一种活性植物烯合成酶的表达及酶的生化特性

Ute Neudert , Isabel M Martı́nez-Férez , Paul D Fraser , Gerhard Sandmann
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引用次数: 34

摘要

从产胡萝卜素的尿路Erwinia uredovora肠杆菌中编码植物烯合成酶的crtB基因在大肠杆菌中过表达,约占细胞总蛋白的20%。活性植物烯合成酶的形成对表达菌株的生长有抑制作用。据推测,生长抑制是由于底物香叶基焦磷酸(GGPP)的耗尽引起的,在大肠杆菌中,GGPP是合成必需的戊烯基焦磷酸衍生物所必需的。为了克服植物烯合成酶表达菌株生长不佳的特点,通过共表达编码Erwinia GGPP合成酶和Rhodobacter异戊烯基焦磷酸异构酶的类异戊二烯生物合成基因crtE和idi,提高了GGPP水平。粗酶制剂经DEAE柱层析部分纯化15倍。开发了一种非放射性试验,使GGPP转化为植物烯。在HPLC体系上用标准液相色谱法对反应产物进行鉴定,并对其光谱特征进行比较。在体外形成的植物烯具有15顺式和全反式两种异构体构型。必需的辅助因子是ATP与Mn2+或Mg2+的结合。GGPP的Km值为41 μM。磷离子和角鲨抑素抑制植物烯的合成。后一种抑制剂的I50值为15 μM。Lineweaver-Burk图显示,在存在或不存在角鲨素的情况下,Km值不变。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Expression of an active phytoene synthase from Erwinia uredovora and biochemical properties of the enzyme

The crtB gene encoding phytoene synthase from the carotenogenic enterobacterium Erwinia uredovora was overexpressed to about 20% of the total cellular protein in Escherichia coli. Formation of the active phytoene synthase had the effect of suppressing the growth of the expressing strain. Presumably inhibition of growth arose from the depletion of the substrate geranylgeranyl pyrophosphate (GGPP) which, in E. coli, is necessary for the synthesis of essential prenylpyrophosphate derivatives. In order to overcome the poor growth characteristics of the phytoene synthase expressing strain, GGPP levels were increased by co-expressing the isoprenoid biosynthetic genes crtE and idi, encoding the Erwinia GGPP synthase and Rhodobacter isopentenyl pyrophosphate isomerase, respectively. The crude enzyme preparation was partially purified 15-fold by chromatography on a DEAE column. A non-radioactive assay was developed that enabled the conversion of GGPP to phytoene. The reaction product was identified by co-chromatography with authentic standards on HPLC systems and comparison of spectral characteristics. The phytoene formed in vitro was present in both a 15-cis and all-trans isomeric configuration. The essential cofactors required were ATP in combinations with either Mn2+ or Mg2+. The Km value for GGPP was determined as 41 μM. Phytoene synthesis was inhibited by phosphate ions and squalestatin. The I50 value for the latter inhibitor was 15 μM. Lineweaver–Burk plots showed constant Km values in the presence or absence of squalestatin.

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