The ascent of man(made oxidoreductases)

Abstract

Though established 40 years ago, the field of de novo protein design has recently come of age, with new designs exhibiting an unprecedented level of sophistication in structure and function. With respect to catalysis, de novo enzymes promise to revolutionise the industrial production of useful chemicals and materials, while providing new biomolecules as plug-and-play components in the metabolic pathways of living cells. To this end, there are now de novo metalloenzymes that are assembled in vivo, including the recently reported C45 maquette, which can catalyse a variety of substrate oxidations with efficiencies rivalling those of closely related natural enzymes. Here we explore the successful design of this de novo enzyme, which was designed to minimise the undesirable complexity of natural proteins using a minimalistic bottom-up approach.

Figure 1

The evolution of C45 from humble beginnings. (a) Evolutionary diagram displaying the broad design strokes and functions of the maquettes from a simple featureless 4-helix bundle (1) [35], to the oxygen binding HP7 maquette (2) [35], to the functional de novo enzyme C45 (3) [22^••]. (b) Sequence alignment of three maquettes from the evolutionary diagram of C45. Heme-ligating histidines are highlighted in blue, CXXC from the c-type cytochrome consensus motif (CXXCH) is highlighted in red, and the interhelical loops are displayed in purple.

Figure 2

Surface tryptophan residues in both C45 (a) and lignin peroxidase (PDB: 1B82) (b) which potentially participate in long-range electron transfer from a surface-bound substrate to the protein-bound heme. Numbers in parentheses represent the edge-to-edge distances between the tryptophan side chains and the conjugated porphyrin system of the bound hemes.

Figure 3

Computational analysis of potential C45-substrate binding sites. While 2,4,6-trichlorophenol (TCP) (a) appears to preferentially bind in one position on C45, the larger ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) molecule (b) appears to bind indiscriminately across the surface. The data presented is derived from docking analysis performed by the Bristol University Docking Engine (BUDE) [22^••] in conjunction with molecular dynamics simulations. Overlays are shown here for representative low energy binding poses for TCP and ABTS.