Structure-Function Studies on Flavonol Dioxygenases

cupin超家族是一组功能多样的蛋白质，在所有三个生命王国中都有发现。这些蛋白具有一个特征性的特征结构域，包括两个含组氨酸的基序，由可变长度的基序间区分开。该结构域由保守的b桶结构中的6条β链组成。虽然大多数杯形蛋白只包含一个杯形结构域，但属于这个家族的大量蛋白质是双杯形蛋白，包含两对基序。虽然一些杯形蛋白具有已知的功能，并已在生化水平上进行了表征，但大多数这些蛋白仅从基因克隆或测序项目中已知。cupin超家族的几个成员已被证明使用氧作为底物或辅助因子，包括黄酮醇2,3-双加氧酶。该酶催化黄酮醇的杂环氧化裂解生成一氧化碳和相应的脱苷，在真菌和其他微生物降解黄酮醇中发挥作用。这种不寻常的转化涉及两个C-C键的裂解和CO的释放。迄今为止，两种黄酮醇2,3-双加氧酶已经被表征，尽管越来越多的假设蛋白质来自大量生物体的基因组测序，它们与这些酶具有高度的序列同源性。对枯草芽孢杆菌黄酮醇2,3-双加氧酶进行了结构、光谱和机理研究。该酶已被选为研究cupin超家族成员金属结合选择性的模型系统，以阐明金属辅助因子的变化如何影响催化中心对O2结合和活化的反应性。智力优势：本研究的长期目标是了解金属酶中蛋白质结构、功能和进化之间的相互关系，利用cupin超家族成员作为模型系统。属于该超家族的大多数酶都需要金属离子才能发挥活性，而cupin基序上的保守残基（3His和1Glu）在具有特征的双桶b -螺旋结构基序中提供了最佳的金属结合环境。杯形支架的构象可塑性对底物和金属离子选择的影响目前尚不清楚，黄酮醇2,3双加氧酶是研究金属离子与酶活性关系的一个很好的模型系统。此外，该酶作为bicupin，也可以作为研究该超家族成员基因复制作用的模型。更广泛的影响：在PI的指导下，本研究为研究生和高级本科生的多学科和多元文化研究环境的教育提供了一个极好的机会，PI是一个未被充分代表的群体的成员，积极参与指导本科和研究生水平的少数民族学生。该项目将鼓励本科生在项目期间参与。重点将放在招收代表性不足的少数民族学生，参与NSF支持的在PI所在机构的LSAMP和AGEP项目。此外，通过与一个国际组织的合作，研究生将有机会接受目前在PIs机构没有的光谱技术培训。

The cupin superfamily is a group of functionally diverse proteins found in all three kingdoms of life. These proteins have a characteristic signature domain comprising two histidine-containing motifs separated by an intermotif region of variable length. This domain consists of six beta strands within a conserved B-barrel structure. While most cupins contain a single cupin domain, a significant number of proteins belonging to this family are bicupins, containing two pairs of motifs. Although some cupins have known functions and have been characterized at the biochemical level, the majority of these proteins are known only from gene cloning or sequencing projects. Several members of the cupin superfamily have been shown to use oxygen as a substrate or cofactor, including flavonol 2,3-dioxygenase. This enzyme, which catalyzes the oxidative cleavage of the heterocyclic ring of flavonols to yield carbon monoxide and the corresponding depside, play a role in the degradation of flavonols by fungi and other microorganisms. This unusual transformation involves the cleavage of two C-C bonds with the release of CO. To date, two flavonol 2,3-dioxygenases have been characterized, although an increasing number of hypothetical proteins from a large number of organisms whose genome have been sequenced share high sequence homology to these enzymes. Structural, spectroscopic and mechanistic studies are proposed for the Bacillus subtilis flavonol 2,3-dioxygenase. This enzyme has been chosen as a model system for the study of the metal-binding selectivity in members of the cupin superfamily to elucidate how variations in metal cofactors influence the reactivity of the catalytic center for O2 binding and activation.Intellectual Merit:The long-term goal of this research is the understanding of the interrelation between protein structure, function and evolution in metalloenzymes, utilizing members of the cupin superfamily as model systems. Most enzymes belonging to this superfamily require a metal ion for activity, and the conserved residues at the cupin motif (3His and 1Glu) provide an optimal metal binding environment in the characteristic double barrel B-helix structural motif. The contribution of conformational plasticity of the cupin scaffold to the choice of substrate and metal ion is currently unclear and the enzyme flavonol 2,3 dioxygenase represents an excellent model system for the study of the relationship between metal ion and enzyme activity. In addition, this enzyme, as a bicupin, also serves as a model for the study of the role of gene duplication in members of this superfamily.Broader Impacts: This research provides an excellent opportunity for the education of graduate and advanced undergraduate students in a multidisciplinary and multicultural research environment under the guidance of the PI, a member of an underrepresented group actively involved in the mentoring of minority students at the undergraduate and graduate levels. The PI will encourage the participation of undergraduate students for the duration of this project. Emphasis will be given to the recruitment of underrepresented minority students with the involvement of NSF supported LSAMP and AGEP programs base at the PI's institution. In addition, graduate students will be provided the opportunity for training in spectroscopic techniques not currently available at the PIs institution through collaboration with an international group.