Structure-Function Studies on Flavonol Dioxygenases

cupin超家族是一组功能多样的蛋白质，存在于生命的所有三个王国中。这些蛋白质具有特征性的签名结构域，其包含由可变长度的基序间区域分隔的两个含组氨酸的基序。该结构域由保守的B桶结构内的六条β链组成。虽然大多数cupin含有一个单一的cupin结构域，但属于这个家族的大量蛋白质是bicupins，含有两对基序。虽然一些cupin具有已知的功能，并已在生物化学水平上进行了表征，但这些蛋白质中的大多数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因子，包括黄酮醇2，3-双加氧酶。这种酶催化黄酮醇的杂环氧化裂解产生一氧化碳和相应的缩酚酸，在真菌和其他微生物降解黄酮醇中发挥作用。这种不寻常的转变涉及两个C-C键的裂解与CO的释放。迄今为止，两个黄酮醇2，3-双加氧酶已被表征，虽然越来越多的假设蛋白质从大量的生物体的基因组已被测序共享这些酶的高序列同源性。对枯草杆菌黄酮醇2，3-双加氧酶进行了结构、光谱和机理研究。这种酶已被选为模型系统的cupin超家族成员的金属结合选择性的研究，以阐明金属辅因子的变化如何影响O2结合和activation.Intellectual优点的催化中心的反应性：这项研究的长期目标是蛋白质的结构，功能和进化之间的相互关系的理解在金属酶，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需要金属离子来进行活性，并且cupin基序（3 His和1Glu）处的保守残基在特征性双桶B-螺旋结构基序中提供了最佳的金属结合环境。cupin支架的构象可塑性对底物和金属离子的选择的贡献目前尚不清楚，并且酶黄酮醇2，3双加氧酶代表了用于研究金属离子与酶活性之间的关系的优良模型系统。此外，这种酶作为一种bicupin，也是研究该超家族成员中基因复制作用的模型。更广泛的影响：这项研究为研究生和高级本科生的教育提供了一个极好的机会，在PI的指导下，在多学科和多元文化的研究环境中，一个代表性不足的群体的成员，积极参与指导本科和研究生阶段的少数民族学生。PI将鼓励本科生参与本项目的持续时间。重点将放在招聘代表性不足的少数民族学生与NSF支持的LSAMP和AGEP方案在PI的机构基地的参与。此外，研究生将通过与一个国际小组的合作，获得目前在PI机构无法获得的光谱技术培训机会。

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.