In vitro reconstitution and substrate specificity of a lantibiotic protease.
Lacticin 481 is a lanthionine-containing bacteriocin (lantibiotic) produced by Lactococcus lactis subsp. lactis. The final steps of lacticin 481 biosynthesis are proteolytic removal of an N-terminal leader sequence from the prepeptide LctA and export of the mature lantibiotic. Both proteolysis and secretion are performed by the dedicated ATP-binding cassette (ABC) transporter LctT. LctT belongs to the family of AMS (ABC transporter maturation and secretion) proteins whose prepeptide substrates share a conserved double-glycine type cleavage site. The in vitro activity of a lantibiotic protease has not yet been characterized. This study reports the purification and in vitro activity of the N-terminal protease domain of LctT (LctT150), and its use for the in vitro production of lacticin 481. The G(-2)A(-1) cleavage site and several other conserved amino acid residues in the leader peptide were targeted by site-directed mutagenesis to probe the substrate specificity of LctT as well as shed light upon the role of these conserved residues in lantibiotic biosynthesis. His 10-LctT150 did not process most variants of the double glycine motif and processed mutants of Glu-8 only very slowly. Furthermore, incorporation of helix-breaking residues in the leader peptide resulted in greatly decreased proteolytic activity by His 10-LctT150. On the other hand, His 10-LctT150 accepted all peptides containing mutations in the propeptide or at nonconserved positions of LctA. In addition, the protease domain of LctT was investigated by site-directed mutagenesis of the conserved residues Cys12, His90, and Asp106. The proteolytic activities of the resulting mutant proteins are consistent with a cysteine protease.
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
L A
LA
Furgerson Ihnken
Champak
C
Chatterjee
Wilfred A
WA
van der Donk
D001430:Bacteriocins / Q000096:biosynthesis / Q000378:metabolism*; D025523:Bacteriophage HK022 / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
D001430:Bacteriocins; C073501:Nun protein, Enterobacteria phage HK022; D012329:RNA, Bacterial; D014157:Transcription Factors; D014764:Viral Proteins; D010447:Peptide Hydrolases