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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.
摘要：
辅因子是许多酶的重要组成部分，因此它们的结构表征，生物物理，生化方法对于理解由此产生的催化和调节机制至关重要。在这一章中,我们提供了一个最近发现的辅因子的案例研究，镍夹式核苷酸（NPN），通过证明我们如何鉴定和彻底表征这种前所未有的含镍辅酶，该辅酶与植物乳杆菌的乳糖酶消旋酶相连。此外,我们描述了如何通过lar操纵子中编码的一组蛋白质生物合成NPN辅因子，并描述了这些新型酶的特性。对含NPN的乳酸消旋酶（LarA）和羧化酶/水解酶（LarB）进行功能和机理研究的综合方案，硫转移酶（LarE），和用于NPN生物合成的金属插入酶(LarC)被提供用于表征相同或同源家族中的酶的潜在应用。
