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Abstract：
Glutamate dehydrogenase (GDH) releases ammonia in a reversible NAD(P)+-dependent oxidative deamination of glutamate that yields 2-oxoglutarate (2OG). In current perception, GDH contributes to Glu homeostasis and plays a significant role at the junction of carbon and nitrogen assimilation pathways. GDHs are members of a superfamily of ELFV (Glu/Leu/Phe/Val) amino acid dehydrogenases and are subdivided into three subclasses, based on coenzyme specificity: NAD+-specific, NAD+/NADP+ dual-specific, and NADP+-specific. We determined in this work that the mitochondrial AtGDH1 isozyme from A. thaliana is NAD+-specific. Altogether, A. thaliana expresses three GDH isozymes (AtGDH1-3) targeted to mitochondria, of which AtGDH2 has an extra EF-hand motif and is stimulated by calcium. Our enzymatic assays of AtGDH1 established that its sensitivity to calcium is negligible. In vivo the AtGDH1-3 enzymes form homo- and heterohexamers of varied composition. We solved the crystal structure of recombinant AtGDH1 in the apo-form and in complex with NAD+ at 2.59 and 2.03 Å resolution, respectively. We demonstrate also that both in the apo form and in 1:1 complex with NAD+, it forms D 3-symmetric homohexamers. A subunit of AtGDH1 consists of domain I, which is involved in hexamer formation and substrate binding, and of domain II which binds coenzyme. Most of the subunits in our crystal structures, including those in NAD+ complex, are in open conformation, with domain II forming a large (albeit variable) angle with domain I. One of the subunits of the AtGDH1-NAD+ hexamer contains a serendipitous 2OG molecule in the active site, causing a dramatic (∼25°) closure of the domains. We provide convincing evidence that the N-terminal peptide preceding domain I is a mitochondrial targeting signal, with a predicted cleavage site for mitochondrial processing peptidase (MPP) at Leu17-Leu18 that is followed by an unexpected potassium coordination site (Ser27, Ile30). We also identified several MPD [(+/-)-2-methyl-2,4-pentanediol] binding sites with conserved sequence. Although AtGDH1 is insensitive to MPD in our assays, the observation of druggable sites opens a potential for non-competitive herbicide design.
摘要：
谷氨酸脱氢酶（GDH）在可逆的NAD（P）依赖性谷氨酸氧化脱氨中释放氨，产生2-酮戊二酸（2OG）。在目前的看法中，GDH有助于Glu稳态，并在碳和氮同化途径的连接处发挥重要作用。GDH是ELFV（Glu/Leu/Phe/Val）氨基酸脱氢酶超家族的成员，并细分为三个亚类，基于辅酶特异性：NAD+特异性，NAD+/NADP+双特定，和NADP+特异性。在这项工作中，我们确定了来自拟南芥的线粒体AtGDH1同工酶是NAD特异性的。总之,拟南芥表达三种针对线粒体的GDH同工酶（AtGDH1-3），其中AtGDH2具有额外的EF手基序，并受到钙的刺激。我们对AtGDH1的酶促测定确定其对钙的敏感性可以忽略不计。在体内,AtGDH1-3酶形成不同组成的同-和异-六聚体。我们以2.59和2.03的分辨率解决了apo形式和与NAD复合的重组AtGDH1的晶体结构，分别。我们还证明了apo形式和与NAD+的1:1复合物，它形成D3对称同数列。AtGDH1的一个亚基由结构域I组成，参与六聚体形成和底物结合，以及结合辅酶的结构域II。我们晶体结构中的大多数亚基，包括NAD+复合体中的那些，处于开放构象，结构域II与结构域I形成大的（尽管可变）角度。AtGDH1-NAD六聚体的一个亚基在活性位点包含一个偶然的2OG分子，导致域的戏剧性(~25°)闭合。我们提供了令人信服的证据，证明结构域I之前的N末端肽是线粒体靶向信号，在Leu17-Leu18处具有线粒体加工肽酶（MPP）的预测切割位点，随后是意外的钾配位位点（Ser27，Ile30）。我们还鉴定了几个具有保守序列的MPD[(+/-)-2-甲基-2,4-戊二醇]结合位点。尽管AtGDH1在我们的检测中对MPD不敏感，对可药用地点的观察为非竞争性除草剂设计开辟了潜力。
