Mapping and identification of the region and secondary structure required for the maturation of the nukacin ISK-1 prepeptide.
The prepeptide NukA of the lantibiotic nukacin ISK-1 consists of an N-terminal leader peptide followed by a propeptide moiety that undergoes post-translational modifications, that is, formation of unusual amino acids by NukM, cleavage of the leader peptide and transport by NukT to yield a mature peptide. To identify the region and conformation required for the maturation of prepeptide, we expressed a series of NukA mutants, mutants with the N-terminus-truncated leader peptide and site-directed mutants with conserved residues in the leader peptide of type A(II) lantibiotics, which were evaluated on the basis of the production of nukacin ISK-1. In addition, the secondary structure data of NukA mutants or fragments were obtained by circular dichroism spectra. The results indicated the importance of the alpha-helical leader peptide with hydrophobic and hydrophilic orientation consisting of the conserved residues in type A(II) lantibiotics. The expression data from various combinations of the chimeric prepeptides consisting of NukA and LctA (the prepeptide of lacticin 481, which shows high identity with NukA) further revealed that the amino acid difference at the C-terminus of the propeptide moiety between NukA and LctA, especially His at position 15 and Phe at position 19, was important for the maturation processes by the nukacin ISK-1 biosynthetic enzymes. Our findings suggest that the determinants in NukA were critically involved in the biosynthesis of nukacin ISK-1 and would thus be important for recognition by the nukacin ISK-1 biosynthetic enzymes.
Nagao
Jun-Ichi
J
Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Morinaga
Yoshiko
Y
Islam
Mohammad R
MR
Asaduzzaman
Sikder M
SM
Aso
Yuji
Y
Nakayama
Jiro
J
Sonomoto
Kenji
K
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001430:Bacteriocins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C421186:nukacin ISK-1; C082508:lacticin 481