Functional involvement of a deoxy-D-xylulose 5-phosphate reductoisomerase gene harboring locus of Synechococcus leopoliensis in isoprenoid biosynthesis
Functional involvement of a deoxy-D-xylulose 5-phosphate reductoisomerase gene harboring locus of Synechococcus leopoliensis in isoprenoid biosynthesis
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DOI：
10.1016/s0014-5793(00)02014-7
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发表时间：
2000-09-22
期刊：
影响因子：
3.5
通讯作者：
Zimmer, W
中科院分区：
文献类型：
--
作者：
Miller, B;Heuser, T;Zimmer, W
本工作旨在证明蓝藻中非甲氧戊酸途径的功能，本工作旨在分离1-脱氧-D-木酮糖5-磷酸还原异构酶基因(Dxr)，该基因编码催化该途径特定的指示性步骤的酶。为此目的，通过聚合酶链式反应从利奥波利聚球藻SAUG1402-1DKA中扩增出DSR片段。DNA测序结果表明，DXR同源基因和相邻的两个开放阅读框被定位在一个操纵子中，该基因在大肠杆菌中表达，并将纯化的基因产物用于底物DXP的NADPH依赖光度测定。虽然类异戊二烯生物合成的中心中间体之一二甲基烯丙基二磷酸的含量显著增加(P小于或等于0.001)，但过表达S的DxP合成酶基因并不会导致DMADP水平的升高。由于即使在具有DXS表达融合的菌株中，DXR的额外过表达也不影响DMADP的含量，因此得出结论，DXS而不是DXR催化非甲戊酸异戊二烯生物合成的速率限制步骤，(C)2000欧洲生化学会联合会。爱思唯尔科学公司出版。版权所有。
The present work aimed to proof the functionality of the non-mevalonate pathway in cyanobacteria, It was intended to isolate the 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase gene (dxr), as this gene encodes the enzyme which catalyzes a pathway-specific, indicative step of this pathway, For this purpose, a segment of dsr was amplified from Synechococcus leopoliensis SAUG 1402-1 DKA via PCR using oligonucleotides for conserved regions, Subsequent hybridization screening of a genomic cosmid library of S, leopoliensis with the PCR segment led to the identification of a 26.5 kbp locus on which a dxr homologous gene and two adjacent open reading frames organized in one operon were localized by DNA sequencing, The functionality of the gene was demonstrated expressing the gene in Escherichia coli and using the purified gene product in a photometrical NADPH dependent test based on the substrate DXP generating system. While the content of one of the central intermediates of the isoprenoid biosynthesis (dimethylallyl diphosphate = DMADP) was significantly (P less than or equal to 0.001) increased in E, coli cells overexpressing the DXP synthase gene (dxs) of S, leopoliensis, overexpression of dxr does not lead to an elevated DMADP level. Since even in strains harboring an expression fusion of dxs the additional overexpression of dxr does not influence the DMADP content, it is concluded that Dxs but not Dxr catalyzes a rate limiting step of the non-mevalonate isoprenoid biosynthesis, (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.