 in 100 mM
phosphate, pH 7.2
NaBH4 reduction of
protein ultrafiltration retentate
(1) EtOAc extraction;
(2.6:1)
4a, 4b, 4c, 4d
(2) NaBH4 treatment of aqueous phase
(2:6:4:1)
hydrolysis, a synthetic sample of 3 was treated with NaBH4
either in MeOH or in the normal pH 7.2 incubation buffer. In
both cases, all four triketide lactones were formed, including
significant proportions of 4c and 4d carrying an L-2-methyl
group. In fact, under these conditions, the bulk of the unreduced
ketolactone 3 was recovered, presumably due to the predomi-
nance of the enol-lactone tautomer. To rule out the alternative
possibility that 4a is being formed by NaBH4 reduction of the
free seco-acid form of prematurely released 2,4-dimethyl-3-keto-
5-hydroxypentanoic acid, the enzymatic incubation mixture was
subjected to ultrafiltration prior to NaBH4 reduction of protein-
bound triketide in the retentate, resulting in exclusive formation
of triketide lactones 4a and 4b. Interestingly, when the incuba-
tion mixture was pre-extracted with EtOAc, in an attempt to
remove any protein-free organic side products, reduction of the
resulting cloudy solution gave all four triketide lactones, but
with 4a and 4b still being the predominant components of the
9
13764 J. AM. CHEM. SOC. VOL. 129, NO. 44, 2007

DEBS Ketoreductase Domains
A R T I C L E S
quenching mixture. Taken together, these experiments establish
firmly that the triketide lactones 4a and 4b result exclusively
from NaBH4 reduction of ACP-bound 2,4-dimethyl-3-keto-5-
hydroxytriketide and that this intermediate carries exclusively
a D-2-methyl group.
the substitution pattern through strict selection of the proper
building block, the particular combination of KR, DH, and ER
domains determines the ultimate oxidation level and stereo-
chemistry of the growing polyketide chain. When only a KR
domain is present in a given module, the reduction stereospeci-
ficity of the KR domain is preserved in the configuration of
the corresponding hydroxyl group in the eventually formed
macrolide or polyether product. For modules carrying additional
tailoring domains, however, the stereochemistry of the â-hy-
droxyacyl-ACP intermediate is obscured by DH-catalyzed
dehydration, while the double bond geometry of the resulting
enoyl-ACP intermediate can be concealed as well by subsequent
ER-catalyzed reduction.
Heterologous expression and in Vitro characterization of
individual PKS modules continues to be an extremely useful
tool for biochemical investigation of PKS-catalyzed chain
elongation, allowing detailed probing of the mechanism, ster-
eochemistry, and substrate specificity of a variety of modules
and their constituent domains. For example, by swapping
heterologous KR domains into DEBS module 2, we first
demonstrated that the stereochemistry of KR-catalyzed reduc-
tions is an intrinsic property of the individual KR domain,
independent of substrate structure or modular context.14 More
recently, using mutants of PICS module 2 in which the DH or
KR domains had been selectively inactivated, we provided direct
evidence for the sequence of biochemical reactions catalyzed
by PICS module 2 and determined the previously cryptic
stereochemistry of the D-hydroxy specific KR2 domain.16
Nonetheless, studies with intact modules still face a number of
experimental obstacles, including the strict substrate specificity
of the constituent AT domains, the labor-intensive effort required
for the construction of mutant or, especially, hybrid modules,
even with improved, structure-based insight into interdomain
boundaries,6,26 and the challenges to analyzing single-turnover
reactions. While recently developed FT-MS-MS methods
allow the detection and structural identification of sub-nanomole
levels of protein-bound polyketide chain elongation intermedi-
ates,27 such protein-MS methods cannot be used for the
determination of the stereochemistry of protein-bound interme-
diates, nor are they yet suited for routine measurements of
enzyme kinetics.
Incubations with DEBS [KR30]. The recently reported
crystal structure of DEBS KR1 revealed a pseudodimeric
domain organization, consisting of an N-terminal “structural”
subdomain and a C-terminal “catalytic” subdomain.17a It was
suggested that this catalytic subdomain might also harbor the
requisite methyl epimerase activity that is associated with DEBS
module 1.17a The epimerized L-methyl group at C-8 of 6-dEB
is installed by DEBS module 3. When the 2-methyl-3-ketoacyl-
ACP triketide intermediate of [KS3][AT3] incubations is
reduced either enzymatically by [KR2] or [KR6] or by reductive
trapping with NaBH4, however, it is clear that the initial product
of KS3-catalyzed chain elongation has the unepimerized 2-D-
methyl configuration. We therefore wished to examine the
possibility that the reductively silent KR30 domain, which lacks
a consensus NADPH binding site,2a,24 might harbor cryptic
methyl epimerase activity. Based on the previously defined
consensus boundaries for KR domains,8,25a we used PCR to
amplify the KR30 component of DEBS module 3 as an
N-terminal His-tag protein, using DNA from plasmid pRSG34,
encoding DEBS module 3 + TE, as a template.5 Expression
and purification as described for the DEBS KR2 and KR6
domains8 gave a 30 mg/L culture of recombinant KR30, whose
purity and molecular weight were verified by SDS-PAGE and
MALDI-TOF MS.
