Structural Chem. of Biomolecular Sys. by X-ray Diffract.

O2-芳基重氮杂多酸 谷胱甘肽S转移酶(GST)是一类催化谷胱甘肽与亲电性化合物结合的酶。在癌前和肿瘤细胞中，特定形式的GSTs高水平表达，并参与细胞对抗癌药物的耐药。同工酶GSTP在生物抗烷化剂方面具有特别重要的作用。旨在抑制GSTP以延长烷化剂疗效的治疗策略一直未获成功。然而，GSTP激活的前药在靶向特定癌细胞方面显示出巨大的潜力。我们已经开发出一种新型的抗癌药物O2-芳基重氮二酸盐，它通过细胞内释放一氧化氮来杀死癌细胞(澳大利亚专利号733590；美国专利号6610660；欧洲专利号04009529.1-2101)。自那以后，两种先导化合物已经被开发出来。 叶酸途径酶 6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(HPPK)是叶酸生物合成途径中的一个关键酶，催化焦磷酸从ATP转移到6-羟甲基-7，8-二氢蝶呤(HP)。叶酸辅助因子是生命所必需的。哺乳动物从它们的饮食中获得叶酸，而大多数微生物必须从头合成叶酸。因此，HPPK是开发新型抗菌剂的理想靶点。HPPK含有158个氨基酸残基，具有热稳定性，是研究酶促焦磷酰化转移机理的良好模型体系。我们通过测定apo-HPPK(无配体酶)、HPPK-GAMATPanalog(HPPK与一种底物的二元络合物)、HPPK-MgATPanalog-HP(HPPK与两种底物的三元络合物)、HPPK-AMP-HPPP(HPPK与两种产物的三元络合物)和HPPK-HPPP(HPPK与一种产物的二元络合物)的三维结构，绘制了HPPK催化反应的轨迹。 RNA加工蛋白 G蛋白ERA参与核糖体RNA(RRNA)的成熟，是细菌生长和细胞周期调控的关键成分。在人类和小鼠中发现的Era同源基因似乎在细胞凋亡的调节中发挥了作用。我们已经在2.4分辨率下测定了无配体ERA(apo-ERA)的晶体结构。三维结构揭示了ERA的两个结构域：一个类似于p21RAS的N-末端结构域和一个独特的C-末端结构域。基于结构的拓扑搜索C-结构域没有发现任何有意义的匹配，尽管序列分析表明它包含一个KH-结构域。KH-结构域是RNA结合基序，通常以串联重复形式出现，除保守的片段VIGxxGxxIK外，序列相似性较低。我们鉴定了一个包含VIGxxGxxIK序列的β-α-α-β折叠，它由ERA的C-结构域和KH-结构域共享。我们认为这个β-α-α-β折叠是RNA结合的基序，这是RNA结合的最低结构要求。年代在水晶中二聚化。二聚体的形成涉及一个严重扭曲的Switch II区域，这可能与ERA蛋白调节下游事件的方式有关。 核糖核酸酶III(RNaseIII)对双链RNA(DsRNA)具有特异性。它在细菌、蠕虫、苍蝇、植物、真菌和哺乳动物中都是保守的。细菌RNase III包含一个内切酶结构域(EndND)和一个dsRNA结合结构域(DsRBD)，它可以通过两种方式影响RNA结构和基因表达：一种是作为切割dsRNA的dsRNA加工酶，另一种是作为结合但不切割dsRNA的dsRNA结合蛋白。我们测定了Aquifex aeolicus RNaseIII(AA-RNaseIII)的内源ND结构，并模拟了全长AA-RNaseIII与dsRNA的催化络合物。我们还测定了一个突变的AA-RNaseIII的晶体结构，它与dsRNA形成络合物，但不切割dsRNA，揭示了非催化组装的结构。RNaseIII-dsRNA的两种功能形式之间的主要区别是蛋白质的构象和dsRNA的方向和位置。RNaseIII家族的每个成员都含有一个或两个endND拷贝和一个dsRBD拷贝。因此，从我们的结构中获得的信息有助于揭示其他RNaseIII蛋白的结构和功能，这随着最近在RNA干扰中发现RNaseIII同源基因“Disher”而变得更加重要。RNaseIII是一种广泛存在于真菌、植物和动物中的RNA沉默现象。

O2-ARYL DIAZENIUMDIOLATES Glutathione S-transferase (GST) is a family of enzymes that catalyze glutathione conjugation with electrophilic compounds. In preneoplastic and neoplastic cells, specific forms of GSTs are expressed at high levels and to participate in the cells' resistance to anticancer drugs. Isozyme GSTP is of particular importance in biological resistance to alkylating agents. Therapeutic strategies aimed at inhibiting GSTP to extend the efficacy of alkylating agents have been unsuccessful. However, GSTP-activated prodrugs have shown great potential in targeting specific cancer cells. We have developed a new type of anticancer agents, O2-aryl diazeniumdiolates, which kill cancer cells by releasing nitric oxide intracellularly (Australia Patent Number 733590; US Patent Number 6610660; and European Patent Number 04009529.1-2101). Two lead compounds have since been developed. FOLATE PATHWAY ENZYMES 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthetic pathway, catalyzing the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). Folate cofactors are essential for life. Mammals derive folates from their diets, whereas most microorganisms must synthesize folate de novo. Therefore, HPPK is an ideal target for the development of novel antimicrobial agents. HPPK contains 158 amino