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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.

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ESI mass spectrum illustrating the phosphorylation of ProcA 2.8 by ProcM-T516A. Expected and experimental m/z values are listed in Table S1, ESI.†
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fig1: ESI mass spectrum illustrating the phosphorylation of ProcA 2.8 by ProcM-T516A. Expected and experimental m/z values are listed in Table S1, ESI.†

Mentions: To obtain a ProcM-derived kinase, we performed an amino acid sequence alignment of LctM and ProcM and identified Arg510 and Thr516 of ProcM as analogous to Arg399 and Thr405 of LctM. These residues of ProcM were mutated to methionine and alanine, respectively. The mutant proteins were expressed in Escherichia coli as N-terminally hexa-histidine tagged fusion proteins for use in in vitro phosphorylation assays. Unfortunately, the R510M variant could not be purified because of solubility problems, and therefore all in vitro phosphorylation experiments were performed with the T516A mutant. Initially, the catalytic competence of T516A was tested with the natural ProcM substrates ProcA 1.1 and ProcA 2.8 (Fig. 1 and Fig. S1, ESI†). Reaction products were analysed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) following purification and proteolytic digestion of the peptide products to remove the leader peptide (Scheme 1B). In each assay, both mono- and diphosphorylated products were observed.


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

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

ESI mass spectrum illustrating the phosphorylation of ProcA 2.8 by ProcM-T516A. Expected and experimental m/z values are listed in Table S1, ESI.†
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: ESI mass spectrum illustrating the phosphorylation of ProcA 2.8 by ProcM-T516A. Expected and experimental m/z values are listed in Table S1, ESI.†
Mentions: To obtain a ProcM-derived kinase, we performed an amino acid sequence alignment of LctM and ProcM and identified Arg510 and Thr516 of ProcM as analogous to Arg399 and Thr405 of LctM. These residues of ProcM were mutated to methionine and alanine, respectively. The mutant proteins were expressed in Escherichia coli as N-terminally hexa-histidine tagged fusion proteins for use in in vitro phosphorylation assays. Unfortunately, the R510M variant could not be purified because of solubility problems, and therefore all in vitro phosphorylation experiments were performed with the T516A mutant. Initially, the catalytic competence of T516A was tested with the natural ProcM substrates ProcA 1.1 and ProcA 2.8 (Fig. 1 and Fig. S1, ESI†). Reaction products were analysed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) following purification and proteolytic digestion of the peptide products to remove the leader peptide (Scheme 1B). In each assay, both mono- and diphosphorylated products were observed.

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