RubisCO的研究进展

1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)是调节光合和光呼吸,决定净光合作用的一个关键酶;也是植物可溶性蛋白质中含量最高的蛋白质.该酶广泛存在于植物及一些微生物体内.综述了近年来有关RubisCO的一些研究进展. 包括RubisCO的基本性质、结构与功能、酶基因工程、酶活性调节及其活化酶等.     

水稻RubisCO的纯化及其与烟草RubisCO性质的比较

利用蔗糖密度梯度离心和DEAE-Sepharose fast flow柱层析等步骤从水稻叶片中纯化了RubisCO。此法不仅快速,而且酶的收得率高,酶的比活达1.15 μmol CO_2 min~(-1) mg~(-1)。 水稻RubisCO的热稳定性比烟草酶差,在活化时对Mg~(2 )较敏感。水稻和烟草RubisCO钝化态时总巯基数和表面巯基数相同,然而当酶活化后,水稻酶表面巯基数增加,而烟草酶则减少。当这些表面巯基被修饰后,水稻酶活性损失60％而烟草酶活力仅损失15％。水稻和烟草RubisCO的远紫外CD光谱有明显的区别,这显示了两者在二级结构、酶比活和性质上的重大差别。     

原核生物Rubisco的研究进展

1 ,5 二磷酸核酮糖羧化酶 /加氧酶 (Rubisco)是卡尔文循环中的关键酶 ,该酶广泛存在于植物和一些原核生物中。由于在结构上 ,原核生物中Rubisco与植物有相似之处 ,因此人们对原核生物中的Rubisco进行了大量研究 ,就原核生物Rubisco的结构、基因调节、装配等方面的最新进展作一综述。     

多能硫杆菌RubisCO基因鉴定以及在大肠杆菌中的表达

多能硫杆菌(Thiobacillus versutus)是兼性化能自养细菌,在生理学和分类学上具有重要的地位,也是研究硫杆菌生理、生化、遗传学的理想材料。该菌通过卡尔文循环固定CO_2,其关键酶是1,5-二磷酸核酮糖羧化酶/加氧酶(简称RubisCO)。我们从多能硫杆菌中分离得到的RubisCO基因片段能够在大肠杆菌细胞中表达,说明自养细菌与异养细菌在基因表达方面是相似的。     

原核生物eno基因在系统进化中应用及水平转移分析

多基因系统发育研究方法是系统发育分析中的一个重要手段,基因树冲突已成为分子系统发育研究中日益突出的问题。烯醇化酶基因(eno)及其编码的蛋白广泛存在于五界系统中,烯醇化酶为糖酵解途径中重要酶类。文章选取原核生物已注释的eno基因序列进行了系统发育分析。对其中的138个模式菌株的eno基因序列进行系统发育分析和同源性搜索,发现19个模式菌株的eno基因是通过水平转移而来;并通过核苷酸组成、密码子偏好性和基因排列等基因特征分析,进一步验证了水平转移基因的外源性。结果表明:原核生物eno序列具有较高保守性,其大小适中,是研究原核生物系统发育的良好材料。文章在对基因水平转移的供体和受体菌株生活习性、进化历史以及烯醇化酶的结构和功能的研究过程中提供重要参考价值。     

多能硫杆菌RubisCO基因同源性分析

以氧化亚铁硫杆菌1,5—二磷酸核酮糖羧化酶/加氧酶(RubisCO)基因为探针,与氧化硫硫杆菌和多能硫杆菌的染色体DNA杂交。结果表明,氧化硫硫杆菌的染色体DNA能够与氧化亚铁硫杆菌RubisCO基因探针杂交。而多能硫杆菌不能与其杂交,然而却能够与球形红杆菌RubisCO基因探针杂交,同源性高。由于RubisCO在进化上的高度保守性,因此认为它们在RubisCO进化关系上应属于不同的类群。     

肝素酶在医药领域应用的研究进展

肝素酶是一类能够特定切割肝素或硫酸乙酰肝素中α-1,4糖苷键并将其裂解成有活性寡糖片段的酶,主要分为真核生物肝素酶(Heparanase)和原核生物肝素酶(Heparinase)。由于原核生物肝素酶是一种高效绿色的生物催化剂,因此近年来在医药领域的应用性研究逐渐被重视。文中结合本课题组相关工作,归纳介绍了原核生物肝素酶通过作用于硫酸肝素蛋白聚糖(HSPGs)生成肝素小分子,抑制肿瘤细胞增殖方面的应用;原核生物肝素酶在制备第三代创新型抗凝血药物低分子量肝素(Low molecular weight heparin,LWMH)和超低分子量肝素(Ultra low molecular weight heparin, ULMWH)方面的应用;原核生物肝素酶作为肝素拮抗药物等医药领域的重要应用;并展望了原核生物肝素酶的未来应用前景及挑战。     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

The rabbit muscle phosphofructokinase gene. Implications for protein structure, function, and tissue specificity

