Carbon monoxide forming dioxygenases: Non-copper quercetinases from Streptomyces sp. and Actinoplanes missouriensis

Basic data for this project

Description

Microbial degradation of quercetin is initiated by 2,4-dioxygenolytic cleavage of the O-heterocyclic ring with formation of carbon monoxide. In contrast to Cu2+-quercetinases from Aspergillus spp., the enzymes from Streptomyces sp. and Actinoplanes missouriensis do not contain copper. Our aim is to characterize these bacterial quercetinases, and to compare them with Cu2+-quercetinase, iron-quercetinase detected recently in Bacillus subtilis, and other CO forming dioxygenases (cofactor-less bacterial 1H-3-hydroxy-(2-methyl-)4-oxoquinoline 2,4-dioxygenases; Ni2+-aci-reductone dioxygenase). Whereas the cofactor-less 2,4-dioxygenases are related to the α/b-hydrolase fold enzymes, fungal as well as Bacillus quercetinases appear to belong to the cupin superfamily. Streptomyces and Actinoplanes quercetinases may share a common fold with known CO forming dioxygenases, or they may belong to an unrelated protein family. CO forming dioxygenases may be mechanistically similar, or they may have evolved different ways to catalyze similar reactions. - Steps towards characterizing non-copper quercetinases involve: 1. Identification, cloning and sequencing of the genes coding for the quercetinases of Streptomyces sp. and A. missouriensis. 2. Expression cloning and purification of (tagged) proteins. 3. Biochemical characterization, metal analysis, and EPR spectroscopy. 4. Attempts to crystallize the quercetinases. To develop an understanding of these enzymes is interesting in terms of enzyme evolution, and in terms of fundamental aspects of catalysis by ring-cleaving dioxygenases.