A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export

Håvarstein, Leiv Sigve; Diep, Dzung B.; Nes, Ingolf F.

doi:10.1111/j.1365-2958.1995.tb02295.x

Cited by 543 publications

(554 citation statements)

References 47 publications

(11 reference statements)

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“…Analogous to similar studies on the protease domain of bifunctional transporters for nonlantibiotic bacteriocins (24)(25)(26), expression in E. coli of the first 150 residues of LctT resulted in an active protease that correctly hydrolyzed the amide bond between Ala-1 and Lys1 in LctA. These findings indicate that the transmembrane transporter domain of LctT is not required for substrate recognition and proteolysis.…”

Section: Discussionmentioning

confidence: 60%

“…Three conserved residues of LctT proposed to be involved in peptide bond cleavage (Cys12, His90, and Asp106) (24) were mutated to investigate their importance in amide bond hydrolysis. The involvement of these residues in catalysis has been suggested, but never experimentally tested for lantibiotic proteases.…”

Section: Proteolytic Activity Of His 10 -Lctt150 Mutant Proteinsmentioning

confidence: 99%

“…This domain is proposed to remove the leader sequence from the modified precursor peptides concomitant with export of the mature species. Håvarstein and co-workers recognized a correlation between bacteriocin transporters that contain this N-terminal proteolytic domain and the presence of a double glycine motif in the leader sequences of their cognate prepeptide substrates (24). They showed that the N-terminal 150 amino acids of the bacteriocin transporter LagD were sufficient for lactococcin G precursor cleavage.…”

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confidence: 99%

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In Vitro Reconstitution and Substrate Specificity of a Lantibiotic Protease

2008

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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 non-conserved 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.

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Section: Discussionmentioning

confidence: 60%

Section: Proteolytic Activity Of His 10 -Lctt150 Mutant Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

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In Vitro Reconstitution and Substrate Specificity of a Lantibiotic Protease

2008

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“…The residual activity of this intracellular bacteriocin precursor, although low, may necessitate the observed co-expression of immunity protein in wild type C. piscicola that is encoded on the same operon. 3 Reduction of antimicrobial properties for precursors of other types of bacteriocins has previously been suggested (20).…”

Section: Discussionmentioning

confidence: 99%

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2

Quadri

Yan²,

Stiles

et al. 1997

Journal of Biological Chemistry

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Carnobacteriocin B2, a 48-amino acid antimicrobial peptide containing a YGNGV motif that is produced by the lactic acid bacterium Carnobacterium piscicola LV17B, was overexpressed as fusion with maltose-binding protein in Escherichia coli. This fusion protein was cleaved with Factor Xa to allow isolation of the mature bacteriocin that was identical in all respects to that obtained from C. piscicola. Similar methodology permitted production of the precursor precarnobacteriocin B2 (CbnB2P), which has an 18-amino acid leader, as well as six mutants of the mature peptide: CbnF3 (Tyr 3 3 Phe), CbnS33 (Phe 33 3 Ser), CbnI34 (Val 34 3 Ile), CbnI37 (Val 37 3 Ile), CbnG46 (Arg 46 3 Gly), and Cbn28 (truncated frameshift mutation: (carnobacteriocin B2 1-28) ؉ ELTHL). Examination of these compounds for antimicrobial activity showed that although CbnI34, CbnI37, and CbnG46 were fully active, CbnB2P, CbnF3, CbnS33, Cbn28, and all of the fusion proteins had greatly reduced or no antimicrobial activity. Expression of the immunity protein that protects against the action of the parent carnobacteriocin B2 in a previously sensitive organism also protects against the active mutants. Because carnobacteriocin B2 also acts as an inducer of bacteriocin production in C. piscicola, the ability of the precursor CbnB2P and the mutants to exert this effect was examined. All were able to induce Bac ؊ cultures and reestablish the Bac ؉ phenotype except for the truncated Cbn28. The results demonstrate that very minor changes in the peptide sequence may drastically alter antimicrobial activity but that the induction of bacteriocin production is much more tolerant of structural modification, especially at the N terminus.

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“…The ABC transporters for production of class IIa bacteriocins have an extra N-terminal cysteine protease domain that is used to cut off the leader after the GG-motif (Figure 3, p30) (Havarstein et al 1995). The GG-leader is consequently removed by the ABC transporters during transmembrane translocation, and the mature bacteriocin is subsequently secreted (Havarstein et al 1995). By exchanging the leader peptides, it is possible to heterologously express class IIa bacteriocin via another secretion system, which may be more efficient (Ennahar et al 2000).…”

Section: Biosynthesismentioning

confidence: 99%