Engineering oxidoreductases: maquette proteins designed from scratch

Abstract

The study of natural enzymes is complicated by the fact that only the most recent evolutionary progression can be observed. In particular, natural oxidoreductases stand out as profoundly complex proteins in which the molecular roots of function, structure and biological integration are collectively intertwined and individually obscured. In the present paper, we describe our experimental approach that removes many of these often bewildering complexities to identify in simple terms the necessary and sufficient requirements for oxidoreductase function. Ours is a synthetic biology approach that focuses on from-scratch construction of protein maquettes designed principally to promote or suppress biologically relevant oxidations and reductions. The approach avoids mimicry and divorces the commonly made and almost certainly false ascription of atomistically detailed functionally unique roles to a particular protein primary sequence, to gain a new freedom to explore protein-based enzyme function. Maquette design and construction methods make use of iterative steps, retraceable when necessary, to successfully develop a protein family of sturdy and versatile single-chain three- and four-α-helical structural platforms readily expressible in bacteria. Internally, they prove malleable enough to incorporate in prescribed positions most natural redox cofactors and many more simplified synthetic analogues. External polarity, charge-patterning and chemical linkers direct maquettes to functional assembly in membranes, on nanostructured titania, and to organize on selected planar surfaces and materials. These protein maquettes engage in light harvesting and energy transfer, in photochemical charge separation and electron transfer, in stable dioxygen binding and in simple oxidative chemistry that is the basis of multi-electron oxidative and reductive catalysis.

Elementary tetrahelical protein design for diverse oxidoreductase functions

“…But the translation of modern mechanistic descriptions of natural enzymes into practical engineering guidelines for construction of man-made enzymes remains elusive 2-4 . The complexity of proteins accumulated during repeated blind natural selection 5,6 obscures the identity of what amino acids support any one function and what functional roles are played by any one amino acid. Hence, the common practice of importing mimicked natural protein sequences or structural motifs into man-made constructions does not assure successful import of a selected function.…”

Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.

“…1 The program to integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs is called the maquette approach. 2,3 By beginning with a generic protein sequence designed only to adopt a simple helical bundle fold and iteratively adding engineering elements onto this stripped-down protein chassis, the maquette approach allows the designer to avoid the layers of complexity that hinder the redesign of natural protein scaffolds. Man-made protein maquettes self-assemble in vitro with a wide range of the redox cofactors seen in nature, including hemes, chlorins, metal ions, flavins and quinones.…”

“…Man-made protein maquettes self-assemble in vitro with a wide range of the redox cofactors seen in nature, including hemes, chlorins, metal ions, flavins and quinones. 2 However synthetic biology requires that functional artificial proteins and enzymes interact productively with natural proteins and substrates. They must also fully and functionally assemble in vivo , meaning that cofactor binding sites must be occupied by the target cofactors to carry out the desired function.…”

Design and engineering of artificial oxygen-activating metalloenzymes

“…35 First and secondary shell interactions were accurately introduced in order to accomplish specific functions, i.e. electron transfer, 169-172 oxygen binding, 173 hydroxylase, 174 oxygenase, 175 peroxydase 176-178 activities in de novo designed proteins.…”