Characterization of the Nickel-inserting Cyclometallase LarC from Moorella thermoacetica and Identification of a CMPylated Reaction Intermediate.

The nickel-pincer nucleotide (NPN) cofactor is required for lactate racemase (LarA) activity , and LarB, LarE, and LarC are needed to generate this coenzyme . LarC catalyzes the last step of the biosynthesis pathway by inserting nickel into pyridinium-3,5-dithiocarboxylic acid mononucleotide (P2TMN), resulting in the mature NPN cofactor . Previously, LarC from Lactobacillus plantarum was shown to be a CTP-dependent, single-turnover nickel insertase or cyclometallase, but a structure was only obtained for its C-terminal domain and its reaction mechanism was not identified. We characterized LarC from Moorella thermoacetica(LarC ) and identified a novel reaction intermediate in which the precursor P2TMN is CMPylated. We propose that LarC uses CTP to generate the CMP-P2TMN species that is properly positioned in the active site for nickel insertion, analogous to the molybdenum insertion step during molybdenum cofactor biosynthesis . LarC variants were characterized to further elucidate the mechanism of the transient CMPylation process. Additionally, we generated preliminary structural data for the full-length LarC through cryo-electron microscopy. Our results highlight the role of CTP in the nickel insertion reaction and provide a better structural and mechanistic understanding of the LarC reaction. 1. Desguin, B. et al. A tethered niacin-derived pincer complex with a nickel-carbon bond in lactate racemase. Science. 349, 66-69 (2015). 2. Desguin, B. et al. Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system. Nat. Commun. 5, 3615 (2014). 3. Desguin, B. et al. Biosynthesis of the nickel-pincer nucleotide cofactor of lactate racemase requires a CTP-dependent cyclometallase. J. Biol. Chem. 293, 12303-12317 (2018). 4. Llamas, A., Mendel, R. R. & Schwarz, G. Synthesis of adenylated molybdopterin: An essential step for molybdenum insertion. J. Biol. Chem. 279, 55241-55246 (2004).