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PMID: 20304931 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Hidden function of catalytic domain in 6-methylsalicylic acid synthase for product release.

The Journal of biological chemistry ·Vol. 285 ·No. 20 ·2010-05-14 ·Pages 15637-15643

Moriguchi T, Kezuka Y, Nonaka T, Ebizuka Y, Fujii I

Abstract

Functional investigation of the proposed dehydratase domain of ATX, a 6-methylsalicylic acid synthase from Aspergillus terreus, revealed that the domain is not involved in dehydration of the beta-hydroxytriketide intermediate tethered on the acyl carrier protein but catalyzes thioester hydrolysis to release the product from the acyl carrier protein. Thus, we renamed this domain the thioester hydrolase (TH) domain. The intermediate bound to the TH domain of mutant H972A formed in the presence of NADPH was released as 6-methylsalicylic acid by both the intact ATX and by THID (a 541-amino acid region containing TH domain and its downstream) protein, in trans. Furthermore, THID showed a catalytic activity to hydrolyze a model substrate, 6-methylsalicylic acid-N-acetylcysteamine. The TH domain is the first example of a product-releasing domain that is located in the middle of a multidomain iterative type I polyketide synthase. Moreover, it is functionally different from serine protease-type thioesterase domains of iterative type I polyketide synthases.

MeSH Terms
Acyltransferases/chemistry,genetics,metabolism Amino Acid Sequence Aspergillus/enzymology Autoradiography Catalytic Domain Electrophoresis, Polyacrylamide Gel Escherichia coli/genetics Hydrolysis Ligases/chemistry,genetics,metabolism Multienzyme Complexes/chemistry,genetics,metabolism Oxidoreductases/chemistry,genetics,metabolism Plasmids
Chemicals
Multienzyme Complexes Oxidoreductases Acyltransferases 6-methylsalicylic acid synthetase Ligases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Moriguchi Tomomi
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033.
Kezuka Yuichiro
School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan.
Nonaka Takamasa
School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan.
Ebizuka Yutaka
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033.
Fujii Isao
School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan. Electronic address: ifujii@iwate-med.ac.jp.
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
1083-351X
DOI
10.1074/jbc.M110.107391
Email
ifujii@iwate-med.ac.jp
Published
2010-05-14
Epub
2010-00-19
Pages
15637-15643
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2865267
Subset
IM
External Links
PubMed source DOI: 10.1074/jbc.M110.107391 PMC Full Text View journal details
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