Institute of Food Sciences, National Research Council, Via Roma 64, 83100 Avellino, Italy.
Department of Agricultural, Food and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy.
Int J Mol Sci. 2021 Jun 13;22(12):6334. doi: 10.3390/ijms22126334.
Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor.
烟酰胺单核苷酸（NMN）是烟酰胺腺嘌呤二核苷酸（NAD+）生物合成的关键中间产物。其补充剂已被证明对多种疾病具有有益作用。本研究的目的是对烟酰胺单核苷酸脱氨酶（PncC）无活性突变体进行表征，用作烟酰胺单核苷酸特异性生物传感器的可能分子识别元件（MRE）。使用热稳定性测定和稳态荧光光谱测量来研究 NMN 和相关代谢物（NaMN、Na、Nam、NR、NAD、NADP 和 NaAD）与 PncC 突变变体的结合。特别是，选择 S29A PncC 和 K61Q PncC 变体形式，因为它们仍然能够在微摩尔范围内结合 NMN，但不能催化酶反应。虽然 S29A PncC 对 NaMN（PncC 催化反应的产物）也表现出相似的亲和力，但 K61Q PncC 与其没有显著相互作用。因此，PncC K61Q 突变体似乎是用作烟酰胺单核苷酸生物传感器的特异性探针的有前途的候选物。
