RUI: Carbonic Anhydrases Involved in Algal Inorganic Carbon Acquisition

碳酸酐酶催化两种形式的无机碳，二氧化碳和碳酸氢盐离子的相互转化，这两种形式的无机碳在生理pH值的水介质中占主导地位。在莱茵衣藻（Chlamydomonas reinhardtii）中，一种或多种形式的碳酸酐酶是无机碳获取和浓缩系统的组成部分。这个系统使这种藻类和其他藻类能够有效地利用低水平的二氧化碳，否则二氧化碳会限制它们的生长。质周碳酸酐酶在无机碳获取中的作用已经得到了很好的证实，并且有大量证据表明细胞内碳酸酐酶活性对于有效利用无机碳也是必不可少的。然而，负责这种细胞内活动的蛋白质尚未被明确鉴定。我们建议利用一种新的碳酸酐酶特异性活性位点定向放射性标记光亲和试剂，作为鉴定和分离生理上重要的碳酸酐酶细胞内形式的工具，这些形式的水平太低，无法通过其他方法容易识别和量化，并且在导致鉴定细胞内碳酸酐酶编码基因的研究中。将这种光亲和试剂应用于蓝藻系统中碳酸酐酶的鉴定和定位的研究也将开始。此外，我们建议继续研究亚基结构和二硫键还原状态对莱茵衣藻主要胞外形式碳酸酐酶的催化和抑制剂结合特性的关系。这些研究的总体目标是增强我们对碳酸酐酶作为藻类细胞能够有效地浓缩无机碳以提供光合作用所需二氧化碳的系统的重要组成部分的作用的理解。单细胞绿藻莱茵衣藻和其他各种水生光合生物已经发展出比大多数陆生植物更有效地获取大气中二氧化碳的系统。这一点很重要，因为光合生物利用二氧化碳进行依赖光的碳水化合物合成，这反过来又导致植物生长和生物质生产。此外，由于大气中二氧化碳含量低，许多植物的生长受到限制。了解莱茵衣藻（Chlamydomonas reinhardtii）等生物体中高效的二氧化碳采集系统是如何工作的，可能对学习如何提高高等植物对二氧化碳的利用有价值。尽管这个系统在莱茵衣藻中运作的所有细节尚不清楚，但很明显，一种特殊的酶，碳酸酐酶，是这个过程的重要组成部分。碳酸酐酶加速二氧化碳和碳酸氢盐的相互转化，这种相互转化对促进这些无机碳在细胞间的转移很重要，并在使二氧化碳进入叶绿体中发挥作用，叶绿体是二氧化碳转化为碳水化合物的细胞内位点。在莱茵衣藻中，有相当多的证据表明，这种酶的几种不同形式对二氧化碳利用过程很重要，并且这些形式存在于藻类细胞的不同部分。这项工作的主要目标是分离存在于叶绿体中的酶的形式。反过来，这可能会导致识别编码这种蛋白质的基因，并了解基因是如何在环境刺激下开启和关闭的。这项工作的其他部分可能会导致其他形式的碳酸酐酶存在于藻类细胞的其他隔间的鉴定。我们相信这一信息将大大提高我们对莱茵衣藻二氧化碳获取有效机制的理解。

The enzyme carbonic anhydrase catalyzes the interconversion of the two forms of inorganic carbon, carbon dioxide and bicarbonate ion, that predominate in aqueous media at physiological pH values. In the alga Chlamydomonas reinhardtii, one or more forms of carbonic anhydrase are components of a system for inorganic carbon acquisition and concentration. This system enables this and other algae to efficiently utilize low levels of carbon dioxide that could otherwise limit their growth. The role of a periplasmic carbonic anhydrase in inorganic carbon acquisition is well established, and there is substantial evidence that intracellular carbonic anhydrase activity is also essential for efficient inorganic carbon u tilization. However, the proteins responsible for this intracellular activity have not been specifically identified. We propose to utilize a novel carbonic anhydrase specific active site directed radiolabeled photoaffinity reagent as a tool both in the identification and isolation of physiologically important intracellular forms of carbonic anhydrase that are present in levels too low to be readily identified and quantified by other methods, and in studies leading to the identification of genes encoding intracellular carbonic anhydrase. Studies to apply this photoaffinity reagent to the identification and localization of carbonic anhydrases in cyanobacterial systems will also be initiated. In addition, we propose to continue studies to investigate the relationship between the subunit structure and disulfide bond reduction status on both the catalytic and inhibitor binding properties of the predominant extracellular form of carbonic anhydrase from Chlamydomonas reinhardtii. The overall objective of these studies is to enhance our understanding of the role of carbonic anhydrases as important components of the systems by which algal cells are able to efficiently concentrate inorganic carbon to provide carbon dioxide for photosynthesis. %%% The unicellular green alga Chlamydomonas reinhtardii and a variety of other aquatic photosynthetic organisms have developed systems that allow them to acquire atmospheric carbon dioxide much more effectively than most terrestrial plants. This is important because photosynthetic organisms utilize carbon dioxide for the light dependent synthesis of carbohydrates, which in turn leads to plant growth and biomass production. Furthermore, many plants can be growth limited because of the low levels of carbon dioxide that are present in the atmosphere. An understanding of how the highly efficient carbon dioxide acquisition systems work in organisms like Chlamydomonas reinhardtii, may be of value in learning how to improve carbon dioxide utilization in higher plants. Alt hough all of the details of the way in which this system functions in Chlamydomonas reinhardtii are not yet understood, it is apparent that a particular enzyme, carbonic anhydrase, is an important component of this process. Carbonic anhydrase accelerates the interconversion of carbon dioxide and bicarbonate, and this interconversion is important to facilitate the transfer of these species of inorganic carbon among cellular compartments, and plays a role in making carbon dioxide available to the chloroplast, the intracellular site of conversion of carbon dioxide to carbohydrates. In Chlamydomonas reinhardtii, there is considerable evidence that several different forms of this enzyme are important for the process of carbon dioxide utilization, and that these forms are found in different parts of the algal cell. It is a primary goal of this work to isolate the form of the enzyme that is present in the chloroplast. This in turn may lead to the identification of the gene that encodes this protein, and to an understanding of how the gene is turned on and off in response to environmental stimuli. Other parts of the work may lead to the identification of other forms of carbonic anhydrase that are present in other compartments of the algal cell. We believe that this information will significantly enhance our understanding of the efficient mechanism for carbon dioxide acquisition in Chlamydomonas reinhardtii.