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

Autor:	Watkins DW; School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK., Armstrong CT; School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK., Beesley JL; School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK; School of Chemistry, University of Bristol, Bristol BS8 1TS, UK., Marsh JE; School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK., Jenkins JMX; School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK., Sessions RB; School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK., Mann S; School of Chemistry, University of Bristol, Bristol BS8 1TS, UK., Ross Anderson JL; School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK. Electronic address: ross.anderson@bristol.ac.uk.
Jazyk:	angličtina
Zdroj:	Biochimica et biophysica acta [Biochim Biophys Acta] 2016 May; Vol. 1857 (5), pp. 493-502. Date of Electronic Publication: 2015 Nov 10.
DOI:	10.1016/j.bbabio.2015.11.003
Abstrakt:	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 L. Koder and J.L. Ross Anderson. (Copyright © 2015 Elsevier B.V. All rights reserved.)
Databáze:	MEDLINE
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