Functional design of enzymes for glutamate synthesis and factors involved in cooperative assimilation of carbon and nitrogen

光合生物的碳氮同化依赖于还原剂和ATP的供应，这些代谢过程的代谢串扰的调节对细胞生长至关重要。本项目主要研究碳氮同化的第一个产物谷氨酸合成过程中的酶和催化还原剂供应系统。取得了如下研究成果：1. Fd和NADPH依赖的谷氨酸合酶的基因克隆从蓝藻Plectonema boryanum。每个基因缺陷的突变体是可行的，但只有缺乏Fd依赖酶的菌株在有效的碳同化条件下遭受氮缺乏。在该菌株中，氮素缺乏导致细胞氨基酸库发生变化，藻胆蛋白的转录水平下降，硝酸盐有效还原为氨.玉米叶片叶肉细胞和维管束鞘细胞在C和N同化中分化，具有独特的铁氧还蛋白同工酶（分别为FdI和FdII）。使用蓝藻细胞的内源Fd基因被替换为每个玉米Fd基因，它被发现，FdI功能主要是作为一个电子载体的非循环光合电子转移和FdII是上级在循环电子转移，导致在一个弱的电子捐赠的N-同化途径。这些结果表明，Fd是C、N同化过程中的一个关键分子。3.测定了Fd与Fd-NADP+还原酶复合物的X-射线晶体结构。获得的辅基和两种蛋白质的相互作用的拓扑结构信息。为了获得C，N和S同化的串扰的结构基础，本研究将扩展到研究Fd与GOGAT和其他S同化氧化还原酶相互作用的分子设计。

Assimilation of carbon and nitrogen in photosynthetic organisms is dependent on the supply of reductant and ATP and the regulation of the metabolic cross-talk of these metabolic processes is crucial for cell growth. This project focused on enzymes involved in glutamate synthesis, which is the first product of C and N assimilation, and reductant supply system for catalysis. Following results have been obtained.1. Genes for Fd- and NADPH-dependent glutamate synthase were cloned from the cyanobacteria Plectonema boryanum. Mutants deficient of each gene is viable, but only the strain lacking Fddependent enzyme is suffered from nitrogen deficiency under an efficient carbon assimilation condition. In this strain, change of cellular amino acid pool, decrease in the transcript level of phycobiliporoteins and efficient reduction from nitrate to ammonia were observed in response to the nitrogen deficiency.2. Mesophyll and bundle sheath cells of maize leaves, which are differentiated in C and N assimilation, possess unique ferredoxin isoproteins (FdI and FdII, respectively). Using cyanobacterial cells whose endogenous Fd gene was replaced by each maize Fd gene, it was found that FdI functions predominantly as an electron carrier for the non-cyclic photosynthetic electron transfer and that FdII was superior in the cyclic electron transfer, resulting in a weak electron donation to the N-assimilation pathway. These results suggest that Fd is a key molecule for the coordination of C and N assimilation.3.X-ray crystal structure of the complex between Fd and Fd-NADP+ reductase has been determined. Structural information on topology of the prosthetic groups and interaction of the two proteins are obtained. To obtain structural basis of the cross-talk of C, N and S assimilation, this study will be extended to investigate molecular designs of Fd for interaction with GOGAT and other S assimilatory redox enzymes.