Biosynthesis of the nickel-pincer nucleotide cofactor of lactate racemase requires a CTP-dependent cyclometallase.

Bacterial lactate racemase is a nickel-dependent enzyme that contains a cofactor, nickel pyridinium-3,5-bisthiocarboxylic acid mononucleotide, hereafter named nickel-pincer nucleotide (NPN). The LarC enzyme from the bacterium participates in NPN biosynthesis by inserting nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide. This reaction, known in organometallic chemistry as a cyclometalation, is characterized by the formation of new metal-carbon and metal-sulfur σ bonds. LarC is therefore the first cyclometallase identified in nature, but the molecular mechanism of LarC-catalyzed cyclometalation is unknown. Here, we show that LarC activity requires Mn-dependent CTP hydrolysis. The crystal structure of the C-terminal domain of LarC at 1.85 Å resolution revealed a hexameric ferredoxin-like fold and an unprecedented CTP-binding pocket. The loss-of-function of LarC variants with alanine variants of acidic residues leads us to propose a carboxylate-assisted mechanism for nickel insertion. This work also demonstrates the synthesis and purification of the NPN cofactor, opening new opportunities for the study of this intriguing cofactor and of NPN-utilizing enzymes.