Copper transfer and activation of the Streptomyces apotyrosinase are mediated through a complex formation between apotyrosinase and its trans-activator MelC1.
The melanin operon (melC) of Streptomyces antibioticus is composed of two genes that encode MelC1 and MelC2 proteins. MelC1 has been suggested as a trans-activator which can facilitate the incorporation of copper into the apotyrosinase (MelC2) (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81). However, the molecular mechanism of the trans-activation or copper-transfer process mediated through MelC1 to MelC2 is not clear yet. In this study, we found apotyrosinase in both the extracellular fraction and cell extract from cells grown in copper-deficient medium. Using gel-filtration and immunoaffinity chromatographies, we demonstrated that apotyrosinase (MelC2) formed a stable complex with MelC1 in the intra- and extracellular fractions. Furthermore, addition of copper ion to the complex generated tyrosinase activity. The MelC1-MelC2 complex was purified to near homogeneity by DE52 and phenyl-agarose chromatographies. In conjunction with fast protein liquid gel filtration chromatography and NH2-terminal sequencing analysis, the results indicated that the stoichiometric ratio of MelC1 and MelC2 in the purified complex was 1:1. Essentially no copper was found in the complex. Addition of copper ion to the purified complex resulted in incorporation of approximately 2 molecules of copper ion and the mature active tyrosinase was gradually released from the complex. Taken together, these results demonstrate that the molecular mechanism of activation of Streptomyces apotyrosinase by its trans-activator MelC1 is initially mediated via a binary complex formation between these two proteins, followed by incorporation of copper ion. This activation mechanism accounts for the essential role of MelC1 in the expression of melanin operon.
Chen
L Y
LY
Institute of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
Leu
W M
WM
Wang
K T
KT
Lee
Y H
YH
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000378:metabolism*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D003300:Copper / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008543:Melanins / Q000235:genetics*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*; D015534:Trans-Activators / Q000378:metabolism*
D001051:Apoenzymes; D008543:Melanins; D015534:Trans-Activators; D003300:Copper; D014442:Monophenol Monooxygenase