Roongsawang Niran, Washio Kenji, Morikawa Masaaki
Division of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Sapporo 060-0810, Japan.
Chembiochem. 2007 Mar 26;8(5):501-12. doi: 10.1002/cbic.200600465.
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 ArfCDeltaTe2 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.
肽或脂肽的大环化发生在合成的最后一步，通常由单个C末端硫酯酶（Te）结构域催化。来自假单胞菌属MIS38的节杆菌素合成酶（Arf）代表了一种新型的非核糖体肽合成酶，它包含独特的串联C末端Te结构域，即ArfC_Te1和ArfC_Te2。为了在体内分析它们的功能，对ArfC_Te1和ArfC_Te2中假定的活性位点残基进行了定点诱变。结果发现两个Te结构域均具有功能。在ArfC_Te1:S89A、ArfC_Te1:S89T和ArfC_Te1:E26G/F27A突变体中，未出现与节杆菌素及其衍生物相对应的峰，并且ArfC_Te2:S92A、ArfC_Te2:S92A/D118A和ArfCDeltaTe2产生的节杆菌素减少了95%，而环状十一肽结构未发生改变。这些结果表明，ArfC_Te1中的Ser89对于大环化的完成和产物的释放至关重要。Glu26和Phe27残基也是ArfC_Te1活性位点的一部分。ArfC_Te2可能是在Arf的进化过程中添加的，以提高大环化效率。
