Limits...
An engineered lantipeptide synthetase serves as a general leader peptide-dependent kinase.

Thibodeaux GN, van der Donk WA - Chem. Commun. (Camb.) (2012)

Bottom Line: Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways.Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, 600 S Mathews Ave, Urbana, IL 61801, USA.

ABSTRACT
Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

Show MeSH
(A) LanM-catalysed introduction of lanthionines and methyllanthionines into the core peptide of their LanA substrates via a cryptic phosphorylation step. Several mutations in these enzymes generate proteins that still phosphorylate but no longer eliminate. (B) General presentation of the strategy used herein. A peptide of interest was expressed fused to the C-terminus of ProcA leader peptides, phosphorylated with a ProcM mutant, and the leader peptide was removed by proteolysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475617&req=5

sch1: (A) LanM-catalysed introduction of lanthionines and methyllanthionines into the core peptide of their LanA substrates via a cryptic phosphorylation step. Several mutations in these enzymes generate proteins that still phosphorylate but no longer eliminate. (B) General presentation of the strategy used herein. A peptide of interest was expressed fused to the C-terminus of ProcA leader peptides, phosphorylated with a ProcM mutant, and the leader peptide was removed by proteolysis.

Mentions: In an effort to develop potentially complementary methods for phosphopeptide synthesis, we have previously exploited the unique catalytic properties of the class II lantipeptide synthetases (termed LanM) for the in vitro synthesis of peptides with homogeneous phosphorylation patterns. LanM enzymes catalyse several steps during the maturation of lantipeptides, including a cryptic ATP-dependent phosphorylation step (Scheme 1). The peptide substrates of LanM enzymes (termed LanA peptides) are composed of an N-terminal leader peptide that plays a role in LanM binding and activation,10 and a C-terminal core peptide that harbors the post-translational modification sites (Scheme 1B). While investigating the mechanism of LctM involved in the biosynthesis of lacticin 481, two mutants (R399M and T405A) were identified that were deficient in the phosphate elimination step.11 These mutants were shown to catalyse in vitro phosphorylation of a variety of peptide substrates attached to the LctA leader peptide.12 Despite our initial success using LctM as a general Ser/Thr kinase, the LctM system has several drawbacks, which include low solubility of the LctA leader peptide, limited tolerance of variations in the sequence context of the Ser/Thr to be phosphorylated, and difficult scalability of an in vitro process that requires purified protein and substrate.


An engineered lantipeptide synthetase serves as a general leader peptide-dependent kinase.

Thibodeaux GN, van der Donk WA - Chem. Commun. (Camb.) (2012)

(A) LanM-catalysed introduction of lanthionines and methyllanthionines into the core peptide of their LanA substrates via a cryptic phosphorylation step. Several mutations in these enzymes generate proteins that still phosphorylate but no longer eliminate. (B) General presentation of the strategy used herein. A peptide of interest was expressed fused to the C-terminus of ProcA leader peptides, phosphorylated with a ProcM mutant, and the leader peptide was removed by proteolysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475617&req=5

sch1: (A) LanM-catalysed introduction of lanthionines and methyllanthionines into the core peptide of their LanA substrates via a cryptic phosphorylation step. Several mutations in these enzymes generate proteins that still phosphorylate but no longer eliminate. (B) General presentation of the strategy used herein. A peptide of interest was expressed fused to the C-terminus of ProcA leader peptides, phosphorylated with a ProcM mutant, and the leader peptide was removed by proteolysis.
Mentions: In an effort to develop potentially complementary methods for phosphopeptide synthesis, we have previously exploited the unique catalytic properties of the class II lantipeptide synthetases (termed LanM) for the in vitro synthesis of peptides with homogeneous phosphorylation patterns. LanM enzymes catalyse several steps during the maturation of lantipeptides, including a cryptic ATP-dependent phosphorylation step (Scheme 1). The peptide substrates of LanM enzymes (termed LanA peptides) are composed of an N-terminal leader peptide that plays a role in LanM binding and activation,10 and a C-terminal core peptide that harbors the post-translational modification sites (Scheme 1B). While investigating the mechanism of LctM involved in the biosynthesis of lacticin 481, two mutants (R399M and T405A) were identified that were deficient in the phosphate elimination step.11 These mutants were shown to catalyse in vitro phosphorylation of a variety of peptide substrates attached to the LctA leader peptide.12 Despite our initial success using LctM as a general Ser/Thr kinase, the LctM system has several drawbacks, which include low solubility of the LctA leader peptide, limited tolerance of variations in the sequence context of the Ser/Thr to be phosphorylated, and difficult scalability of an in vitro process that requires purified protein and substrate.

Bottom Line: Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways.Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, 600 S Mathews Ave, Urbana, IL 61801, USA.

ABSTRACT
Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

Show MeSH