Department of Biochemistry and Molecular Pharmacology , UMass Medical School , 364 Plantation Street , Worcester , Massachusetts 01605 , United States.
Program in Chemical Biology , UMass Medical School , 364 Plantation Street , Worcester , Massachusetts 01605 , United States.
ACS Chem Biol. 2018 Sep 21;13(9):2663-2672. doi: 10.1021/acschembio.8b00578. Epub 2018 Aug 8.
Nicotinamide- N-methyltransferase (NNMT) catalyzes the irreversible methylation of nicotinamide (NAM) to form N-methyl nicotinamide using S-adenosyl methionine as a methyl donor. NNMT is implicated in several chronic disease conditions, including cancers, kidney disease, cardiovascular disease, and Parkinson's disease. Although phosphorylation of NNMT in gastric tumors is reported, the functional effects of this post-translational modification has not been investigated. We previously reported that citrullination of NNMT by Protein Arginine Deiminases abolished its methyltransferase activity. Herein, we investigate the mechanism of inactivation. Using tandem mass spectrometry, we identified three sites of citrullination in NNMT. With this information in hand, we used a combination of site-directed mutagenesis, kinetics, and circular dichoism experiments to demonstrate that citrullination of R132 leads to a structural perturbation that ultimately promotes NNMT inactivation.
烟酰胺-N-甲基转移酶（NNMT）以 S-腺苷甲硫氨酸作为甲基供体，催化烟酰胺（NAM）不可逆甲基化生成 N-甲基烟酰胺。NNMT 与多种慢性疾病相关，包括癌症、肾病、心血管疾病和帕金森病。尽管已经报道了胃肿瘤中 NNMT 的磷酸化，但这种翻译后修饰的功能影响尚未得到研究。我们之前报道了蛋白精氨酸脱亚氨酶（Protein Arginine Deiminases）对 NNMT 的瓜氨酸化会使其甲基转移酶活性丧失。在此，我们研究了失活的机制。通过串联质谱分析，我们在 NNMT 中鉴定了三个瓜氨酸化位点。有了这些信息，我们使用定点突变、动力学和圆二色性实验的组合，证明了 R132 的瓜氨酸化导致结构扰动，最终促进 NNMT 失活。
