Roles of copper ligands in the activation and secretion of Streptomyces tyrosinase.
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 CuA and CuB) 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 MelC1.MelC2 binary complex function, six single substitution mutations were introduced into the CuA and CuB 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 MelC1. MelC2 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.
Institute of Biochemistry, National Yang-Ming University, Taipei 112, Taiwan, Republic of China.
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Lee
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D004789:Enzyme Activation; D008024:Ligands; D046911:Macromolecular Substances; D008543:Melanins / Q000235:genetics; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism*; D009876:Operon; D011487:Protein Conformation; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D008024:Ligands; D046911:Macromolecular Substances; C082891:MelC1 protein, Streptomyces; D008543:Melanins; D018832:Molecular Chaperones; D015534:Trans-Activators; D003300:Copper; D014442:Monophenol Monooxygenase