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

光合生物对碳和氮的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。本项目重点研究谷氨酸合成过程中涉及的酶，这是C和N同化的第一个产物，以及催化的还原剂供应系统。得到了以下结果：从蓝藻Plectonema boryanum中克隆了Fd-和nadph依赖性谷氨酸合成酶基因。每个基因缺失的突变体都可以存活，但在有效的碳同化条件下，只有缺乏独立依赖酶的突变体缺氮。该菌株在缺氮条件下，细胞氨基酸池发生变化，藻脂蛋白转录物水平下降，硝酸盐高效还原为氨氮。玉米叶片叶肉细胞和束鞘细胞在C和N同化中分化，具有独特的铁氧还蛋白同工蛋白（FdI和FdII）。利用内源Fd基因被每个玉米Fd基因取代的蓝藻细胞，发现FdI主要作为非循环光合电子转移的电子载体，而FdII在循环电子转移中更优，导致n同化途径的电子捐赠较弱。这些结果表明，Fd是协调碳氮同化的关键分子。测定了Fd与Fd- nadp +还原酶复合物的x射线晶体结构。获得了假体基团拓扑结构信息和两种蛋白的相互作用。为了获得C、N和S同化串扰的结构基础，本研究将扩展到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.