Artificial Metalloproteins: Design and Engineering

Abstract

Engineering of artificial metalloproteins is an expanding field with potential impacts in many areas from fundamental understanding of protein structure and function to industrial production of specialty chemicals such as chiral drug intermediates. Incorporation of unnatural amino acids and non‐native metal cofactors into proteins is an emerging field in the area of protein design, as it offers the tantalizing prospect of introducing new functionality and provides exquisite probes for and fine‐tuning of native protein properties. Although it is a relatively young field, engineering of non‐native metalloproteins boasts a myriad of techniques for design, construction, and/or expression of the desired artificial protein. Additionally, many groundbreaking studies exist in this field that have enhanced basic scientific understanding of protein function or generated promising artificial enzymes. Discussion of the techniques prominent for incorporation of unnatural amino acids and non‐native cofactors is followed by some of the most interesting applications of these techniques reported to date.

Redesigning the Blue Copper Azurin into a Redox-Active Mononuclear Nonheme Iron Protein: Preparation and Study of Fe(II)-M121E Azurin

“…Rational design of metalloproteins is an exciting field where our understanding of native proteins can not only be tested and expanded, but also applied toward engineering less expensive and more robust biocatalysts for many applications. − While much progress has been made in designing metalloproteins that structurally mimic native enzymes, the design of proteins with new and desired functions is still largely limited to heme enzymes or proteins containing small inorganic catalysts. ,,− Recent successes in the de novo design of enzymes based on helix bundles bearing diiron , or dirhodium centers , have been reported. In contrast, limited success has been achieved in the design of mononuclear nonheme iron enzymes, , even though they belong to a large and important family of enzymes that are active in oxidative stress defenses, oxidation, and oxygenation reactions. − Such diversity in reaction, combined with the relative abundance of iron and its wide range of accessible oxidation states, has made these enzymes attractive targets for biophysical investigations and synthetic modeling, with the aim of understanding them and developing efficient biomimetic catalysts with earth-abundant metal ions. ,− …”

Azurin as a Protein Scaffold for a Low-coordinate Nonheme Iron Site with a Small-molecule Binding Pocket

“…Nature uses proteins as scaffolds for controlling transition-metal behavior, and chemists have learned to adapt naturally occurring scaffolds for artificial uses. , This research effort has uncovered routes to functional and spectroscopic models of metalloenzymes, using both rational design and directed evolution strategies. − Despite many successes in metalloprotein engineering, artificial complexes containing unsaturated iron sites remain a relatively unexplored area. ,− Preparation of such complexes could further our understanding of nonheme iron enzymes that activate small molecules, − broaden the applications of iron chemistry, and facilitate the use of unsaturated iron complexes in a water-soluble scaffold.…”

Engineering of Enzymes for Selective Catalysis

“…Others like Reetz and Ward have created hybrid enzymes, i.e., metalloenzymes with nonnatural metal centers [88,89], thereby trying to combine the interesting chemistry afforded by organometallic catalysts with the high specificity offered by enzymes. Lu and coworkers have combined the two aforementioned ideas to generate proteins with unnatural amino acids as well as heavy metal centers [90]. Catalytic antibodies are another avenue that has been explored to impart catalytic activity to the innately highly selective antibodies [91].…”