In vitro reconstitution and substrate specificity of a lantibiotic protease

L. A. Furgerson Ihnken, Champak Chatterjee, Wilfred A. Van Der Donk

Research output: Contribution to journalArticlepeer-review

Abstract

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. His10-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 His10-LctT150. On the other hand, His10-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.

Original languageEnglish (US)
Pages (from-to)7352-7363
Number of pages12
JournalBiochemistry
Volume47
Issue number28
DOIs
StatePublished - Jul 15 2008

ASJC Scopus subject areas

  • Biochemistry

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