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J. Biol. Chem., Vol. 277, Issue 50, 48028-48034, December 13, 2002
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From the The pikromycin biosynthetic gene
cluster contains the pikAV gene encoding a type II
thioesterase (TEII). TEII is not responsible for polyketide termination
and cyclization, and its biosynthetic role has been unclear. During
polyketide biosynthesis, extender units such as methylmalonyl acyl
carrier protein (ACP) may prematurely decarboxylate to generate the
corresponding acyl-ACP, which cannot be used as a substrate in the
condensing reaction by the corresponding ketosynthase domain, rendering
the polyketide synthase module inactive. It has been proposed that TEII
may serve as an "editing" enzyme and reactivate these modules by
removing acyl moieties attached to ACP domains. Using a purified
recombinant TEII we have tested this hypothesis by using in
vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and
methylmalonyl-ACP substrates derived from either PikAIII or the loading
didomain of DEBS1 (6-deoxyerythronolide B synthase;
ATL-ACPL). The pikromycin TEII exhibited high
Km values (>100 µM) with all
substrates and no apparent ACP specificity, catalyzing cleavage of
methylmalonyl-ACP from both ATL-ACPL
(kcat/Km 3.3 ± 1.1 M
Biochemical Evidence for an Editing Role of Thioesterase II
in the Biosynthesis of the Polyketide Pikromycin*
§,,
Department of Medicinal Chemistry and
Institute for Structural Biology and Drug Discovery, Virginia
Commonwealth University, Richmond, Virginia 23219 and ¶ Department
of Microbiology and Biotechnology Institute, University of
Minnesota, Minneapolis, Minnesota 55455
1 s1) and PikAIII
(kcat/Km 2.9 ± 0.9 M1 s1). The TEII exhibited some
acyl-group specificity, catalyzing hydrolysis of propionyl
(kcat/Km 15.8 ± 1.8 M1 s1) and butyryl
(kcat/Km 17.5 ± 2.1 M1 s1) derivatives of
ATL-ACPL faster than acetyl
(kcat/Km 4.9 ± 0.7 M1 s1), malonyl
(kcat/Km 3.9 ± 0.5 M1 s1), or methylmalonyl
derivatives. PikAIV containing a TEI domain catalyzed cleavage of
propionyl derivative of ATL-ACPL at a
dramatically lower rate than TEII. These results provide the first
unequivocal in vitro evidence that TEII can hydrolyze
acyl-ACP thioesters and a model for the action of TEII in which the
enzyme remains primarily dissociated from the polyketide synthase,
preferentially removing aberrant acyl-ACP species with long half-lives.
The lack of rigorous substrate specificity for TEII may explain the
surprising observation that high level expression of the protein in
Streptomyces venezuelae leads to significant (>50%) titer decreases.
*
This research was supported by National Institute of Health
Grant GM48562.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute for
Structural Biology and Drug Discovery, 800 East Leigh St., Richmond, VA
23219. Tel.: 804-828-5679; Fax: 804-827-3664; E-mail:
kareynol@hsc.vcu.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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