Overnight incubation of recombinant [KR30] with NADPH
and trans-1-decalone, a useful surrogate substrate for DEBS
KR domains,25 confirmed the complete absence of detectable
KR30 reductase activity, as assayed by both UV and GC-MS.
Attempted monitoring of any epimerase activity by incubation
of [KR30] with trans-1-decalone in deuterated 100 mM sodium
phosphate (pD 7.2) did not lead to any detectable deuterium
exchange, as determined by GC-MS.
When recombinant [KR30] was coincubated with [KS6][AT6]
and [ACP6] in the presence of diketide 2 and methylmalonyl-
CoA, followed by quenching with NaBH4, a mixture of triketide
lactones 4a and 4b was obtained, without any detectable forma-
tion of L-methyl products 4c and 4d. Alternatively, 45-min incu-
bation of KR30 with the product of [KS6][ACP6]-catalyzed
chain extension, followed by enzymatic reduction with [KR6]
and NADPH, resulted in exclusive formation of triketide lactone
4a. Identical results were obtained using the reconstituted [KS3]-
[AT3] and [ACP3] mixture. The recombinant KR30 therefore
has no detectable methyl epimerase activity, although we cannot
rigorously rule out the possibility that native KR30 in its natural
DEBS module 3 context might still serve as an epimerase.
More recently, the ready availability of recombinant PKS
domains and didomains has opened up a wealth of exciting
opportunities that has already dramatically deepened our
understanding of PKS structure and mechanism. By reconstitut-
ing chimeric PKS systems from combinations of heterologous
domains, it has become possible to systematically probe the
substrate specificity and stereochemistry of a variety of PKS
domains.6-8,25a We have recently reported the results of
individual incubations of a reconstituted PKS system consisting
of the DEBS [KS3][AT3] didomain and its cognate acyl carrier
partner [ACP3] with recombinant DEBS ketoreductase domains
[KR1], [KR2], and [KR6] in the presence of diketide-SNAC 2
and methylmalonyl-CoA.8 Although KR1, which normally
reduces a diketide substrate, failed to reduce the ACP-bound
Discussion
Each module in a modular polyketide synthase is responsible
for a discrete round of polyketide chain elongation and
functional group modification. While the AT domain controls
(26) Tang, Y.; Kim, C. Y.; Mathews, I. I; Cane, D. E.; Khosla, C. Proc. Natl.
Acad. Sci. U.S.A. 2006.
(27) (a) Hicks, L. M.; Mazur, M. T.; Miller, L. M.; Dorrestein, P. C.; Schnarr,
N. A.; Khosla, C.; Kelleher, N. L. ChemBioChem 2006, 7, 904-907. (b)
Chan, Y. A.; Boyne, M. T., II.; Podevels, A. M.; Klimowicz, A. K.;
Handelsman, J.; Kelleher, N. L.; Thomas, M. G. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 14349-14354.
(24) Donadio, S.; Staver, M. J.; Mcalpine, J. B.; Swanson, S. J.; Katz, L. Science
1991, 252, 675-679.
(25) (a) Siskos, A. P.; Baerga-Ortiz, A.; Bali, S.; Stein, V.; Mamdani, H.;
Spiteller, D.; Popovic, B.; Spencer, J. B.; Staunton, J.; Weissman, K. J.;
Leadlay, P. F. Chem. Biol. 2005, 12, 1145-1153. (b) Bali, S.; O’Hare, H.
M.; Weissman, K. J. ChemBioChem 2006, 7, 478-484.
9
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A R T I C L E S
Castonguay et al.
