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Publication - Dr Ross Anderson

    A suite of de novo c-type cytochromes for functional oxidoreductase engineering


    Watkins, DW, Armstrong, CT, Beesley, J, Marsh, JE, Jenkins, JM, Sessions, RB, Mann, S & Anderson, JLR, 2016, ‘A suite of de novo c-type cytochromes for functional oxidoreductase engineering’. Biochimica et Biophysica Acta (BBA) - Bioenergetics, vol 1857., pp. 493-502


    Central to the design of an efficient de novo enzyme is a
    robust yet mutable protein scaffold. The maquette approach to protein
    design offers precisely this, employing simple four-α-helix bundle
    scaffolds devoid of evolutionary complexity and with proven tolerance
    towards iterative protein engineering. We recently described the design
    of C2, a de novo designed c-type cytochrome maquette that undergoes post-translational modification in E. coli to covalently graft heme onto the protein backbone in vivo. This de novo
    cytochrome is capable of reversible oxygen binding, an obligate step in
    the catalytic cycle of many oxygen-activating oxidoreductases. Here we
    demonstrate the flexibility of both the maquette platform and the
    post-translational machinery of E. coli by creating a suite of functional de novo designed c-type
    cytochromes. We explore the engineering tolerances of the maquette by
    selecting alternative binding sites for heme C attachment and creating
    di-heme maquettes either by appending an additional heme C binding motif
    to the maquette scaffold or by binding heme B through simple
    bis-histidine ligation to a second binding site. The new designs retain
    the essential properties of the parent design but with significant
    improvements in structural stability. Molecular dynamics simulations aid
    the rationalization of these functional improvements while providing
    insight into the rules for engineering heme C binding sites in future
    iterations. This versatile, functional suite of de novo c-type cytochromes shows significant promise in providing robust platforms for the future engineering of de novo
    oxygen-activating oxidoreductases. This article is part of a Special
    Issue entitled Biodesign for Bioenergetics—the design and engineering of
    electron transfer cofactors, proteins and protein networks, edited by
    Ronald Koder and J.L. Ross Anderson

    Full details in the University publications repository