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

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 essent...