Scheme 7. Reduction of 2-Methyl-3-ketodiketide-SNAC 13 by
Recombinant KR Domains
KR6 resulted in each case in both an apparent erosion in the
intrinsic stereospecifity of ketoreduction, as evidenced by the
formation of 2-20% of the unnatural D-(3R)-hydroxydiketide-
SNAC products along with the expected L-(3S)-hydroxy-
diastereomers, and a significant reversal in diastereoselectivity,
unnatural L-(2S)-methyl diketides being generated in 3-fold
excess over the predicted D-(2R)-methyl diketide-SNAC
diastereomers.25a A similar degradation in stereoselectivity was
observed when 13 was incubated with intact DEBS module 2
or with DEBS3, harboring both modules 5 and 6.30 Since each
of these modules is known to act completely stereospecifically
when processing both natural and unnatural enzyme-bound
substrates,5,22b it is likely that the loss of stereochemical control
in the reduction of the ketodiketide-SNAC analogue 13 may
reflect the requirement that the substrate be covalently anchored
to the ACP domain for proper positioning and orientation in
the KR active site. Indeed, the results reported here confirm
that recombinant DEBS KR2 and KR6 both catalyze completely
stereospecific reductions of ACP-bound 2-methyl-3-keto-
triketides that have been generated in situ by the action of a
[KS][AT] didomain. Whether the observed ACP requirement
reflects the existence of an ACP-specific binding site on the
surface of the KR domain, as has been reported for the
interaction of the Type II ketoreductase FabG with ACP,31 or
whether the size of the ACP protein simply prevents the
misbinding observed with the SNAC analogues, is not yet clear,
although recombinant KR domains exhibit no apparent prefer-
ence for ACP domains from different DEBS modules.8
The exclusive formation of a single triketide lactone 4a in
reductions catalyzed by DEBS KR2 or KR6 in combination with
either the [KS3][AT3] or [KS6][AT6] didomains does not
establish whether either KR2 or KR6 is capable of discriminat-
ing against ACP-bound substrates with the unnatural L-2-methyl
configuration, since only the D-2-methyl substrate generated by
either KS3 or KS6 is presented to the KR domain, as established
conclusively by the reductive quenching with NaBH4. The same
is also true of the diastereoselectivity of KR5, as deduced from
the formation of triketide lactone 4a by the chimeric [KS5]-
[AT5][KR5][ACP5][TE] module. Similarly, the reported gen-
eration of the syn-(2R,3S)-2-methyl-3-hydroxypentanoic acid by
the chimeric module DKS1-2, corresponding to DEBS [ATL]-
[ACPL][KS1][AT2][KR2][ACP2][TE], established that epimer-
ization occurs after KS-catalyzed condensation (pathway B of
Scheme 1) and that KR2 can stereospecifically reduce the
initially generated intermediate D-2-methyl-3-ketopentanoyl-
ACP.32 No conclusions could be drawn as to whether KR2
exercises any selection against the diastereomeric L-2-methyl-
3-ketopentanoyl-ACP, which may or may not have been
transiently formed by the hybrid DKS1-2 enzyme.
2-methyl-3-keto triketide that was generated by [KS3][AT3],
both [KR2] and [KR6] catalyzed the NADPH-dependent reduc-
tion to give, after base-catalyzed release from the ACP domain,
triketide lactone 4. In experiments with [KR6], triketide lactone
4 was also accompanied by unreduced triketide ketolactone 3.
Similar results were also obtained using the same set of KR
domains in combination with DEBS [KS6][AT6] and [ACP6].
We have now established the stereochemistry of each of these
reductions and shed light on the diastereoselectivity of each
ketoreductase, using a sensitive method that can in principle
be extended to any recombinant KR domain capable of reducing
an ACP-bound 3-ketoacyl-ACP triketide.
GC-MS Analysis of Triketide Lactone Diastereomers.
The synthetic reference samples of each of the four triketide
lactone diastereomers 4a-4d can be readily separated and
analyzed by capillary GC-MS, with sensitivity down to 1 nmol
or better using chemical ionization (CI) and extracted ion
monitoring (XIM) of the parent [M + H]+ ion at m/z 245 for
the corresponding TMS derivatives.28 At this scale, each enzyme
incubation can be carried out in a convenient volume of 500
µL, using 10-20 nmol of [KS][AT] didomain in combination
with 100-150 nmol of each of the other PKS domains. Even
[KS][AT] didomains with exceptionally low turnover numbers
of 0.01 min-1 readily generate the requisite amounts of triketide
lactone in a 1-2-h incubation. With sufficient quantities of
product from a given incubation, both the free lactones and the
corresponding TMS derivatives can be subjected to the cali-
brated GC-MS analysis to reinforce to the reliability of the
analysis.
