Hidden Function of Catalytic Domain in 6-Methylsalicylic Acid Synthase for Product Release

Tomomi Moriguchi,Yuichiro Kezuka,Takamasa Nonaka,Yutaka Ebizuka,Isao Fujii

doi:10.1074/jbc.m110.107391

Tomomi Moriguchi, Yuichiro Kezuka + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.107391

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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.

Highlights

19310139 from the Japan Society for the Promotion of Science
The conserved DH domain motif, HXXXGXXXXP, has been identified in fatty acid synthase (FAS) DH, in which the histidine residue is a catalytic residue for dehydration, [11] and the corresponding sequence H972XXXGXXXXP981 was found in ATX
We previously proposed that (i) the DH domain in Methylsalicylic acid synthase (MSAS) is involved in isomerization of a 2-transenoyl intermediate to the 3-cis-enoyl form required for ring cyclization or (ii) the DH domain catalyzes the hydrolytic release of 6-methylsalicylic acid (6MSA) tethered as thioester on the MSAS acyl carrier protein (ACP) [9]

Summary

Introduction

19310139 from the Japan Society for the Promotion of Science We demonstrated that the DH-like domain of ATX hydrolyzes the thioester bond between tetraketide intermediate and the phosphopantetheinyl arm of ACP to release the product 6MSA and that the 541-amino acid fragment of the DH-like domain region between the AT and KR domain has a thioester-hydrolyzing activity.

Results

Conclusion