Roles of Copper Ligands in the Activation and Secretion ofStreptomyces Tyrosinase

Abstract

The expression of the melanin operon (melC) of Streptomyces antibioticus requires the chaperone-like protein MelC1 for the incorporation of two copper ions (designated as Cu A and Cu B ) and the secretion of the apotyrosinase (MelC2) via a transient binary complex formation between these two proteins. To investigate whether the copper ligand of tyrosinase is involved in this MelC1⅐MelC2 binary complex function, six single substitution mutations were introduced into the Cu A and Cu B sites. These mutations led to differential effects on the stability, copper content, and export function of binary complexes but a complete abolishment of tyrosinase activity. The defects in the tyrosinase activity in mutants were not because of the impairment of the formation of MelC1⅐MelC2 complex but rather the failure of MelC2 to be discharged from the copper-activated binary complex. Moreover, the impairments on the discharge of the mutant MelC2 from all the mutant binary complexes appeared to result from the structural changes in their apoforms or copper-activated forms of the complexes, as evidenced by the fluorescence emission and circular dichroism spectral analysis. Therefore, each of six copper ligands in Streptomyces tyrosinase binuclear copper sites plays a pivotal role in the final maturation and the discharge of tyrosinase from the binary complex but has a less significant role in its secretion.

The Tat Substrate CueO Is Transported in an Incomplete Folding State

“…In Streptomyces species, this type 3 copper-containing protein is transported by the aid of a Tat-signal peptidecontaining chaperone, and there is evidence that copper assembly induces the release of the active enzyme from its chaperone (44). Although not concluded at that time, these data clearly agree with the view that copper is incorporated after transport also in streptomycetes and, therefore, possibly in most or all bacteria.…”

The pro-enzyme C-terminal processing domain of Pholiota nameko tyrosinase is responsible for folding of the N-terminal catalytic domain

“…Although streptomyces tyrosinases do not usually carry a C-terminal domain, their production is assisted by co-expression of the caddie protein MelC1, which is essential for appropriate expression of tyrosinase and forms an inactive complex with it (Chen et al 1993;Liaw and Lee 1995;Claus and Decker 2006). Release of the caddie protein from the complex activates this tyrosinase (Tsai and Wu 1998). The three-dimensional structure of Streptomyces castaneoglobisporus tyrosinase in complex with its caddie protein ORF378 has also been reported (Matoba et al 2006).…”

Involvement of a novel copper chaperone in tyrosinase activity and melanin synthesis in Marinomonas mediterranea

“…Specific interactions between copper proteins and their copper chaperones have been described in both prokaryotes and eukaryotes. One example is the tyrosinase of Streptomyces, whereby the tyrosinase MelC2 interacts with its copper chaperone MelC1 (Tsai & Lee, 1998). Another system recently described in Streptomyces is the tyrosinase homologue copper enzyme GriE with o-aminophenol oxidase activity, which is encoded in Streptomyces griseus on the same operon as its copper chaperone GriF, which is very similar to MelC1 (Suzuki et al, 2006).…”

“…MelC1 is not a membrane protein, but a small soluble protein that forms a binary complex with the tyrosinase. The formation of this complex is necessary for both copper delivery (Chen et al, 1992) and secretion of tyrosinase (Tsai & Lee, 1998). The proteins are exported by using the twin-arginine translocation system and a signal peptide present in MelC1 (Schaerlaekens et al, 2001).…”