Biochemistry. 2019 Feb 19;58(7):951-964. doi: 10.1021/acs.biochem.8b01253. Epub 2019 Jan 31.
Cobamides are coenzymes used by cells from all domains of life but made de novo by only some bacteria and archaea. The last steps of the cobamide biosynthetic pathway activate the corrin ring and the lower ligand base, condense the activated intermediates, and dephosphorylate the product prior to the release of the biologically active coenzyme. In bacteria, a phosphoribosyltransferase (PRTase) enyzme activates the base into its α-mononucleotide. The enzyme from Salmonella enterica ( SeCobT) has been extensively biochemically and structurally characterized. The crystal structure of the putative PRTase from the archaeum Methanocaldococcus jannaschii ( MjCobT) is known, but its function has not been validated. Here we report the in vivo and in vitro characterization of MjCobT. In vivo, in vitro, and phylogenetic data reported here show that MjCobT belongs to a new class of NaMN-dependent PRTases. We also show that the Synechococcus sp. WH7803 CobT protein has PRTase activity in vivo. Lastly, results of isothermal titration calorimetry and analytical ultracentrifugation analysis show that the biologically active form of MjCobT is a dimer, not a trimer, as suggested by its crystal structure.
钴胺素是所有生命领域的细胞都使用的辅酶，但只有一些细菌和古菌可以从头合成。钴胺素生物合成途径的最后几步是激活钴胺环和较低的配体碱基，缩合活化的中间体，并在释放生物活性辅酶之前对产物进行去磷酸化。在细菌中，磷酸核糖基转移酶（PRTase）酶将碱基激活成其α-单核苷酸。来自沙门氏菌（SeCobT）的酶已被广泛进行生物化学和结构表征。已知产甲烷球菌（MjCobT）的假定 PRTase 的晶体结构，但尚未验证其功能。在这里，我们报告了 MjCobT 的体内和体外特征。这里报道的体内、体外和系统发育数据表明，MjCobT 属于一种新的 NaMN 依赖的 PRTase 类。我们还表明，鱼腥藻 sp. WH7803 CobT 蛋白在体内具有 PRTase 活性。最后，等温滴定量热法和分析超速离心分析的结果表明，生物活性形式的 MjCobT 是二聚体，而不是其晶体结构所暗示的三聚体。
