Abstract
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-1 S-1) and PikAIII (kcat/Km 2.9 ± 0.9 m-1 S-1). The TEII exhibited some acylgroup specificity, catalyzing hydrolysis of propionyl (kcat/Km 15.8 ± 1.8 M-1 S-1) and butyryl (kcat/Km 17.5 ± 2.1 M-1 S-1) derivatives of ATL-ACPL faster than acetyl (kcat/Km 4.9 ± 0.7 M-1 S-1), malonyl (kcat/Km 3.9 ± 0.5 M-1 s-1, 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.
| Original language | English |
|---|---|
| Pages (from-to) | 48028-48034 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 277 |
| Issue number | 50 |
| DOIs | |
| Publication status | Published - 2002 Dec 13 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin. / Kim, Beom Seok; Cropp, T. Ashton; Beck, Brian J.; Sherman, David H.; Reynolds, Kevin A.
In: Journal of Biological Chemistry, Vol. 277, No. 50, 13.12.2002, p. 48028-48034.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin
AU - Kim, Beom Seok
AU - Cropp, T. Ashton
AU - Beck, Brian J.
AU - Sherman, David H.
AU - Reynolds, Kevin A.
PY - 2002/12/13
Y1 - 2002/12/13
N2 - 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-1 S-1) and PikAIII (kcat/Km 2.9 ± 0.9 m-1 S-1). The TEII exhibited some acylgroup specificity, catalyzing hydrolysis of propionyl (kcat/Km 15.8 ± 1.8 M-1 S-1) and butyryl (kcat/Km 17.5 ± 2.1 M-1 S-1) derivatives of ATL-ACPL faster than acetyl (kcat/Km 4.9 ± 0.7 M-1 S-1), malonyl (kcat/Km 3.9 ± 0.5 M-1 s-1, 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.
AB - 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-1 S-1) and PikAIII (kcat/Km 2.9 ± 0.9 m-1 S-1). The TEII exhibited some acylgroup specificity, catalyzing hydrolysis of propionyl (kcat/Km 15.8 ± 1.8 M-1 S-1) and butyryl (kcat/Km 17.5 ± 2.1 M-1 S-1) derivatives of ATL-ACPL faster than acetyl (kcat/Km 4.9 ± 0.7 M-1 S-1), malonyl (kcat/Km 3.9 ± 0.5 M-1 s-1, 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0037073685&partnerID=8YFLogxK
U2 - 10.1074/jbc.M207770200
DO - 10.1074/jbc.M207770200
M3 - Article
C2 - 12368286
AN - SCOPUS:0037073685
VL - 277
SP - 48028
EP - 48034
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 50
ER -