Sequence homologies between bacterial and rabbit muscle phosphofructokinases and between the amino- and carboxyl-terminal halves of the latter suggest that the mammalian enzyme evolved from a prokaryotic progenitor by gene duplication and divergence (Poorman, R. A., Randolph, A., Kemp, R. G., and Heinrikson, R. L. (1984) Nature 309, 467-469). We have isolated the gene for the rabbit enzyme and determined the nucleotide sequence for all the exons and most of the introns. This represents the first eukaryotic phosphofructokinase gene ever sequenced. The cloned gene is 17 kilobase pairs long. The coding sequence for 780 amino acids is split into 22 exons ranging in size from 15 to 63 codons. Sequence analysis shows that 75% of the bases at the third position of the codons in these exons are either G or C. Exons XV and XVI code for the 30 amino acid residues which were left unidentified in the published primary structure for this enzyme. When overlaid on the structure of the protein, most of the introns are located between or near the ends of the secondary structural elements but not at analogous positions in the two protein-coding halves of the gene.     

Interleukin-1βconverting enzyme: A novel cysteine protease required for IL-1β production and implicated in programmed cell death

Interleukin-1β converting enzyme is the first member of a new class of cysteine proteases. The most distinguishing feature of this family is a nearly absolute specificity for cleavage at aspartic acid. This enzyme has been the subject of intense research because of its role in the production of IL-1β, a key mediator of inflammation. These studies have culminated in the design of potent inhibitors and determination of its crystal structure. The structure secures the relationship of the enzyme to CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans, suggesting that members of this family function in cell death in vertebrates.     

Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis

The discovery of the 2-C-methyl-D-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this pathway. CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1 mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The beta-glucuronidase expression pattern driven from the CLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in the CLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of the CLA1 gene mutation in mesophyll tissue formation.     

酯酶基因扩增及突变与昆虫抗药性

有机磷和氨基甲酸酯类杀虫剂的大量使用导致昆虫对其产生抗药性。酯酶是昆虫体内重要的解毒代谢酶,酯酶基因表达量上升和点突变使其代谢或结合杀虫剂的能力增强是昆虫对常用农药产生抗药性的2个重要原因。文章概述昆虫酯酶基因扩增及突变所导致的抗药性,进一步分析了酯酶突变对蛋白结构和功能的影响。     

A role in true-late gene expression for the T4 bacteriophage 5' polynucleotide kinase 3' phosphatase.

Two bacteriophage T4-induced, nucleic acid-modifying activities, 5′ polynucleotide kinase and 3′ phosphatase, are both coded by the pseT gene. Therefore, the product of this gene is an enzyme which can remove phosphates from 3′ termini and add them to 5′-hydroxyl termini and thus could be said to “shuttle” phosphates on polynucleotides. This enzyme is sometimes required for T4 true-late gene expression, probably by helping establish the required intracellular DNA structure. Our data suggest that a host gene product normally can substitute for the product of the pseT gene, making it non-essential for phage multiplication on most laboratory strains of Escherichia coli.     

Structure and function of the E. coli dihydroneopterin triphosphate pyrophosphatase: a Nudix enzyme involved in folate biosynthesis

Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the committed step of folate synthesis in bacteria. That this dihydroneopterin hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed in vivo: knockout of this gene in E. coli leads to a marked reduction in folate synthesis that is completely restored by a plasmid carrying the gene. We also determined the crystal structure of this enzyme using data to 1.8 A resolution and studied the kinetics of the reaction. These results provide insight into the structural bases for catalysis and substrate specificity in this enzyme and allow the definition of the dihydroneopterin triphosphate pyrophosphatase family of Nudix enzymes.     

Recent developments on the regulation and structure of glutamine synthetase enzymes from selected bacterial groups

Abstract: The structure of glutamine synthetase (GS) enzymes from diverse bacterial groups fall into three distinct classes. GSI is the typical bacterial GS, GSII is similar to the eukaryotic GS and is found together with GSI in plant symbionts and Streptomyces , while GSIII has been found in two unrelated anaerobic rumen bacteria. In most cases, the structural gene for GS enzyme is regulated in response to nitrogen. However, different regulatory mechanisms, to ensure optimal utilization of nitrogen substrates, control the GS enzyme in each class.     

Full-size form of human liver AMP-deaminase?

AMP-deaminase from human liver was purified by two-step phosphocellulose chromatography, and SDS-PAG electrophoresis of the most active enzyme fraction eluted has been performed. The largest of the protein fragments revealed had a size (92 kDa) of an apparent full-size enzyme subunit, and reacted positively with antibodies produced against specific human ampd2 gene product. Three-day storage at cold room temperature modified significantly the electrophoretical pattern of the enzyme, evidencing continuous and progressive degradation of its structure. This is a first report evidencing the presence of apparent full-size form of human liver AMP-deaminase in preparation obtained from endogenous source.