In vivo characterization of tandem C-terminal thioesterase domains in arthrofactin synthetase.
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.
Roongsawang
Niran
N
Division of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Sapporo 060-0810, Japan.
Washio
Kenji
K
Morikawa
Masaaki
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010453:Peptide Synthases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013869:Thiolester Hydrolases / Q000737:chemistry*
D010455:Peptides; D012694:Serine; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C486863:arthrofactin synthetase