Stereospecificity of the dehydratase domain of the erythromycin polyketide synthase

Article abstract of DOI:10.1021/ja107344h

The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ～3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS β-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3- hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4- dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.

Full text of DOI:10.1021/ja107344h

Published on Web 10/06/2010
Stereospecificity of the Dehydratase Domain of the Erythromycin Polyketide
Synthase
†
†
†
§
‡
Chiara R. Valenzano, Young-Ok You, Ashish Garg, Adrian Keatinge-Clay, Chaitan Khosla, and
,
†
David E. Cane*
Department of Chemistry, Box H, Brown UniVersity, ProVidence, Rhode Island 02912-9108, Departments of
Chemical Engineering, Chemistry, and Biochemistry, Stanford UniVersity, Stanford, California 94305, and
Department of Chemistry and Biochemistry, The UniVersity of Texas at Austin, 1 UniVersity Station A5300,
Austin, Texas 78712-0165
Received August 15, 2010; E-mail: David_Cane@brown.edu
Abstract: The dehydratase (DH) domain of module 4 of the
6
-deoxyerythronolide B synthase (DEBS) has been shown to
catalyze an exclusive syn elimination/syn addition of water.
Incubation of recombinant DH4 with chemoenzymatically
prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-
ACP) gave the dehydration product 3-ACP. Similarly, incuba-
tion of DH4 with synthetic 3-ACP resulted in the reverse
enzyme-catalyzed hydration reaction, giving an ∼3:1 equilbrium
mixture of 2a-ACP and 3-ACP. Incubation of a mixture of
propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the
DEBS ꢀ-ketoacyl synthase-acyl transferase [KS6][AT6] dido-
main, DEBS ACP6, and the ketoreductase domain from
tylactone synthase module 1 (TYLS KR1) generated in situ
anti-2a-ACP that underwent DH4-catalyzed syn dehydration
to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP,
syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ
by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7
Figure 1. Proposed tetraketide substrate and pentaketide intermediates of
DEBS module 4. The module has a KR, a DH, and an ER domain in addition
to the obligate KS, AT, and ACP domains.
6
,7
tion of a derivative of the corresponding (E)-∆ -anhydro-6-dEB
by mutants of the erythromycin producer Saccharopolyspora
(
(
(
[
RIFS KR7), respectively. Similarly, incubation of a mixture of
2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteaminethioester
2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS
KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-
-ACP that underwent syn dehydration catalyzed by DEBS
4
erythraea. Although the stereochemistry of the substrate for the
DEBS DH4 dehydratase is not known, the responsible ketoreduc-
tase, DEBS KR4, is predicted to generate the (3R)-diastereomer of
the 2-methyl-3-hydroxyacyl-ACP pentaketide, as deduced from the
presence of a Leu-Ala-Asp triad closely correlated with the
6
DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-
ACP (7-ACP). The structure and stereochemistry of 7 were
established by GC-MS and LC-MS comparison of the derived
methyl ester 7-Me to a synthetic sample of 7-Me.
5
formation of (3R)-3-hydroxyacyl-ACP polyketide intermediates.
Indeed, the vast majority of KR domains that are paired with a
5a,b
DH domain appear to harbor a conserved “Leu-Asp-Asp” motif.
DEBS KR4 is also predicted to belong to the class of nonepimer-
izing ketoreductases, which would give rise to a (2R)-methyl group
5
c
in the reduced product.
Of the more than 2000 nonaromatic polyketides, the vast
majority contain one or more disubstituted or trisubstituted
double bonds, most of which have E (trans) geometry.
To establish the substrate specificity and stereochemical course
of the DEBS DH4-catalyzed dehydration we used a chemoenzy-
matic strategy to prepare the requisite ACP-bound substrate and
product analogues, 2a-ACP and 3-ACP. To this end the free acids
1
Moreover, essentially all polyketides that do not themselves
display a double bond are biosynthesized by way of one or more
unsaturated polyketide chain elongation intermediates. Thus
although 6-deoxyerythronolide B (1, 6-dEB), the parent aglycone
of the erythromycin family of antibiotics, does not have any
double bonds in the final macrolactone, the responsible modular
polyketide synthase (PKS), 6-dEB synthase (DEBS), does in fact
harbor a dehydratase domain in module 4, termed DEBS DH4
2
a and 3 were each converted to the corresponding -SCoA
thioesters, 2a-SCoA and 3-SCoA, and thence to anti-(2R,3R)-2-
methyl-3-hydroxypentanoyl-ACP6 (2a-ACP) and the expected
dehydration product, (E)-2-methylpent-2-enoyl-ACP (3-ACP), from
DEBS apo-ACP6 using the phosphopantetheinyl transferase Sfp
6
(
Scheme 1A). The two ACP derivatives, which were readily
distinguished by reversed-phase LC-ESI(+)-MS, both exhibited the
2
,3
(
Figure 1).
Direct evidence for the intermediacy of an unsaturated polyketide
7
expected molecular weights. The structures were each confirmed
2
by the MS phosphopantetheinate (PPant) ejection method which
in erythromycin biosynthesis first came from disruption of the
NADPH-binding motif of the ER4 domain, resulting in accumula-
gave 2a-pant, m/z 375.33, and 3-pant, m/z 357.3, each with the
3
predicted MW, as well as MS analysis of each of the characteristic
8
PPant ejection fragments.
†
Brown University.
9
‡
Incubation of recombinant DEBS DH4 with 2a-ACP resulted
Stanford University.
§
The University of Texas at Austin.
in formation of the predicted dehydration product 3-ACP, as
10.1021/ja107344h  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 14697–14699 9 14697

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
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JA107344H
J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 14699