Catalytically functional flavocytochrome chimeras of P450 BM3 and nitric oxide synthase

S Fuziwara, Ikuko Sagami, E Rozhkova, D Craig, Michael A Noble, Andrew W Munro, Stephen K Chapman, Toru Shimizu

    Research output: Contribution to journalArticle

    19 Citations (Scopus)

    Abstract

    P450 BM3 and the nitric oxide synthases are related classes of flavocytochrome mono-oxygenase enzymes, containing NADPH-dependent FAD- and FMN-containing oxidoreductase modules fused to heme b-containing oxygenase domains. Domain-swap hybrids of these two multi-domain enzymes were created by genetic engineering of different segments of reductase and heme domains from neuronal nitric oxide synthase and P450 BM3, as a means of investigating the catalytic competence and substrate-binding properties of the fusions and the influence of tetrahydrpbiopterin and calmodulin binding regions on the electron transfer kinetics of the chimeras. Despite marked differences in hybrid stability and solubility, four catalytically functional chimeras were created that retained good reductase activity and which could be expressed successfully in Escherichia coli and purified. All of the BM3 reductase domain chimeras (chimeras I-III) exhibited inefficient flavin-to-heme inter-domain electron transfer, diminishing their oxygenase activity. However, the chimera containing the neuronal nitric oxide synthase reductase domain (chimera IV) showed good oxygenase domain activity, indicating that the flavin-to-heme electron transfer reaction is relatively efficient in this case. The data reinforce the importance of the nature of inter-domain linker constitution in multi-domain enzymes, and the difficulties posed in attempts to create chimeric enzymes with enhanced catalytic properties. (C) 2002 Elsevier Science Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)515-526
    Number of pages12
    JournalJournal of Inorganic Biochemistry
    Volume91
    Issue number4
    DOIs
    Publication statusPublished - 20 Sep 2002
    Event12th International Conference on Advances in the Inorganic Biochemistry of Cytochrome P450 - La Grande Motte, France
    Duration: 11 Sep 200115 Sep 2001

    Keywords

    • flavocytochromes
    • P-450 BM3
    • chimeras
    • electron transfer
    • nNOS
    • BINDING-SITE
    • CYTOCHROME P450BM-3
    • BACILLUS-MEGATERIUM
    • CRYSTAL-STRUCTURE
    • ELECTRON-TRANSFER
    • REDUCTASE
    • P450BM3
    • FUSION PROTEIN
    • DOMAIN
    • FATTY-ACID SUBSTRATE

    Cite this