acid residues and is thermostable, which makes it an excellent model system for the mechanistic study of the enzymatic pyrophosphoryl transfer. We have mapped out the trajectory of HPPK-catalyzed reaction by determining the three-dimensional structures of apo-HPPK (ligand-free enzyme), HPPK-MgATPanalog (binary complex of HPPK with one substrate), HPPK-MgATPanalog-HP (ternary complex of HPPK with both substrates), HPPK-AMP-HPPP (ternary complex of HPPK with both products), and HPPK-HPPP (binary complex of HPPK with one product). RNA-PROCESSING PROTEINS G Protein ERA is involved in the maturation of ribosomal RNA (rRNA) and is a key component that couples the regulation of growth and cell cycle in bacteria. ERA homologues identified in human and mouse appear to play a role in the regulation of apoptosis. We have determined the crystal structure of ligand-free ERA (apo-ERA) at 2.4- resolution previously. The three-dimensional structure revealed a two-domain arrangement of ERA: an N-terminal domain that resembles p21 Ras and a C-terminal domain that is unique. Structure-based topological search of the C-domain fails to reveal any meaningful match, although sequence analysis suggests that it contains a KH-domain. KH-domains are RNA-binding motifs that usually occur in tandem repeats and exhibit low sequence similarity except for the well-conserved segment VIGxxGxxIK. We identified a beta-alpha-alpha-beta fold that contains the VIGxxGxxIK sequence and is shared by the C-domain of ERA and the KH-domain. We proposed that this beta-alpha-alpha-beta fold is the RNA-binding motif, the minimum structural requirement for RNA-binding. ERA dimerizes in crystal. The dimer formation involves a significantly distorted switch II region, which may relate to how ERA protein regulates downstream events. Ribonuclease III (RNase III) shows specificity toward double-stranded RNA (dsRNA). It is conserved in bacteria, worms, flies, plants, fungi, and mammals. Bacterial RNase III, containing an endonuclease domain (endoND) and a dsRNA-binding domain (dsRBD), can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA-binding protein that binds but does not cleave dsRNA. We have determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. We have also determined the crystal structure of a mutant Aa-RNase III, which binds but does not cleave dsRNA, in complex with dsRNA, revealing the architecture of a non-catalytic assembly. The major differences between the two functional forms of RNase III-dsRNA are the conformation of the protein and the orientation and location of dsRNA. Every member of the RNase III family contains one or two copies of the endoND and one copy of the dsRBD. Therefore, the information derived from our structures sheds light on the structure and function of other RNase III proteins, which has gained added importance with the recent discovery of the RNase III ortholog "Dicer" in RNA interference, a broad class of RNA silencing phenomena found in fungi, plants, and animals.