Abstract
Cofactors are essential components of numerous enzymes, therefore their characterization by structural, biophysical, and biochemical approaches is crucial for understanding the resulting catalytic and regulatory mechanisms. In this chapter, we present a case study of a recently discovered cofactor, the nickel-pincer nucleotide (NPN), by demonstrating how we identified and thoroughly characterized this unprecedented nickel-containing coenzyme that is tethered to lactase racemase from Lactiplantibacillus plantarum. In addition, we describe how the NPN cofactor is biosynthesized by a panel of proteins encoded in the lar operon and describe the properties of these novel enzymes. Comprehensive protocols for conducting functional and mechanistic studies of NPN-containing lactate racemase (LarA) and the carboxylase/hydrolase (LarB), sulfur transferase (LarE), and metal insertase (LarC) used for NPN biosynthesis are provided for potential applications towards characterizing enzymes in the same or homologous families.