Engineering oxidoreductases : maquette proteins designed from scratch Bruce

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 inwhich 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 threeand 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. Introduction All too commonly, it is inferred that a specific biochemical function is necessarily linked with either a singular enzyme structure or a particular primary sequence. This ignores the apparent ‘memory-less’ random Markovian process of evolution that occurs down to the codon level [1]. An unknown and probably large variety of evolutionary pressures have contributed to the selection of the observed primary sequence and molecular structure, obfuscating and overlapping the multiplicity of biological roles that each amino acid plays [2,3]. These facts impair broad scientific goals directed at understanding and exploiting the fundamental principles and requirements for effective biological activity of natural enzymes. In some tantalizing cases, functionally identical enzymes from a variety of biological sources preserve ‘active-site’ residues; however, these residues may only afford the observed catalytic activity within their particular structural contexts. This makes it