ROLES OF COPPER LIGANDS IN THE ACTIVATION AND SECRETION OF STREPTOMYCES TYROSINASE

The expression of the melanin operon (melC) of Streptomyces antibiotic us 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 betwee n these two proteins. To: investigate whether the copper ligand of tyr osinase is involved in this MelC1 MelC2 binary complex function, six s ingle substitution mutations were introduced into the Cu-A and CUB sit es. These mutations led to differential effects on the stability, copp er content, and export function of binary complexes but a complete abo lishment of tyrosinase activity. The defects in the tyrosinase activit y in mutants were not because of the impairment of the formation of Me lC1 MelC2 complex but rather the failure of MelC2 to be discharged fro m the copper-activated binary complex. Moreover, the impairments on th e discharge of the mutant MelC2 from all the mutant binary complexes a ppeared to result from the structural changes in their apoforms or cop per-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 tyro sinase from the binary complex but has a less significant role in its secretion.