Analysis of KR Stereospecificity. Recombinant DEBS KR1
has been shown to reduce racemic 2-methyl-3-ketopentanoyl-
SNAC (13) exclusively to the syn-(2S,3R)-diketide-SNAC 2,
thereby demonstrating that this reductase is completely specific
not only for reduction on the natural diastereotopic face of the
ketone carbonyl but also for the natural L-configuration of the
adjacent 2-methyl group, as expected based on the overall
stereospecificity of DEBS module 1 (Scheme 7).25a These results
were also completely consistent with earlier results obtained
using monomodular and bimodular DEBS proteins harboring
KR1.29 Recombinant KR1 from the tylactone synthase reduces
13 exclusively to the anti-(2R,3R)-diketide-SNAC 14, again
completely consistent with the expected diastereoselectivity for
the D-2-methyl substrate.25a Unfortunately, reduction of 3-ke-
todiketide-SNAC 13 with recombinant DEBS KR2, KR5, and
Timing of the Epimerization of the 2-Methyl Group. The
exclusive formation of triketide lactone 4a, whether resulting
from mixed incubations of [KS3][AT3] with either [KR2] or
(30) A hybrid triketide synthase based on DEBS3, in which the native KS5
domain was replaced by fusion with the DEBS ATL-ACPL loading didomain
and KS1, has been reported to generate a mixture of the expected triketide
lactone and a diastereomer of unknown absolute configuration whose
mechanism of formation has not yet been satisfactorily explained. Cf.
Holzbaur, I. E.; Ranganathan, A.; Thomas, I. P.; Kearney, D. J.; Reather,
J. A.; Rudd, B. A.; Staunton, J.; Leadlay, P. F. Chem. Biol. 2001, 8, 329-
340.
(31) Zhang, Y. M.; Wu, B.; Zheng, J.; Rock, C. O. J. Biol. Chem. 2003, 278,
52935-52943.
(32) Bohm, I.; Holzbaur, I. E.; Hanefeld, U.; Cortes, J.; Staunton, J.; Leadlay,
P. F. Chem. Biol. 1998, 5, 407-412.
(28) Although GC-MS has previously been used to characterize tetrasubstituted
polyketide δ-lactones, the diastereomers cannot be differentiated on the
basis of their MS fragmentation patterns. Cf. Weissman, K. J.; Kearney,
G. C.; Leadlay, P. F.; Staunton, J. Rapid Commun. Mass. Spectrom. 1999,
13, 2103-2108.
(29) Ostergaard, L. H.; Kellenberger, L.; Cortes, J.; Roddis, M. P.; Deacon,
M.; Staunton, J.; Leadlay, P. F. Biochemistry 2002, 41, 2719-2726.
Holzbaur, I. E.; Harris, R. C.; Bycroft, M.; Cortes, J.; Bisang, C.; Staunton,
J.; Rudd, B. A.; Leadlay, P. F. Chem. Biol. 1999, 6, 189-195.
9
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DEBS Ketoreductase Domains
A R T I C L E S
[KR6] or when [KS3] is paired with KR5 in the chimeric
module, establishes firmly that the methyl group epimerization
normally associated with DEBS module 3, which ultimately
leads to generation of the L-8-methyl of 6-dEB (1), must occur
subsequent to and not prior to the KS3-catalyzed condensation
reaction. The absence of epimerization by the [KS3][AT3]
didomain, either alone or in combination with a KR domain,
suggests that the epimerization associated with DEBS module
3 is not mediated by the KS3 domain. Based on analysis of the
KR1 crystal structure, it was recently suggested that epimer-
ization might in fact be catalyzed by the KR1 domain itself.17a
A mechanism involving enolization of the â-ketoacyl-ACP ester
and conjugate reduction of the resulting â-hydroxyenoyl-ACP
was advanced to account for diastereoselective formation of the
observed diketide. Subsequent bioinformatics analysis has
claimed to support this proposal, but direct experimental
evidence is so far lacking.33 It was also suggested that
reductively silent domains such as DEBS KR30, which lacks a
functional NADPH binding site, might in fact play a role in
methyl group epimerization. Since addition of recombinant KR30
to incubation mixtures containing either the [KS3][AT3] or
[KS6][AT6] didomains did not result in any detectable epimer-
ization, however, the proposed role for KR30 in mediating
epimerization is so far unsupported.
and configurationally stable has a number of important implica-
tions, both practical and theoretical: (1) The ACP-bound
5-hydroxyacylthioester is considerably more resistant to buffer-
catalyzed lactonization than the corresponding free thioester.
