Mutational study of Streptomyces tyrosinase trans-activator MelC1. MelC1 is likely a chaperone for apotyrosinase

L. Y. Chen, M. Y. Chen, W. M. Leu, T. Y. Tsai, Yan-Hwa Wu Lee*

*Corresponding author for this work

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

The melanin operon (melC) of Streptomyces antibioticus contains two genes, melC1 and melC2 (apotyrosinase). Our previous studies indicated that MelC1 forms a transient binary complex with the downstream apotyrosinase MelC2 to facilitate the incorporation of copper ion and the secretion of tyrosinase. In this study, we investigated the role of histidine residues in the function of MelC1 by examining a series of substitution or deletion mutants. Of eight mutants only the substitution of His-117 with Asp in the mutant M-117D rendered the complete abolishment of the intracellular tyrosinase activity in both Streptomyces and Escherichia coli. Replacement of His-102 by Leu in the mutant M-102L also caused a 64-70% reduction of tyrosinase activity in Streptomyces and E. coli. These two mutations also affected the secretion of both MelC1 and MelC2 proteins. In vitro copper activation of the purified MelC1·MelC2 binary complex from these two mutants regained only 20-30% tyrosinase activity of the wild type. Biochemical characterization of the 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 two mutants the copper activation process was defective, very likely due to the incompetent MelC1·MelC2 binary complex formed: reduced association in M-117D and elevated association in M-102L. Furthermore, the conformation of MelC2 in the binary complex or in the mature enzyme form 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 that MelC1 is indispensable in the incorporation of copper ion into MelC2 apotyrosinase via a transient, competent binary complex formation, during which a conformational transition of MelC2 has occurred. This strongly suggests that MelC1 is a chaperone for the apotyrosinase MelC2.

Original languageEnglish
Pages (from-to)18710-18716
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number25
StatePublished - 1 Jan 1993

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