Yu-Chieh Cheng1, Wan-Ju Ke2, Shih-Tung Liu3. 1. Department of Microbiology and Immunology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 2. Department of Microbiology and Immunology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Microbiology and Immunology, Research Center for Bacterial Pathogenesis, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 3. Department of Microbiology and Immunology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Medical Research and Development Chiayi Branch, Chang Gung Memorial Hospital, Chiayi, Taiwan. Electronic address: cgliu@mail.cgu.edu.tw.
Abstract
BACKGROUND: Fengycin is a lipopeptide antibiotic synthesized nonribosomally by five fengycin synthetases. These enzymes are linked in a specific order to form the complex. This study investigates how these enzymes interact in the complex and analyzes the regions in the enzymes that are critical to the interactions. METHODS: Deletions were generated in the fengycin synthetases. The interaction of these mutant proteins with their partner enzymes in the complex was analyzed in vitro by a glutathione S-transferase (GST) or nickel pulldown assay. RESULTS: The communication-mediating donor (COM-D) domains of the fengycin synthetases, when fused to GST, specifically pulled down their downstream partner enzymes in the GST-pulldown assays. The communication-mediating acceptor (COM-A) domains were required for binding between two partner enzymes, although the domains alone did not confer specificity of the binding to their upstream partner enzymes. This study found that the COM-A domain, the condensation domain, and a portion of the adenylation domain in fengycin synthetase B (FenB) were required for specific binding to fengycin synthetase A (FenA). CONCLUSION: The interaction between the COM-D and COM-A domains in two partner enzymes is critical for nonribosomal peptide synthesis. The COM-A domain alone is insufficient for interacting with its upstream partner enzyme in the enzyme complex with specificity; a region that contains COM-A, condensation, and a portion of adenylation domains in the downstream partner enzyme is required.
BACKGROUND:Fengycin is a lipopeptide antibiotic synthesized nonribosomally by five fengycin synthetases. These enzymes are linked in a specific order to form the complex. This study investigates how these enzymes interact in the complex and analyzes the regions in the enzymes that are critical to the interactions. METHODS: Deletions were generated in the fengycin synthetases. The interaction of these mutant proteins with their partner enzymes in the complex was analyzed in vitro by a glutathione S-transferase (GST) or nickel pulldown assay. RESULTS: The communication-mediating donor (COM-D) domains of the fengycin synthetases, when fused to GST, specifically pulled down their downstream partner enzymes in the GST-pulldown assays. The communication-mediating acceptor (COM-A) domains were required for binding between two partner enzymes, although the domains alone did not confer specificity of the binding to their upstream partner enzymes. This study found that the COM-A domain, the condensation domain, and a portion of the adenylation domain in fengycin synthetase B (FenB) were required for specific binding to fengycin synthetase A (FenA). CONCLUSION: The interaction between the COM-D and COM-A domains in two partner enzymes is critical for nonribosomal peptide synthesis. The COM-A domain alone is insufficient for interacting with its upstream partner enzyme in the enzyme complex with specificity; a region that contains COM-A, condensation, and a portion of adenylation domains in the downstream partner enzyme is required.