In-planta analyses of the three Arabidopsis isoforms of the bifunctional RIBA with enzymatic domains for GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase

核黄素作为黄素辅酶（FMN和FAD）的前体，是所有生物体所必需的。核黄素生物合成的最初两个关键酶促步骤在植物中通过双功能RIBA酶进行，所述双功能RIBA酶包括GTP环化水解酶II（GCHII）和3，4-二羟基-2-丁酮-4-磷酸合酶（DHBPS）。在被子植物中，RIBA基因家族由三个成员组成。开始我们的研究在植物核黄素的生物合成，我们发现，强烈减少AtRIBA 1表达的拟南芥rfd 1突变体（T-DNA插入RIBA 1）和RIBA 1反义线不能由同时表达的AtRIBA 2 AtRIBA 3的补充。关于植物核黄素代谢的生物化学和控制的信息很少。通过所提出的工作计划，其目的是验证所有三种RIBA同工型在核黄素生物合成中的功能。它的目的是在植物中检查的酶的性质和生理重要性的三个二分野生型和多个工程化的RIBA变体，这将在一个致命的核糖体突变体中表达。此外，转基因拟南芥植物与沉默和过表达的RIBA 1基因将被分析的修改内容的黄素和黄素辅酶依赖性蛋白质和它们的细胞过程。RIBA相互作用伴侣的筛选旨在解码核黄素生物合成的功能，组织和控制以及与其他亚细胞过程的相互关系。在初步实验中，用质体提取物实现了RIBA 1和RIBA 2磷酸化。接下来，计划鉴定RIBA蛋白的磷酸化位点，并揭示拟南芥中RIBA磷酸化的生理后果。

Riboflavin serves as precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The initial two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprising GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). In Angiosperms the RIBA gene family consists of three members. Starting our studies in plant riboflavin biosynthesis, we found that strongly reduced AtRIBA1 expression in an Arabidopsis thaliana rfd1 mutant (T-DNA insertion in RIBA1) and in RIBA1 antisense lines cannot be complemented by the simultaneously expressed AtRIBA2 AtRIBA3. Only little information is available on biochemistry and control of plant riboflavin metabolism. By means of the proposed working program it is aimed to verify the function of all three RIBA isoforms in riboflavin biosynthesis. It is intended to examine in planta the enzymatic properties and the physiological importance of the three bipartite wild-type and multiple engineered RIBA variants, which will be expressed in a lethal riba mutant. In addition, transgenic Arabidopsis plants with silenced and overexpressed RIBA1 genes will be analysed on the modified content of flavins and the flavocoenzyme-dependent proteins and their cellular processes. Screens for RIBA-interaction partners are aimed at to decode the function, organisation and control of riboflavin biosynthesis and the interrelation to other subcellular processes. In preliminary experiments RIBA1 and RIBA2 phosphorylation was achieved with plastid extracts. In continuation it is planned to identify the phosphorylation site of the RIBA proteins and unravel the physiological consequences of RIBA phosphorylation in Arabidopsis plants.