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

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。过去气候的预测、限制当今温室气体排放命运的模型、复杂生态系统中营养转移的推断，以及对初级生产力的酶和细胞控制的阐明，都使用了稳定的碳同位素分馏数据（δ 13C值）。尽管这种分馏信息具有广泛的适用性，但这些数据几乎总是假设碳是固定的，其同位素被分馏，就像核酮糖- 1,5 -二磷酸一样。菠菜的羧化酶/加氧酶（RubisCO）是罪魁祸首！这可能是一个巨大的错误，可能会使所有后续的数据解释无效，例如上面提到的问题。在已研究的少数情况下，rubiscos的动力学同位素效应（KIEs），即固定碳的δ 13C与所使用的co2底物不同的程度，已被证明差异很大。因此，确定不同RubisCOs的KIEs对于海洋学中三角洲13C值的理解、解释和建模至关重要。因此，它是一个长？从自养细胞中各种形式的RubisCO中确定KIEs的长期研究目标。为了确定IA型RubisCOs的KIEs，将从具有代表性的海洋浮游植物和趋化自养细菌中分离纯化这些固定碳酶。模式种聚球藻WH7803、原绿球藻med4和两个硫？利用氧化化自养生物、沿海和深海双壳类的共生生物、软蚌和嗜热深海生物。分离得到的蛋白质将根据氨基酸序列、组成、Vmax和Km （CO2）进行表征。最后，用高精度方法测定动力学同位素效应。确定IA型rubisco的KIEs将使我们能够评估δ 13C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。

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.