ATP-dependent leader peptide cleavage by NukT, a bifunctional ABC transporter, during lantibiotic biosynthesis.
NukT, a possible ABC transporter maturation and secretion (AMS) protein, may contribute to the cleavage of the leader peptide of NukA, which is the prepeptide of the lantibiotic nukacin ISK-1, and to nukacin ISK-1 transport. In this study, we reconstituted in vitro peptidase activity of the full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that the presence of unusual amino acids in NukA is required for leader peptide cleavage. Furthermore, NukT peptidase activity was inhibited by phenylmethylsulfonyl fluoride, a serine/cysteine protease inhibitor; this finding strongly suggests that NukT, like other AMS proteins, is a cysteine protease. Interestingly, NukT peptidase activity depended on ATP hydrolysis. These results suggest that the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain. This is the first in vitro study on lantibiotics that reports the processing mechanism of a full-length bifunctional ABC transporter.
Nishie
Mami
M
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.
Shioya
Kouki
K
Nagao
Jun-Ichi
J
Jikuya
Hiroyuki
H
Sonomoto
Kenji
K
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001430:Bacteriocins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000235:genetics; D017124:Conserved Sequence; D057056:Cysteine Proteases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001430:Bacteriocins; D010455:Peptides; D021382:Protein Sorting Signals; C421186:nukacin ISK-1; D000255:Adenosine Triphosphate; D057056:Cysteine Proteases