C O M M U N I C A T I O N S
Scheme 1. (A) Synthesis and Analysis of ACP-Bound Substrates
and (B) DEBS DH4-Catalyzed Interconversion of 2a-ACP and
3-ACP
gave exclusively E-7-ACP (Scheme 2). The structure and stereo-
chemistry of 7-ACP were determined by chiral GC-MS and LC-
MS analysis of the derived methyl ester 7-Me, obtained by basic
hydrolysis and treatment of the liberated acid with TMS-diazo-
methane, and comparison with an authentic synthetic standard of
1
4
7
-Me.
Sequence alignments of the DEBS DH4 domain with numerous
2
409
PKS and FAS DH domains reveal conserved
HXXXGXXXXP
2571
2
and D(A/V)(V/A)(A/L)(Q/H) motifs. Site-directed mutagenesis
of the conserved active site His2409 of the DEBS DH4 domain
1
5a
abolished DEBS activity in Sac. erythraea while the analogous
His mutation also inactivates the homologous DH2 domain of the
1
5b
picromycin synthase.
Together the conserved His and Asp
residues comprise the catalytic dyad of the dehydratase, in which
the active site His acts as a general base while the Asp2571, located
4.1 Å from H2409 at the base of the substrate tunnel, is thought to
3
established by direct monitoring by LC-ESI(+)-MS , including
detection of the corresponding intact acyl-ACP and PPant ejection
fragments for both 2a-ACP and 3-ACP (Scheme 1B). Similarly,
incubation of DEBS DH4 with 3-ACP resulted in the reverse
enzyme-catalyzed hydration reaction, giving an ∼3:1 equilibrium
9
,16,17
serve as a general acid.
Our results establish definitively that the DEBS DH4 domain
catalyzes a syn elimination of water during erythromycin
biosynthesis. The prototype dehydration catalyzed by the DH
domain of the yeast FAS to give the characteristic disubstituted
(E)-enoyl-ACP intermediates of fatty acid biosynthesis also takes
1
0
mixture of 2a-ACP and 3-ACP.
We also carried out combinatorial incubations using mixtures
of recombinant PKS domains in order to generate in situ each
of the four diastereomers of 2a-2d-ACP (Scheme 2). In this
manner, a mixture of the DEBS [KS6][AT6] didomain, DEBS
ACP6, and TYLS KR1, the ketoreductase domain from module
1
1
18
place with net syn stereochemistry, as do the dehydrations
catalyzed by the DH domains of module 2 of nanchangmycin
1
9
11b
synthase and module 2 of tylactone synthase.
Indeed, the
1
of the tylactone synthase, was incubated with propionyl-SNAC
significant levels of overall sequence identity (>40%) and
similarity (>55%) and the presence of the conserved motifs
containing the catalytic dyad in more than 50 DH domains from
a wide range of modular PKS systems strongly suggest that the
formation of all (E)-unsaturated polyketide intermediates in-
volves a common syn dehydration mechanism.
(
4), methylmalonyl-CoA, and NADPH to produce anti-(2R,3R)-
1
1b
2
a-ACP.
Addition of recombinant DEBS DH4, either simul-
taneously with or subsequent to the formation of 2a-ACP,
resulted in dehydration to yield exclusively the predicted (E)-
2
-methylpent-2-enoyl-ACP (3-ACP), as confirmed by GC-MS
analysis of the corresponding acid 3 and comparison with
1
2
Acknowledgment. This work was supported by NIH Grants
GM22172 (D.E.C.) and CA66736 (C.K.) and a Welch Foundation
Grant (A.K.C.).
synthetic 3. By contrast, DEBS DH4 did not dehydrate either
syn-(2S,3R)-2b-ACP or syn-(2R,3S)-2c-ACP generated by DEBS
1
1a,c
KR1 or KR6, respectively,
to either E-3-ACP or the
corresponding Z-isomer 5-ACP, nor did DEBS DH4 dehydrate
anti-(2S,3S)-2d-ACP produced by recombinant RIFS KR7, the
KR domain from module 7 of the rifamycin synthase.
Supporting Information Available: Experimental procedures, LC-
ESI(+)-MS , and GC-MS data. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
3
3
In further confirmation of the stereochemistry of the dehydration
reaction, incubation of DEBS DH4 with anti-(2R,3R,4S,5R)-2,4-
dimethyl-3,5-dihydroxyheptanoyl-ACP (6-ACP) generated in situ
from 2b-SNAC, methylmalonyl-CoA, and NADPH by DEBS
References
(
1) Shiomi, K.; Omura, S. In Macrolide Antibiotics. Chemistry, Biology, and
Practice, 2nd ed.; Omura, S., Ed.; Academic Press: San Diego, CA, 2002;
pp 1-56.
1
1b
[
KS6][AT6] + ACP6 + TYLS KR1, as previously described,
(
2) (a) Donadio, S.; Staver, M. J.; McAlpine, J. B.; Swanson, S. J.; Katz, L.
Science 1991, 252, 675–679. Donadio, S.; Staver, M. J.; McAlpine, J. B.;
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S. F.; Roberts, G. A.; Bevitt, D. J.; Leadlay, P. F. Nature 1990, 348, 176–
Scheme 2. Stereochemistry of DEBS DH4-Catalyzed Dehydration
1
78.
(3) Each PKS module typically has three core catalytic domains: the ꢀ-ketoacyl-
ACP synthase (KS), the acyltransferase (AT), and the acyl carrier protein
(
(
ACP). Most modules also carry specific combinations of ketoreductase
KR), dehydratase (DH), and enoylreductase (ER) tailoring domains.
(
(
4) Donadio, S.; McAlpine, J. B.; Sheldon, P. J.; Jackson, M.; Katz, L. Proc.
Natl. Acad. Sci. U.S.A. 1993, 90, 7119–7123.
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003, 42, 72–79. (b) Caffrey, P. ChemBioChem 2003, 4, 654–657. (c)
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(
6) Quadri, L. E.; Weinreb, P. H.; Lei, M.; Nakano, M. M.; Zuber, P.; Walsh,
C. T. Biochemistry 1998, 37, 1585–1595.
(
7) Although it is not essential to the application of the PPant ejection
methodology, 2a-ACP and 3-ACP were cleanly resolved under the LC
conditions used.
(
8) Dorrestein, P. C.; Bumpus, S. B.; Calderone, C. T.; Garneau-Tsodikova,
S.; Aron, Z. D.; Straight, P. D.; Kolter, R.; Walsh, C. T.; Kelleher, N. L.
Biochemistry 2006, 45, 12756–12766. (b) Meluzzi, D.; Zheng, W. H.;
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1
8, 3107–3111.
(
9) Keatinge-Clay, A. J. Mol. Biol. 2008, 384, 941–953.
(
10) The equilibrium for the FAS catalyzed dehydration to a disubstituted enoyl-
ACP strongly also favors the hydrated form by ∼3:1. Cf. Brown, A.;
Affleck, V.; Kroon, J.; Slabas, A. FEBS Lett. 2009, 583, 363–368.
1
4698 J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010