title: ε-Poly-L-lysine peptide chain length regulated by the linkers connecting the transmembrane domains of ε-Poly-L-lysine synthetase.
abstract: ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.
authors.affiliation: Department of Bioscience, Fukui Prefectural University, Fukui, Japan hamano@fpu.ac.jp.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Hamano
authors.affiliation: Department of Bioscience, Fukui Prefectural University, Fukui, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Kito
authors.affiliation: Department of Bioscience, Fukui Prefectural University, Fukui, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kita
authors.affiliation: Department of Bioscience, Fukui Prefectural University, Fukui, Japan.
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Imokawa
authors.affiliation: Yokohama Research Center, JNC Corporation, Yokohama, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Yamanaka
authors.affiliation: Department of Bioscience, Fukui Prefectural University, Fukui, Japan.
authors.forename: Chitose
authors.initials: C
authors.lastname: Maruyama
authors.affiliation: Department of Bioscience, Fukui Prefectural University, Fukui, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Katano
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011107:Polylysine / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011107:Polylysine; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase