RUI: Carbonic Anhydrases Involved in Algal Inorganic Carbon Acquisition

RUI：参与藻类无机碳获取的碳酸酐酶

• 批准号: 9418502
• 负责人: H. David Husic
• 金额: $ 11.8万
• 依托单位: Lafayette College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-15 至 1999-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9418502&HistoricalAwards=false
• 关键词: RUI; Carbonic; Anhydrases; Involved; Algal

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.

碳酸酐酶催化在生理pH值下在水性介质中占主导地位的两种形式的无机碳（二氧化碳和碳酸氢根离子）的相互转化。在莱茵衣藻中，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分。该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，否则会限制它们的生长。周质碳酸酐酶在无机碳获取中的作用已被充分确立，并且有大量证据表明细胞内碳酸酐酶活性对于有效的无机碳利用也是必不可少的。然而，负责这种细胞内活性的蛋白质尚未被具体鉴定。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的光亲和试剂作为工具，在识别和分离的生理重要的细胞内形式的碳酸酐酶，存在的水平太低，很容易识别和定量的其他方法，并在研究中导致识别的基因编码的细胞内碳酸酐酶。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，以调查的亚基结构和二硫键还原状态的催化和抑制剂结合性能的主要细胞外形式的碳酸酐酶从莱茵衣藻之间的关系。这些研究的总体目标是提高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。 %%% 单细胞绿色藻类莱茵衣藻和各种其他水生光合生物已经开发出了比大多数陆生植物更有效地获取大气二氧化碳的系统。这是重要的，因为光合生物利用二氧化碳进行碳水化合物的光依赖性合成，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。了解高效的二氧化碳采集系统如何在生物体中工作，如莱茵衣藻，可能对学习如何提高高等植物的二氧化碳利用率有价值。尽管这个系统在莱茵衣藻中发挥作用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，并且这种相互转化对于促进这些无机碳物质在细胞隔室之间的转移是重要的，并且在使二氧化碳可用于叶绿体（二氧化碳转化为碳水化合物的细胞内位点）中起作用。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位。这项工作的主要目标是分离叶绿体中存在的酶的形式。这反过来可能导致识别编码这种蛋白质的基因，并了解基因如何响应环境刺激而打开和关闭。 这项工作的其他部分可能导致对存在于藻类细胞其他区室中的其他形式的碳酸酐酶的鉴定。我们相信，这一信息将显着提高我们的理解的有效机制，二氧化碳收购莱茵衣藻。