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.