Earlier attempts in our laboratory to prepare 2,4-dimethyl-3,5-
dihydroxyheptanoyl-SNAC were repeatedly thwarted by spon-
taneous formation of the corresponding δ-lactone 4a. Although
precise kinetic measurements were not carried out, we estimated
the t1/2 for lactonization as <1-2 min. By comparison, although
buffer-catalyzed release of triketide lactones 3 or 4 from the
ACP is measurable, it generally does not exceed 5-10% of the
recovered product even after 1 h of incubation.6 Indeed DEBS
and related macrolide synthases ordinarily generate little if any
detectable triketide lactones such as 4 as coproducts of complete
polyketide chain elongation.37 (2) The remarkable configura-
tional stability of the ACP-bound 2-methyl-3-ketoacylthioester
intermediates, which retain their configurational integrity even
after 1 h at pH 7.2, conditions under which free â-ketoesters
undergo complete exchange of R-protons, makes the enzymatic
and chemical reductive trapping of these otherwise configura-
tionally labile intermediates possible. The enhanced stability of
these species might also facilitate eventual NMR determination
of the structure and conformation of the ACP-bound polyketide
intermediates.36
Role of the ACP Domain. For many years it has been
assumed that the acyl carrier protein in fatty acid and polyketide
biosynthesis merely serves to solubilize the growing hydropho-
bic fatty acid or polyketide chain while acting as an essentially
passive platform for the flexible, 18-Å phosphopantetheinate
prosthetic group whose role is to deliver attached chain
elongation intermediates to the successive FAS or PKS reaction
centers. Recent structural studies of both the DEBS [KS3][AT3]
and [KS5][AT5] didomains2a,26,34 show that the active sites of
the constituent KS and AT domains are separated by more than
80 Å. The simplistic “swinging-arm” model for the ACP must
therefore be discarded. A structure-based, dynamic model of
ACP biochemistry is now required, in which the ACP protein
itself undergoes significant segmental motion in order to deliver
the phosphopantetheinyl arm and its tethered substrates to the
various active sites of each module. Considerable support for
the notion of a dynamic ACP has also come from the recently
reported crystal structure of the topologically distinct yeast FAS
and a closely related fungal FAS.35 Furthermore, although all
DEBS ACP domains appear to share a modest level of sequence
similarity and are predicted to have a common 4-helix
topology,2a,36 there is a growing body of evidence that ACP
domains are not simply interchangeable and that individual ACP
domains preferentially interact with the KS domains from the
same module.7,34
The simplest explanation of the remarkable chemical and
configurational stability of the ACP-bound polyketides is that
the phosphopantetheinate-tethered substrate does not swing
freely in solution but is bound by the acyl-carrier protein itself.
An extended binding conformation of the substrate would
suppress spontaneous lactonization or other intramolecular
reactions. Similarly, an appropriately placed kink in the
polyketide chain can prevent the â-ketone and thioester carbonyl
groups from becoming coplanar, thereby raising the apparent
pKa by 10-15 orders of magnitude, more than sufficient to
retard or completely suppress undesirable loss of stereochemical
integrity during the normal assembly line synthesis of the
polyketide.38 In fact, there have been several recent reports of
ACP binding of fatty acid chain elongation intermediates. E.
coli ACP contains a cavity in which acyl chains 6, 7, or 10
carbon atoms in length can be observed interacting with
hydrophobic amino acid residues, well shielded from the
buffer.39 The crystal structure of a heptanoyl-ACP clearly shows
the conformation of the acyl chain in which the â-methylene
and the thioester carbonyl are out of plane. The recently reported
crystal structure of the Saccharomyces cereVisiae FAS exhibits
a stalled ACP at the active site of KS, with ACP-bound substrate
well buried in a groove on the ACP with only the reactive
portion of the substrate exposed.35a This observation has led to
a proposed “switch-blade” model, in which the phosphopan-
tetheinyl residue folds back on the protein in order to allow the
attached substrate to be bound in a groove in the ACP, with
The unexpected finding that KS-generated 2,4-dimethyl-3-
keto-5-hydroxyheptanoyl-ACP triketides are both chemically
(33) Starcevic, A.; Jaspars, M.; Cullum, J.; Hranueli, D.; Long, P. F. Chem-
BioChem 2007, 8, 28-31. The specific mechanism suggested for enoyl-
ACP reduction is unlikely, since it posits nucleophilic attack of NADPH
on a carbon bearing a negatively-charged enolate oxygen. It is also
implausible that different KR domains would reduce 2-methyl-3-ketoacyl-
ACP substrates by two distinct mechanisms, depending on the reaction
stereospecificity.
