Structural Genomics Consortium, University of Oxford OX3 7DU, United Kingdom.
J Biol Chem. 2010 Dec 3;285(49):38204-13. doi: 10.1074/jbc.M110.177717. Epub 2010 Sep 28.
Vitamin B(12) (cobalamin, Cbl) is essential to the function of two human enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). The conversion of dietary Cbl to its cofactor forms, methyl-Cbl (MeCbl) for MS and adenosyl-Cbl (AdoCbl) for MUT, located in the cytosol and mitochondria, respectively, requires a complex pathway of intracellular processing and trafficking. One of the processing proteins, MMAA (methylmalonic aciduria type A), is implicated in the mitochondrial assembly of AdoCbl into MUT and is defective in children from the cblA complementation group of cobalamin disorders. To characterize the functional interplay between MMAA and MUT, we have crystallized human MMAA in the GDP-bound form and human MUT in the apo, holo, and substrate-bound ternary forms. Structures of both proteins reveal highly conserved domain architecture and catalytic machinery for ligand binding, yet they show substantially different dimeric assembly and interaction, compared with their bacterial counterparts. We show that MMAA exhibits GTPase activity that is modulated by MUT and that the two proteins interact in vitro and in vivo. Formation of a stable MMAA-MUT complex is nucleotide-selective for MMAA (GMPPNP over GDP) and apoenzyme-dependent for MUT. The physiological importance of this interaction is highlighted by a recently identified homoallelic patient mutation of MMAA, G188R, which, we show, retains basal GTPase activity but has abrogated interaction. Together, our data point to a gatekeeping role for MMAA by favoring complex formation with MUT apoenzyme for AdoCbl assembly and releasing the AdoCbl-loaded holoenzyme from the complex, in a GTP-dependent manner.
维生素 B(12)（钴胺素，Cbl）对人类两种酶的功能至关重要，即蛋氨酸合成酶（MS）和甲基丙二酰辅酶 A 变位酶（MUT）。膳食 Cbl 向其辅因子形式的转化，即 MS 的甲基-Cbl（MeCbl）和 MUT 的腺苷酰-Cbl（AdoCbl），分别位于细胞质和线粒体中，需要一个复杂的细胞内加工和运输途径。加工蛋白之一，MMAA（甲基丙二酸尿症 A 型），与 AdoCbl 在线粒体中组装到 MUT 中有关，并且在钴胺素代谢障碍的 cblA 互补组的儿童中存在缺陷。为了表征 MMAA 和 MUT 之间的功能相互作用，我们已经结晶了 GDP 结合形式的人 MMAA 和apo、全酶和底物结合的三元形式的人 MUT。两种蛋白质的结构都揭示了高度保守的结构域结构和催化机制用于配体结合，但与它们的细菌对应物相比，它们显示出截然不同的二聚体组装和相互作用。我们表明，MMAA 表现出 GTPase 活性，该活性受 MUT 调节，并且这两种蛋白质在体外和体内相互作用。形成稳定的 MMAA-MUT 复合物对 MMAA 具有核苷酸选择性（GMPPNP 优于 GDP），并且对 MUT 依赖于脱辅基酶。最近发现的 MMAA 同型等位基因突变患者 G188R 突出了这种相互作用的生理重要性，我们表明，它保留了基础 GTPase 活性，但已消除了相互作用。总的来说，我们的数据表明 MMAA 具有门控作用，通过有利于与 MUT 脱辅基酶形成复合物来组装 AdoCbl，并以 GTP 依赖性方式从复合物中释放加载 AdoCbl 的全酶。
