Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum

Fellner, Matthias; Rankin, Joel A.; Desguin, Benoît; Hu, Jian; Hausinger, Robert P.

doi:10.1021/acs.biochem.8b00601

Title: Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum

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Abstract

LarE from Lactobacillus plantarum is an ATP-dependent sulfur transferase that sacrifices its Cys176 sulfur atom to form a dehydroalanine (Dha) side chain during biosynthesis of the covalently linked nickel-pincer nucleotide (NPN) cofactor (pyridinium 3-thioamide-5-thiocarboxylic acid mononucleotide) of lactate racemase. Coenzyme A (CoA) stabilizes LarE and forms a CoA-Cys176 mixed disulfide with the protein. This study presents the crystal structure of the LarE/CoA complex, revealing protein interactions with CoA that mimic those for binding ATP. CoA weakly inhibits LarE activity, and the persulfide of CoA is capable of partially regenerating functional LarE from the Dha176 form of the protein. The physiological relevance of this cycling reaction is unclear. A new form of LarE was discovered, an NPN-LarE covalent adduct, explaining prior results in which activation of the lactate racemase apoprotein required only the isolated LarE. The crystal structure of the inactive C176A variant revealed a fold essentially identical to that of wild-type LarE. Additional active site variants of LarE were created and their activities characterized, with all LarE variants analyzed in terms of the structure. Finally, the L. plantarum LarE structure was compared to a homology model of Thermoanaerobacterium thermosaccharolyticum LarE, predicted to contain three cysteine residues at the active site,more »« less

Authors:

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Michigan State Univ., East Lansing, MI (United States); Univ. of Otago, Dunedin (New Zealand)
Michigan State Univ., East Lansing, MI (United States)
Univ. catholique de Louvain, Louvain-La-Neuve (Belgium)

Publication Date:: Wed Aug 29 00:00:00 EDT 2018

Research Org.:: Argonne National Lab. (ANL), Argonne, IL (United States)

Sponsoring Org.:: National Science Foundation (NSF)

OSTI Identifier:: 1476089

Grant/Contract Number:: CHE-1516126; CHE-1807073

Resource Type:: Accepted Manuscript

Journal Name:: Biochemistry

Additional Journal Information:: Journal Volume: 57; Journal Issue: 38; Journal ID: ISSN 0006-2960

Publisher:: American Chemical Society (ACS)

Country of Publication:: United States

Language:: ENGLISH

Subject:: 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; disulfides; sulfur; peptides and proteins; monomers; cluster chemistry

Citation Formats


                    Fellner, Matthias, Rankin, Joel A., Desguin, Benoît, Hu, Jian, and Hausinger, Robert P. Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum.  United States: N. p., 2018. 
Web.  doi:10.1021/acs.biochem.8b00601.

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                    Fellner, Matthias, Rankin, Joel A., Desguin, Benoît, Hu, Jian, & Hausinger, Robert P. Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum.  United States.  https://doi.org/10.1021/acs.biochem.8b00601

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                    Fellner, Matthias, Rankin, Joel A., Desguin, Benoît, Hu, Jian, and Hausinger, Robert P. Wed .  
"Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum".  United States.  https://doi.org/10.1021/acs.biochem.8b00601.  https://www.osti.gov/servlets/purl/1476089.

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@article{osti_1476089,

  title        = {Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum},

  author       = {Fellner, Matthias and Rankin, Joel A. and Desguin, Benoît and Hu, Jian and Hausinger, Robert P.},

  abstractNote = {LarE from Lactobacillus plantarum is an ATP-dependent sulfur transferase that sacrifices its Cys176 sulfur atom to form a dehydroalanine (Dha) side chain during biosynthesis of the covalently linked nickel-pincer nucleotide (NPN) cofactor (pyridinium 3-thioamide-5-thiocarboxylic acid mononucleotide) of lactate racemase. Coenzyme A (CoA) stabilizes LarE and forms a CoA-Cys176 mixed disulfide with the protein. This study presents the crystal structure of the LarE/CoA complex, revealing protein interactions with CoA that mimic those for binding ATP. CoA weakly inhibits LarE activity, and the persulfide of CoA is capable of partially regenerating functional LarE from the Dha176 form of the protein. The physiological relevance of this cycling reaction is unclear. A new form of LarE was discovered, an NPN-LarE covalent adduct, explaining prior results in which activation of the lactate racemase apoprotein required only the isolated LarE. The crystal structure of the inactive C176A variant revealed a fold essentially identical to that of wild-type LarE. Additional active site variants of LarE were created and their activities characterized, with all LarE variants analyzed in terms of the structure. Finally, the L. plantarum LarE structure was compared to a homology model of Thermoanaerobacterium thermosaccharolyticum LarE, predicted to contain three cysteine residues at the active site, and to other proteins with a similar fold and multiple active site cysteine residues. Lastly, these findings suggest that some LarE orthologs may not be sacrificial but instead may catalyze sulfur transfer by using a persulfide mechanism or from a labile site on a [4Fe-4S] cluster at this position.},

  doi          = {10.1021/acs.biochem.8b00601},

  journal      = {Biochemistry},

  number       = 38,

  volume       = 57,

  place        = {United States},

  year         = {Wed Aug 29 00:00:00 EDT 2018},

  month        = {Wed Aug 29 00:00:00 EDT 2018}

}

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