Bacteria establish copper homeostasis chiefly by exporting excess copper and by sequestering cytoplasmic copper with copper chaperones for safe delivery to copper exporters and copper-requiring proteins (Solioz et al., 2010). The genes involved in copper homeostasis are regulated by copper-responsive transcriptional regulators. Lactococcus lactis has been used recently as a model organism for the study of bacterial copper homeostasis. It was found that a set Mitomycin C purchase of widely diverse genes are under the control of the CopR copper-responsive repressor (Magnani et al., 2008). This so-called CopR regulon encompasses 14 genes: two monocistronic genes (lctO, copB) and four operons (ydiDE,

yahCD-yaiAB, ytjDBA and copRZA). Some of the proteins encoded by these genes, such as the CopR repressor, the CopZ copper chaperone and the two copper ATPases, CopA and CopB, play an evident role in copper homeostasis by dealing directly with copper ions (Solioz & Vulpe, 1996; Solioz & Stoyanov,

2003; Solioz et al., 2011). Two more proteins of the CopR regulon have been studied in detail: LctO is a lactate oxidase that converts lactate to pyruvate under the use of molecular oxygen, presumably to reduce oxygen tension and thus oxygen-associated stress (Barréet al., 2007). learn more CinD, on the other hand, is a nitroreductase (encoded by ytjD) that can detoxify nitro compounds that exacerbate copper stress (Mermod et al., 2010). In the present study, we investigated the function of another member gene of the CopR regulon, yahD. By sequence comparison, this gene is predicted to encode an α/β serine hydrolase of 206 amino acids. The α/β-hydrolase fold is one of the most versatile and widespread folds known (Nardini & Dijkstra, 1999). Even though all the members of this superfamily have a similar fold and a conserved catalytic triad, they exhibit wide substrate specificity. Serine hydrolases use a nucleophilic serine to hydrolyze amidic, ester and thioester bonds in small molecules or proteins (Simon & Cravatt, 2010). YahD was found to be induced Mirabegron by copper, cadmium and silver, but not by other metals or by oxidative or

nitrosative stress-inducing chemicals. The three-dimensional structure of YahD was resolved by X-ray crystallography to a resolution of 1.88 Å and was found to exhibit an α/β-hydrolase fold with the characteristic Ser-His-Asp catalytic triad. YahD did not catalyze any of the known α/β serine hydrolase reactions and appears to represent a novel subclass of serine hydrolases. Lactococcus lactis IL1403 was grown semi-anaerobically (air-saturated media in sealed bottles), in M17 media (Terzaghi & Sandine, 1975) at 30 °C or on plates containing M17 media with 1.5% agar (AppliChem, Darmstadt, Germany). Escherichia coli DH5α (Stratagene, La Jolla, CA) and ER2566 (Invitrogen life Technologies) cells used for cloning were transformed according to the manufacturer’s instructions.