FUNCTIONAL IDENTIFICATION OF AL-3 FROM NEUROSPORA-CRASSA AS THE GENE FOR GERANYLGERANYL PYROPHOSPHATE SYNTHASE BY COMPLEMENTATION WITH CRT GENES, INVITRO CHARACTERIZATION OF THE GENE-PRODUCT AND MUTANT ANALYSIS
FUNCTIONAL IDENTIFICATION OF AL-3 FROM NEUROSPORA-CRASSA AS THE GENE FOR GERANYLGERANYL PYROPHOSPHATE SYNTHASE BY COMPLEMENTATION WITH CRT GENES, INVITRO CHARACTERIZATION OF THE GENE-PRODUCT AND MUTANT ANALYSIS
复制标题
DOI：
10.1016/1011-1344(93)80071-g
复制
发表时间：
1993-05-01
影响因子：
5.4
通讯作者：
MACINO, G
中科院分区：
文献类型：
--
作者：
SANDMANN, G;MISAWA, N;MACINO, G
本工作对粗面脉孢菌al-3基因的功能进行了测定。采用体外合成法测定了al-2 FGSC 313和al-3 Rp100 FGSC 2082突变株的香叶基焦磷酸香叶素合成酶活性。本实验表明，al-3Rp100突变体表达的GGPP合酶活性降低。对突变的al-3基因进行克隆和测序，发现1个错义突变将丝氨酸转化为天冬酰胺。利用厄文氏菌CRT基因簇转化大肠杆菌进行遗传互补。当粗面新菌al-3基因取代产生胡萝卜素的CRT簇中的GGPP合成酶基因(CrtE)时，在大肠杆菌中观察到类胡萝卜素的积累。对大肠杆菌转化子进行的无细胞研究直接证明了al-3蛋白作为GGPP合成酶的功能，并表明短链戊烯基焦磷酸是真正的底物。
In this work the Neurospora crassa al-3 gene function was determined. Geranylgeranyl pyrophosphate (GGPP) synthase activity was measured in al-2 FGSC 313 and al-3 RP100 FGSC 2082 mutant strains by in vitro synthesis methods. This experiment showed that al-3 RP100 mutant expresses a reduced GGPP synthase activity. The mutated al-3 gene was cloned and sequenced; a single missense mutation was found changing serine into asparagine. Genetic complementation was performed by Escherichia coli transformation, with clusters of crt genes from Erwinia uredovora. Carotenoid accumulation was observed in E. coli transformants when the N. crassa al-3 gene substitutes the GGPP synthase gene (crtE) in the carotenogenic crt cluster. Cell-free studies with E. coli transformants gave direct evidence of the function of the al-3 protein as GGPP synthase and indicated that a short-chain prenylpyrophosphate, such as dimethylallyl pyrophosphate, is the genuine substrate.