(37) Aromatic polyketide synthases generate long-chain, poly-â-ketoacyl-ACP
intermediates that do not undergo premature aldol cylizations, in contrast
to the highly reactive protein-free substrates which rapidly cyclize at the
triketide or tetraketide stage unless prevented by masking of the ketone
groups. Cf. Harris, T. M.; Harris, C. M.; Hindley, K. B. Fortschr. Chem.
Org. Naturst. 1974, 31, 217-282.
(34) Tang, Y.; Kim, C. Y.; Chen, A. Y.; Cane, D. E.; Khosla, C. Chem. Biol.
2007, 14, 931-943.
(38) The Evans â-ketoimides 5 and 9 are widely used for alkylation due to
their high enantiomeric purity and configurational stability which due to
conformational constraints that suppress epimerization of the C-2 methyl
group; cf. ref 21.
(35) (a) Leibundgut, M.; Jenni, S.; Frick, C.; Ban, N. Science 2007, 316, 288-
290. (b) Jenni, S.; Leibundgut, M.; Boehringer, D.; Frick, C.; Mikolasek,
B.; Ban, N. Science 2007, 316, 254-261.
(36) Alekseyev, V. Y.; Liu, C. W.; Cane, D. E.; Puglisi, J. D.; Khosla, C. Protein
Sci. 2007, 16, 2093-2107.
(39) Roujeinikova, A.; Simon, W. J.; Gilroy, J.; Rice, D. W.; Rafferty, J. B.;
Slabas, A. R. J. Mol. Biol. 2007, 365, 135-145.
9
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A R T I C L E S
Castonguay et al.
agitated under nitrogen for 10 min. The sample was then used directly
for GC-MS analysis.
the tethered substrate being flicked out upon binding to the
appropriate FAS domain.
Incubation of DEBS Domains and Extraction of Enzymatic
Products. [KS][AT] didomain (40 µM) was incubated with diketide-
SNAC (2) (5 mM) and TCEP (5 mM) in 100 mM sodium phosphate,
pH 7.2 at 30 °C for 1 h in a typical volume of 500 µL. [ACP] (200
µM), [KR2] (200 µM) or [KR6] (300 µM), methylmalonyl-CoA (300
µM), and NADPH (2 mM) were added, and the mixture was incubated
at 30 °C for 1.5 h. Alternatively, all enzymes and substrates were
combined for 1.5 h or there was a delay of 20-45 min between the
addition of the [ACP] and [KR] proteins along with their respective
substrates. The triketide was hydrolytically released from the ACP by
addition of 200 µL of aq 0.5 M NaOH and incubation at 65 °C for 20
min, followed by addition of 100 µL of 1.5 M HCl. Alternatively, the
mixture was treated with DEBS TE (100 µM) for 50 min at 30 °C.
The reaction products were isolated by extraction with ethyl acetate (4
× 700 µL), the organic phase was concentrated in Vacuo, and the residue
was derivatized prior to GC-MS analysis.
Conclusions
In summary, we have described a convenient, sensitive, and
robust system for the analysis of PKS ketoreductase stereospeci-
ficity. In principle, the method can be extended to any
combination of [KS][AT] and [KR] domains, provided that the
KS domain be able to utilize the diketide-SNAC 2 or related
substrate analogue and that the paired recombinant KR domain
be capable of processing the KS-generated, ACP-bound inter-
mediate. The experimental system can be further expanded by
using the Sfp phosphopantetheinyl transferase to generate
unnatural malonyl-ACP derivatives. In each case, the relevant
triketide lactone standards can be readily prepared using variants
of the Evans oxazolidinone chemistry or related methods for
acyclic stereoselection. The use of reconstituted PKS systems
made up of dissected domains has already established the
stereochemical course of KS and KR-catalyzed reactions, shed
light on the relative timing of methyl group epimerization, and
revealed unexpected configurational stability of ACP-bound
polyketide intermediates. Further studies will contribute to the
solution of critical questions in PKS enzymology and should
provide valuable guidelines for the rational design of combi-
natorial biosynthetic strategies.
NMR Analysis of Ethyl 2-Methylacetoacetate Racemization. Ethyl
2-methylacetoacetate was dissolved in 100 mM sodium phosphate in
1
deuterated water, pD 7.2. H NMR spectra were recorded at 1, 3, 6,
10, 14, and 20 min. The intensity of the R proton signal was measured
as a function of time, and the data were fit to the first-order decay
1
expression to calculate the t1/2 for proton exchange. H, 13C, COSY,
and HSQC spectra NMR in CDCl3 on a 35 mM sample of ethyl
1
2-methylacetoacetate were used to fully assign the H and 13C NMR
spectra.
