MUTATIONAL STUDY OF STREPTOMYCES TYROSINASE TRANSACTIVATOR MELC1 - MELC1 IS LIKELY A CHAPERONE FOR APOTYROSINASE

The melanin operon (melC) of Streptomyces antibioticus contains two ge nes, melC1 and melC2 (apotyrosinase). Our previous studies indicated t hat MelC1 forms a transient binary complex with the downstream apotyro sinase MelC2 to facilitate the incorporation of copper ion and the sec retion of tyrosinase. In this study, we investigated the role of histi dine residues in the function of MelC1 by examining a series of substi tution or deletion mutants. Of eight mutants only the substitution of His-117 with Asp in the mutant M-117D rendered the complete abolishmen t of the intracellular tyrosinase activity in both Streptomyces and Es cherichia coli. Replacement of His-102 by Leu in the mutant M-102L als o caused a 64-70% reduction of tyrosinase activity in Streptomyces and E. coli. These two mutations also affected the secretion of both MelC 1 and MelC2 proteins. In vitro copper activation of the purified MelC1 . MelC2 binary complex from these two mutants regained only 20-30% ty rosinase activity of the wild type. Biochemical characterization of th e tyrosinases from these two mutants revealed that they were different in several aspects. The intracellular tyrosinase activity in M-117D, but not in M-102L, could be partially reactivated by copper ion or by the cell extract containing MelC1. The copper content and the specific activity of the tyrosinase purified from the culture supernatant from M-117D were only 40% of those in wild type and M-102L. Additionally, fast protein liquid chromatography analysis indicated that in these tw o mutants the copper activation process was defective, very likely due to the incompetent MelC1 . MelC2 binary complex formed: reduced assoc iation in M-117D and elevated association in M-102L. Furthermore, the conformation of MelC2 in the binary complex or in the mature enzyme fo rm in wild type could be differentiated by the proteinase K digestion pattern, and so did the conformation of MelC2 found in those of M-102L , but not in M-117D mutant. Taken together, our results demonstrate th at MelC1 is indispensable in the incorporation of copper ion into MelC 2 apotyrosinase via a transient, competent binary complex formation, d uring which a conformational transition of MelC2 has occurred. This st rongly suggests that MelC1 is a chaperone for the apotyrosinase MelC2.