Structures of Ferredoxin Reductase and Old Yellow Enzyme

Karplus博士建议研究两种黄酶的结构和结构-功能关系：铁氧还蛋白：NADP+还原酶（FNR），这是一种依赖于fad的酶，在光合作用过程中产生NADPH；老黄酶（OYE），一种依赖于FMN的NADPH氧化酶，生理功能未知。在这些研究中使用的主要方法是单晶x射线衍射结合蛋白质化学进行结构测定，纯化单个酶，制备相关的晶体和配合物，以及基于结构检验假设。研究这两种酶的时机已经成熟，因为它们在去年已经被克隆和表达，因此定点诱变研究可以用来探测晶体学所揭示的结构的功能方面。总的目标是阐明参与这两种酶催化作用的具体结构因素。由于FNR是唯一结构已知的NAD(P)依赖性脱氢酶-电子转移酶，并且OYE具有与其他特征黄酶相比独特的黄素环境，因此该结果将对黄蛋白催化的一般特征提供重要的见解。

Dr. Karplus proposes to investigate the structure and structure- function relationships of two flavoenzymes: ferredoxin:NADP+ reductase (FNR), which is an FAD-dependent enzyme which produces NADPH during photosynthesis, and Old Yellow Enzyme (OYE), an FMN- dependent NADPH oxidase of unknown physiological function. The main methods to be used in these studies are single crystal x- ray diffraction for structure determination combined with protein chemistry, for purifying the individual enzymes, preparing relevant crystals and complexes, and testing hypotheses based on the structures. The time is ripe for the study of both of these enzymes as they have been cloned and expressed within the last year, so that site-directed mutagenesis studies can be used to probe functional aspects of the structure as revealed by crystallography. %%% The overall goal is to elucidate the specific structural factors involved in catalysis for both of these enzymes. The results will give significant insight into general features of flavoprotein catalysis because FNR is the only structurally known NAD(P) dependent dehydrogenase-electron transferase and OYE has a flavin environment which is unique with respect to other characterized flavoenzymes.