Incubation with Hybrid Module [KS3][AT3][KR5][ACP5][TE].
The plasmid pAYC46 harboring the hybrid module [KS3][AT3][KR5]-
[ACP5][TE] was transformed into E. coli BAP1 cells (incorporating
the sfp gene for in vivo phosphopantetheinylation of ACP)43 and used
for expression and purification of the module as previously described.7
The hybrid protein (40 µM), in a volume of 15 mL, was incubated
with diketide-SNAC 2 (4 mM), methylmalonyl-CoA (1.5 mM, added
in three portions separated by 1 h), and NADPH (2 mM) in 100 mM
sodium phosphate, pH 7.2 overnight at 30 °C. The reaction was
quenched by addition of 2 mL of 1 M HCl. The protein was removed
by centrifugation, the supernatant was extracted with ethyl acetate (5
× 20 mL), and the organic extract was concentrated in Vacuo. The
residue was dissolved in BSTFA (100 µL) and TMS-imidazole (1 µL)
and analyzed by GC-MS.
Sodium Borohydride Reduction of ACP-Bound 3-Keto-2-methyl-
triketide. Incubations using mixtures of DEBS [KS][AT] and [ACP]
were carried out as described above without addition of KR domain.
Following addition of [ACP] and methylmalonyl-CoA, the mixture was
incubated for 45 min before quenching with sodium borohydride (0.3
mg) which was added directly to the buffered solution followed by
slow rotation for 15 min at room temperature. The resultant mixture
of triketide lactones was released from the ACP by the usual basic
hydrolysis-acidification protocol. In a control incubation, 45 min after
the addition of [ACP] and methylmalonyl-CoA protein-free side
products were removed by ultrafiltration using an UltraFree-MC unit
(5000 MWCO). The reaction mixture was concentrated to a final
volume of ∼40 µL, and the retentate was diluted to a volume of 500
µL with 100 mM sodium phosphate buffer, pH 7.2 before reductive
quenching with NaBH4 for 15 min. Protein-free side products were
also removed by extraction with ethyl acetate. After 45 min of
incubation with [ACP] and methylmalonyl-CoA, the aqueous phase
was extracted with 2 × 700 µL of ethyl acetate, followed by reduction
of the aqueous phase with NaBH4 and workup in the usual manner. In
another control reaction, synthetic triketide ketolactone 3 (0.25 mg) in
100 µL of methanol or phosphate buffer (100 mM, pH 7.2) was reacted
with NaBH4 at room temperature under agitation for 30 min. The
Materials and Methods
Material. Reagents, solvents, and substrates purchased from Sigma-
Aldrich were of the highest quality available and were used without
further purification. Restriction enzymes were from Promega. N,O-
(Bistrimethylsilyl)trifluoroacetamide (BSTFA) was purchased from
Supelco, and trimethylsilyl (TMS)-imidazole was from Pierce. Isopro-
pylthio-D-galactopyranoside (IPTG) was purchased from Invitrogen.
Preparations of plasmids pAYC02 ([KS3][AT3]),7 pAYC11 ([KS6]-
[AT6]),7 pVYA05 ([ACP3]),7 pPK223 ([ACP6]),7 pAYC60 ([KR2]),8
pAYC62 ([KR6]),8 pRSG34 (DEBS module 3 + TE),5 and pAYC46
([KS3][AT3][KR5][ACP5][TE])7 have been previously described. Ni-
NTA affinity resin was from Qiagen. PD-10 columns were from GE
Healthcare and ultrafiltration devices (UltraFree-MC 5000 MWCO and
Amicon Ultra-15 10 000 and 50 000 MWCO) were purchased from
Millipore. (2S,3R)-2-Methyl-3-hydroxypentanoic acid N-acetylcysteam-
ine thioester (2) was synthesized as described.40
Methods. All DNA manipulations were performed following
standard procedures.41 The sequence of plasmid pRCD1 was verified
directly (U. C. Davis Sequencing Facility, Davis, CA). GC-CI-MS
(CH4) and GC-EI-MS spectra were recorded on a Jeol JMS-600H
mass spectrometer using an HP-5MS capillary column (30 m × 0.25
mm, 0.25 µm film thickness) with the injector port at 250 °C and a
temperature program of 60 °C for 1 min, then 25 °C/min up to
280 °C, and 280 °C for 5 min. Previously published procedures were
employed for the purification of [KS3][AT3], [KS6][AT6], [ACP3],
[ACP6], [KR2], and [KR6] proteins and for DEBS TE.7,8,9a Protein
concentrations were determined by Bradford assay using bovine serum
albumin as the standard.42
Derivatization and Mass Spectrometric Analysis of Triketide
Lactones 4a-4d. Triketide lactones 4a-4d (0.5 mg) were dissolved
directly in 100 µL of BSTFA containing 1 µL of TMS-imidazole and
(40) Cane, D. E.; Kudo, F.; Kinoshita, K.; Khosla, C. Chem. Biol. 2002, 9,
131-142.
