Molecular and Biochemical Basis for Stable Carbon Isotope Ratios in Marine Autotrophs using Form IA RubisCO

使用 IA RubisCO 形式确定海洋自养生物稳定碳同位素比率的分子和生化基础

• 批准号: 0002460
• 负责人: Colleen Cavanaugh
• 金额: $ 39.79万
• 依托单位: Harvard University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0002460&HistoricalAwards=false
• 关键词: Molecular; Biochemical; Basis; Stable; Carbon

Stable carbon isotope data are used widely in ecology and earth sciences. These data on isotope fractionations influence our understandings of processes occurring on molecular to global scales. Hindcasts of past climates, models constraining the fates of present day greenhouse gas emissions, inferences of trophic transfers within complex ecosystems, and elucidation of enzyme and cellular controls on primary productivity have all used stable carbon isotope fractionation data (delta 13C values). Despite the wide applicability of such fractionation information, such data are nearly always interpreted assuming carbon is fixed and its isotopes fractionated as if the ribulose?1,5?bisphosphate?carboxylase/oxygenase (RubisCO) of spinach was responsible! This is probably a huge mistake and may substantially invalidate all of the subsequent data interpretations for problems such as those mentioned above.In the few cases that have been studied, the kinetic isotope effects (KIEs) forRubisCOs, the degree to which the delta 13C of the carbon fixed differs from the C02 substrate used, have been demonstrated to vary broadly. Thus, determining the KIEs of different RubisCOs is critical to understanding, delta 13C interpretation, and modeling of delta 13C values in oceanography. As a result, it is a long?term research goal to determine KIEs from all forms of RubisCO in autotrophs. In order to determine the KIEs of Form IA RubisCOs, these carbon-fixing enzymes will be isolated and purified from representative marine picophytoplankton and chemoautotrophic bacteria. Model species Synechococcus WH7803, Prochlorococcus MED 4, and two sulfur?oxidizing chemoautotrophs, symbionts of the coastal and vent bivalves, Solemya velum and Bathymodiolus thermophilus will be used. The isolated proteins will be characterized with respect to amino acid sequence, composition, Vmax, and Km (CO2). Finally, the kinetic isotope effects will be determined by high precision methods. Determining the KIEs for Form IA RubisCOs will allow us to evaluate a fundamental assumption of delta 13C analyses, and to infer the factors affecting oceanic primary productivity with a substantial improvement in accuracy.

稳定碳同位素数据在生态学和地球科学中有着广泛的应用。这些同位素分馏的数据影响了我们对发生在分子到全球尺度上的过程的理解。过去的气候，模型约束的命运，今天的温室气体排放，复杂的生态系统中的营养转移的推论，酶和细胞控制初级生产力的阐明，都使用了稳定的碳同位素分馏数据（δ 13 C值）。尽管这种分馏信息的广泛适用性，这些数据几乎总是解释假设碳是固定的，其同位素分馏，如果核酮糖？一点五？二磷酸盐？菠菜的羧化酶/加氧酶（RubisCO）负责！这可能是一个巨大的错误，并可能实质上无效的所有后续数据解释的问题，如上面提到的。在少数情况下，已被研究，动力学同位素效应（KIE）forRubisCOs，在何种程度上的δ 13 C固定的碳不同于CO2底物，已被证明有很大的差异。 因此，确定KIE的不同RubisCO的理解，三角洲13 C的解释，并在海洋学三角洲13 C值的建模是至关重要的。因此，这是一个长期的？长期的研究目标是确定KIE从所有形式的RubisCO在自养生物。 为了确定IA型RubisCOs的KIE，这些碳固定酶将从代表性的海洋微型浮游植物和化能自养细菌中分离和纯化。模式种聚球藻WH 7803，原绿球藻MED 4，和两个硫？将使用氧化性化能自养生物、沿海和喷口双壳类的共生体、Solemya velum和Bathymodiolus thermophilus。将对分离的蛋白质的氨基酸序列、组成、Vmax和Km（CO2）进行表征。最后，动力学同位素效应将通过高精度方法确定。确定IA型RubisCOs的KIE将使我们能够评估三角洲13 C分析的基本假设，并推断影响海洋初级生产力的因素，从而大大提高准确性。