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

Aiko Turmo (Michigan State University), Dexin Sui (Michigan State University), Kelly Kim (Michigan State University), Jian Hu (Michigan State University), Robert Hausinger (Michigan State University)

The nickel-pincer nucleotide (NPN) cofactor is required for lactate racemase (LarA) activity1, and LarB, LarE, and LarC are needed to generate this coenzyme 2. 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 cofactor3. 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 (LarCMt) and identified a novel reaction intermediate in which the precursor P2TMN is CMPylated. We propose that LarCMt 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 biosynthesis4. LarCMt variants were characterized to further elucidate the mechanism of the transient CMPylation process. Additionally, we generated preliminary structural data for the full-length LarCMt 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. amp; Schwarz, G. Synthesis of adenylated molybdopterin: An essential step for molybdenum insertion. J. Biol. Chem. 279, 55241–55246 (2004).

Funding in support of this work was provided by the National Science Foundation [CHE1807073] and the National Institutes of Health [GM128959] to R.P.H. and J.H., MSU Start-Up Funds for K.H.K, and from the National Science Foundation Graduate Research Fellowship Program [DGE1848739] to A.T.