Bringing Artificial Enzymes Closer to Nature

The artificial metalloenzyme, termed biot-Ru–SAV, is based on the biotin-streptavidin technology that is extensively used to generate metalloenzymes by combining organometallic compounds, which are fused to biotin, with streptavidin, a protein with high affinity for biotin. Organometallic compounds are molecules containing at least one bond between a metal and a carbon atom, and are often used as catalysts in industrial chemical reactions. However, organometallic catalysts perform poorly, if at all, in aqueous solutions or cellular-like environments, and need to be incorporated into protein scaffolds like streptavidin to overcome these limitations.

"The goal was to create an artificial metalloenzyme that can catalyse olefin metathesis, a reaction mechanism that is not present among natural enzymes," says Thomas Ward, Professor at the Department of Chemistry, University of Basel, and one of the two senior authors of the study. The olefin metathesis reaction is a method for the formation and redistribution of carbon-carbon double bonds widely used in laboratory research and large-scale industrial productions of various chemical products. Biot-Ru–SAV catalyses a ring-closing metathesis to produce a fluorescent molecule for easy detection and quantification.

However, the environment inside a living cell is far from ideal for the proper functioning of organometallic-based enzymes. "The main breakthrough was the idea to use the periplasm of Escherichia coli as a reaction compartment, whose environment is much better suited for an olefin metathesis catalyst," says Markus Jeschek from the ETH Zurich Department of Biosystems Science and Engineering team. The periplasm, the space between the inner cytoplasmic membrane and the bacterial outer membrane in Gram-negative bacteria, contains low concentrations of metalloenzymes inhibitors, such as glutathione.

Having found ideal in vivo conditions, the authors went a step forward and decided to optimise biot-Ru–SAV by applying principles of directed evolution, a method that mimics the process of natural selection to evolve proteins with enhanced properties or activities. They developed a simple and robust screening method that allowed to test thousands of biot-Ru–SAV mutants and identify the most active variant. Not only could the authors markedly improve the catalytic properties of biot-Ru–SAV, but they could also show that organometallic-based enzymes can be engineered and optimised for different substrates, thus producing a variety of different chemical products.

Ultimately, artificial metalloenzymes such as biot-Ru–SAV can be used to produce novel biomolecules and to envision completely novel, artificial biochemistries in cells – which might then be rightly called "synthetic".