(41) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning, A Laboratory
Manual, Second Edition; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, NY, 1989.
(43) Pfeifer, B. A.; Admiraal, S. J.; Gramajo, H.; Cane, D. E.; Khosla, C. Science
2001, 291, 1790-1792.
(42) Bradford, M. Anal. Biochem. 1976, 72, 248-254.
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13768 J. AM. CHEM. SOC. VOL. 129, NO. 44, 2007

DEBS Ketoreductase Domains
A R T I C L E S
reaction was then quenched with 1 M HCl (50 µL), the reaction mixture
was extracted with ethyl acetate, and the organic residue derivatized
with BSTFA containing 1% (v/v) TMS-imidazole.
250 mM imidazole, 10 column volumes). The eluted protein was
concentrated by ultrafiltration, the buffer was exchanged for storage
buffer (100 mM sodium phosphate, pH 7.2, 10% glycerol) on a PD-10
column, and the protein was concentrated by ultrafiltration and flash
frozen at -78 °C. MALDI-TOF of the purified protein (bovine serum
albumin as internal standard): predicted (without N-terminal Met)
51 111 Da; observed, doublet of peaks at 51 040 and 51 191 Da.
Incubations with Added DEBS [KR30] Protein. [KS][AT] dido-
main was incubated with diketide-SNAC 2 for 1 h as described above.
[ACP] and [KR30] were then added along with methylmalonyl-CoA
and NADPH. After 45 min, [KR6] was added and the incubation
continued for 1.5 h. Alternatively, the incubation was quenched with
NaBH4 for 15 min, and the resulting products were isolated and
derivatized as described above.
Cloning, Expression, and Purification of DEBS [KR30] from
DEBS. Plasmid pRSG34 harboring DEBS module 3 + TE was used
as a template for amplification of [KR30] by PCR using the primers
5′-CACACACATATGGCCTCCGACGAGCTGGCCTACC-3′ (NdeI
restriction site underlined) and 5′-CACACAGAATTCTTAAAGCC-
CCGCGAGCCGTTGTGCC-3′ (EcoRI underlined and stop codon in
italics). The resulting PCR amplicon was digested with NdeI and EcoRI
and cloned into the corresponding restriction sites in the expression
vector pET28a. The resulting plasmid, pRCD1, encodes the [KR30]
with an N-terminal His-tag and the amino acids from the YRVSW to
the RLAGL consensus sequences.5 The plasmid pRCD1 was trans-
formed into E. coli BL21(DE3) for protein expression. Cells were grown
with 50 µg/mL kanamycin in a superbroth (SB) medium at 37 °C up
to an OD600 of 0.6, then cooled to 18 °C, and induced with 0.2 mM
IPTG for 20 h. The cells were harvested by centrifugation (2,700 g,
10 min), then washed, and resuspended in lysis buffer (50 mM sodium
phosphate, pH 8.0, 300 mM NaCl, 10 mM â-mercaptoethanol, 10%
glycerol, 10 mM imidazole) supplemented with 1 µg/mL each of
pepstatin and leupeptin. The cells were lysed by incubation with
lysozyme (1 mg/mL) for 30 min on ice and then sonicated (3 × 30 s
cycles), and cellular debris was removed by centrifugation (20 000 g,
45 min). The supernatant was mixed with Ni-NTA resin (2 mL/L
culture) for 1.5 h at 4 °C, and the resin was poured into a column
fitted with a fritted filter. The resin was washed with lysis buffer (10
column volumes) and then eluted with elution buffer (lysis buffer with
Acknowledgment. This work was supported by NIH Grants
GM22172 (D.E.C.) and CA66736 (C.K.). R.C. is a recipient of
an NSERC postdoctoral fellowship, and A.Y.C. is a recipient
of a Stanford Graduate Fellowship. We thank Dr. Tun-Li Shen
for assistance with the mass spectrometric analysis.
Supporting Information Available: Synthesis and spectro-
scopic data for triketide lactones 3 and 4a-4d and SDS-PAGE
of recombinant DEBS [KR30]. This material is available free
of charge via the Internet at http://pubs.acs.org.
JA0753290
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