期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 109, 期 27, 页码 10793-10797出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1208718109
关键词
amine N-oxygenation; copper enzymes; oxygen activation; resonance Raman spectroscopy; molecular mechanism
资金
- National Institutes of Health [DK31450, GM085914]
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [A2]
- Bureau of Science, Technology, and Innovation Policy, Cabinet Office, Government of Japan [GS006]
The terminal step of 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis is performed by NspF, a mono-oxygenase that converts o-aminophenols to the corresponding nitroso product (hydroxyanilinase activity). Previous biochemical characterization of the resting form of NspF suggested that this enzyme belonged to the coupled binuclear copper enzyme (CBC) family. Another member of this enzyme family, tyrosinase, is able to mono-oxygenate monophenols (monophenolase activity) but not o-aminophenols. To gain insight into the unique reactivity of NspF, we have generated and characterized the oxy form of its active site. The observation of spectral features identical to those of oxytyrosinase indicates that oxy-NspF contains a Cu2O2 core where peroxide is coordinated in a mu-eta(2):eta(2) mode, confirming that NspF is a CBC enzyme. This oxy form is found to react with monophenols, indicating that, like tyrosinase, NspF also possesses monophenolase activity. A comparison of the two electrophilic mechanisms for the monophenolase and hydroxyanilinase activity indicates a large geometric change between their respective transition states. The potential for specific interactions between the protein pocket and the substrate in each transition state is discussed within the context of the differential reactivity of this family of enzymes with equivalent mu-eta(2):eta(2) peroxy bridged coupled binuclear copper active sites.
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