Limits...
The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold.

Dong SH, Tang W, Lukk T, Yu Y, Nair SK, van der Donk WA - Elife (2015)

Bottom Line: We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM.Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology.The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States.

ABSTRACT
The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

No MeSH data available.


Related in: MedlinePlus

MALDI-TOF mass spectra of phosphorylated CylLS intermediates incubated with CylM in the absence (black trace) or presence of ADP (magenta trace).M = unmodified CylLs; P = phosphorylation. Not only are the phosphates eliminated from pSer/pThr, ADP also is used to dehydrate non-phosphorylated Ser/Thr to afford fully, fourfold dehydrated peptide. See ‘Materials and methods’ for further discussion.DOI:http://dx.doi.org/10.7554/eLife.07607.023
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fig6s2: MALDI-TOF mass spectra of phosphorylated CylLS intermediates incubated with CylM in the absence (black trace) or presence of ADP (magenta trace).M = unmodified CylLs; P = phosphorylation. Not only are the phosphates eliminated from pSer/pThr, ADP also is used to dehydrate non-phosphorylated Ser/Thr to afford fully, fourfold dehydrated peptide. See ‘Materials and methods’ for further discussion.DOI:http://dx.doi.org/10.7554/eLife.07607.023

Mentions: Non-hydrolyzable adenosine derivatives were used for analysis of the elimination activity because we determined that CylM could use both ATP and ADP to dehydrate its substrates (i.e., both ATP and ADP can be used for phosphorylation). Hence, when the mixture of CylLS peptides that carry 1–3 phosphate esters were supplied to CylM in the presence of ATP or ADP, both elimination and dehydration reactions proceeded, which complicated the outcome and precluded data interpretation. For example, when ADP was supplied instead of non-hydrolyzable ADP, only fully (fourfold) dehydrated CylLS was observed (Figure 6—figure supplement 2). Since the phosphorylated peptides carried only 1–3 phosphate esters, the additional dehydrations resulted from conversion of non-phosphorylated Ser/Thr to Dha/Dhb. Therefore, to study the elimination reaction in isolation, non-hydrolyzable ATP and ADP analogs were used.


The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold.

Dong SH, Tang W, Lukk T, Yu Y, Nair SK, van der Donk WA - Elife (2015)

MALDI-TOF mass spectra of phosphorylated CylLS intermediates incubated with CylM in the absence (black trace) or presence of ADP (magenta trace).M = unmodified CylLs; P = phosphorylation. Not only are the phosphates eliminated from pSer/pThr, ADP also is used to dehydrate non-phosphorylated Ser/Thr to afford fully, fourfold dehydrated peptide. See ‘Materials and methods’ for further discussion.DOI:http://dx.doi.org/10.7554/eLife.07607.023
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4550811&req=5

fig6s2: MALDI-TOF mass spectra of phosphorylated CylLS intermediates incubated with CylM in the absence (black trace) or presence of ADP (magenta trace).M = unmodified CylLs; P = phosphorylation. Not only are the phosphates eliminated from pSer/pThr, ADP also is used to dehydrate non-phosphorylated Ser/Thr to afford fully, fourfold dehydrated peptide. See ‘Materials and methods’ for further discussion.DOI:http://dx.doi.org/10.7554/eLife.07607.023
Mentions: Non-hydrolyzable adenosine derivatives were used for analysis of the elimination activity because we determined that CylM could use both ATP and ADP to dehydrate its substrates (i.e., both ATP and ADP can be used for phosphorylation). Hence, when the mixture of CylLS peptides that carry 1–3 phosphate esters were supplied to CylM in the presence of ATP or ADP, both elimination and dehydration reactions proceeded, which complicated the outcome and precluded data interpretation. For example, when ADP was supplied instead of non-hydrolyzable ADP, only fully (fourfold) dehydrated CylLS was observed (Figure 6—figure supplement 2). Since the phosphorylated peptides carried only 1–3 phosphate esters, the additional dehydrations resulted from conversion of non-phosphorylated Ser/Thr to Dha/Dhb. Therefore, to study the elimination reaction in isolation, non-hydrolyzable ATP and ADP analogs were used.

Bottom Line: We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM.Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology.The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States.

ABSTRACT
The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

No MeSH data available.


Related in: MedlinePlus