首页> 外文学位 >Isoprenoid biosynthesis: Identification and characterization of isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Rhodobacter, characterization of a eukaryotic isopentenyl diphosphate:dimethylallyl diphosphate isomerase and phylogenetic analysis of pathway enzymes.
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Isoprenoid biosynthesis: Identification and characterization of isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Rhodobacter, characterization of a eukaryotic isopentenyl diphosphate:dimethylallyl diphosphate isomerase and phylogenetic analysis of pathway enzymes.

机译:类异戊二烯的生物合成:鉴定和鉴定来自红细菌的异戊烯基二磷酸:二甲基烯丙基二磷酸异构酶,鉴定真核异戊烯基二磷酸:二甲基烯丙基二磷酸异构酶和通路酶的系统进化分析。

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摘要

The discovery and initial characterization of the first known bacterial IPP:DMAPP isomerase are presented in these studies. A putative Rhodobacter capsulatus gene for IPPIase was identified in the photosynthetic gene cluster by searching protein sequence databases with an amino acid probe based on the active site region of the Schizosaccharomyces pombe IPPIase. The purification and characterization of the cloned gene are reported in these studies. The purified protein was demonstrated to catalyze the conversion of IPP to DMAPP. The Mn++ and Mg++ dependencies were examined in these studies. The R. capsulatus IPPIase was shown to have a higher activity in the presence of Mn++ as compared to Mg++. Furthermore, the kcat and kcat/Km values were 4-fold higher in the Mn ++ containing buffer.; The IPP:DMAPP isomerase from S. pombe was the subject of kinetic studies. The KM and kcat values for IPP were determined by standard steady state kinetics which yielded values of 81 μM and 5 s−1 respectively. Product inhibition studies were used to determine the same values for IPP (KM = 78 μM and k cat = 4.8 s−1) and also the KM for DMAPP which was 86 μM. This allowed the calculation of the kcat value for DMAPP (1.6 s−1) using the Haldane relationship. The Mn++ and Mg++ dependencies were examined in these studies. It was demonstrated Mg++ was the preferred divalent metal ion cofactor for the eukaryotic enzyme.; A phylogenetic analysis of the isoprenyl diphosphate synthase family of proteins allowed for the categorization and description of this family of proteins. Seven groups of phylogenetically distinct enzymes were identified in these studies including bifunctional FPP/GGPP synthases from archeabacteria, FPP synthases from eukaryotes, GGPP synthases from eukaryotes, FPP synthases from bacteria, GGPP synthases from bacteria, chromatoplastic GGPP synthases from plants and long chain synthases. The chromatoplastic synthases from plants were shown to have evolved from a bacterial like FPP synthase. This work represents the first comprehensive classification of the trans isoprenyl diphosphate synthase family of enzymes.
机译:这些研究提出了第一个已知的细菌IPP:DMAPP异构酶的发现和初步表征。通过基于粟酒裂殖酵母 IPPIase的活性位点区域的氨基酸探针搜索蛋白质序列数据库,在光合基因簇中鉴定出IPPIase的假定的荚膜红假单胞菌基因。这些研究报道了克隆基因的纯化和鉴定。已证明纯化的蛋白质可催化IPP向DMAPP的转化。在这些研究中检查了Mn ++ 和Mg ++ 的依赖性。 R。结果表明,与Mg ++ 相比,荚膜中的IPPIase在Mn ++ 的存在下具有更高的活性。此外,在Mn ++ 中,k cat 和k cat / K m 值高4倍。包含缓冲区。来自 S的IPP:DMAPP异构酶。 pombe 是动力学研究的主题。 IPP的K M 和k cat 值通过标准稳态动力学确定,分别得出81μM和5 s -1 的值。乘积抑制研究用于确定相同的IPP值(K M = 78μM和k cat = 4.8 s -1 ),并且DMAPP的K M 为86μM。这允许使用Haldane关系计算DMAPP的k cat 值(1.6 s -1 )。在这些研究中检查了Mn ++ 和Mg ++ 的依赖性。证明Mg ++ 是真核生物酶优选的二价金属离子辅因子。对异戊二烯基二磷酸合酶家族蛋白进行系统发育分析,可以对该蛋白家族进行分类和描述。在这些研究中鉴定出七类系统发育不同的酶,包括古细菌的双功能FPP / GGPP合成酶,真核生物的FPP合成酶,真核生物的GGPP合成酶,细菌的FPP合成酶,细菌的GGPP合成酶,植物的色塑性GGPP合成物和长链合成酶。显示来自植物的色塑性合酶已经从细菌如FPP合酶进化而来。这项工作代表了 trans 异戊二烯基二磷酸合酶家族的首次全面分类。

著录项

  • 作者

    Baker, Jonathan A.;

  • 作者单位

    The University of Utah.;

  • 授予单位 The University of Utah.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2002
  • 页码 198 p.
  • 总页数 198
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

  • 入库时间 2022-08-17 11:46:18

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