Purification, crystallization and catalytic activity of the ETHE1 and DUF442 domains of the BLH protein from Xylella fastidiosa and Agrobacterium tumefaciens

Abstract

Xylella fastidiosa and Agrobacterium tumefaciens are plant pathogenic bacteria with a wide host range. X. fastidiosa colonizes the vascular tissue occluding the xylem vessels, causing the development of diseases such as Citrus Variegated Chlorosis (CVC). A. tumefaciens is a root-associated bacterium that also colonizes the vascular tissues, and it induces a kind of cell hyperplasia known as the Crown Gall disease. We have shown previously that both Xylella and Agrobacterium cells have an operon denominated "bigR" which is required for the detoxification of hydrogen sulfide, a toxic compound that inhibits cytochrome C oxidase, thus blocking aerobic respiration. We found that the bigR operon catalyses the formation and secretion of sulfite from hydrogen sulfide, allowing the cells to grow in oxygen-limited environments. Although much has been learned about the transcriprional regulation of the bigR operon, little is known of the function of BLH (beta-lactamase-like hydrolase), an enzyme thought to convert hydrogen sulfide into sulfite. BLH has two functional domains, DUF442, thought to display a rhodanese activity, and ETHE1, with a sulfur dioxygenase activity. Although a few crystall structures related to the DUF442 and ETHE1 domains have been described, how hydrogen sulfide is converted to sulfite is not known. In addition, how the activity of these two domains is connected remains a mystery. The aim of this project is to express, purify and crystallize the ETHE1 and DUFF442 domains from BLH proteins from X. fastidiosa and A. tumefaciens for structural and biochemical analysis. We expect to find enzymatic activities for the DUFF442 and ETHE1 domains associated with rhodanese and sulfur dioxygenase, respectively.