title: In vitro catalytic activity of N-terminal and C-terminal domains in NukM, the post-translational modification enzyme of nukacin ISK-1.
abstract: Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme.
authors.affiliation: Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: momiwo94@gmail.com.
authors.forename: Chinatsu
authors.initials: C
authors.lastname: Shimafuji
authors.affiliation: Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: n.megumi3224@gmail.com.
authors.forename: Megumi
authors.initials: M
authors.lastname: Noguchi
authors.affiliation: Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: mami.nishie@gmail.com.
authors.forename: Mami
authors.initials: M
authors.lastname: Nishie
authors.affiliation: Section of Infection Biology, Department of Functional Bioscience, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan. Electronic address: jungao@college.fdcnet.ac.jp.
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Nagao
authors.affiliation: Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: raleigh.blackpearl@gmail.com.
authors.forename: Kouki
authors.initials: K
authors.lastname: Shioya
authors.affiliation: Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: zendo@agr.kyushu-u.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Zendo
authors.affiliation: Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: nakayama@agr.kyushu-u.ac.jp.
authors.forename: Jiro
authors.initials: J
authors.lastname: Nakayama
authors.affiliation: Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: sonomoto@agr.kyushu-u.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Sonomoto
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D001430:Bacteriocins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D003500:Cyclization; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D009561:Nisin / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013210:Staphylococcus / Q000378:metabolism; D013379:Substrate Specificity; D013440:Sulfides / Q000737:chemistry
chemical_list: D001430:Bacteriocins; D004798:Enzymes; D013440:Sulfides; C421186:nukacin ISK-1; D009561:Nisin; C102526:nisin A; C001520:lanthionine; D000409:Alanine
keywords: Lantibiotic; Modification enzyme; Nukacin ISK-1; Peptide engineering; Unusual amino acids