In Vivo Characterization of Tandem C-Terminal Thioesterase Domains in Arthrofactin Synthetase
✍ Scribed by Niran Roongsawang; Kenji Washio; Masaaki Morikawa
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 849 KB
- Volume
- 8
- Category
- Article
- ISSN
- 1439-4227
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✦ Synopsis
Abstract
Macrocyclization of a peptide or a lipopeptide occurs at the last step of synthesis and is usually catalyzed by a single C‐terminal thioesterase (Te) domain. Arthrofactin synthetase (Arf) from Pseudomonas sp. MIS38 represents a novel type of nonribosomal peptide synthetase that contains unique tandem C‐terminal Te domains, ArfC_Te1 and ArfC_Te2. In order to analyze their function in vivo, site‐directed mutagenesis was introduced at the putative active‐site residues in ArfC_Te1 and ArfC_Te2. It was found that both Te domains were functional. Peaks corresponding to arthrofactin and its derivatives were absent in ArfC_Te1:S89A, ArfC_Te1:S89T, and ArfC_Te1:E26G/F27A mutants, and the production of arthrofactin by ArfC_Te2:S92A, ArfC_Te2:S92A/D118A, and ArfCΔTe2 was reduced by 95 % without an alteration of the cyclic lipoundecapeptide structure. These results suggest that Ser89 in ArfC_Te1 is essential for the completion of macrocyclization and the release of product. Glu26 and Phe27 residues are also part of the active site of ArfC_Te1. ArfC_Te2 might have been added during the evolution of Arf in order to improve macrocyclization efficiency.