4 publications

4 publications

Design Strategies for Redox Active Metalloenzymes: Applications in Hydrogen Production

Review

Ghirlanda, G.

Methods Enzymol. 2016, 389-416, 10.1016/bs.mie.2016.06.001

The last decades have seen an increased interest in finding alternative means to produce renewable fuels in order to satisfy the growing energy demands and to minimize environmental impact. Nature can serve as an inspiration for development of these methodologies, as enzymes are able to carry out a wide variety of redox processes at high efficiency, employing a wide array of earth-abundant transition metals to do so. While it is well recognized that the protein environment plays an important role in tuning the properties of the different metal centers, the structure/function relationships between amino acids and catalytic centers are not well resolved. One specific approach to study the role of proteins in both electron and proton transfer is the biomimetic design of redox active peptides, binding organometallic clusters in well-understood protein environments. Here we discuss different strategies for the design of peptides incorporating redox active FeS clusters, [FeFe]-hydrogenase organometallic mimics, and porphyrin centers into different peptide and protein environments in order to understand natural redox enzymes.


Notes: Book chapter

Metal-Directed Design of Supramolecular Protein Assemblies

Review

Tezcan, F.A.

Methods Enzymol. 2016, 10.1016/bs.mie.2016.05.009

Owing to their central roles in cellular signaling, construction, and biochemistry, protein–protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive noncovalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, metal-directed protein self-assembly (MDPSA) and metal-templated interface redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution.


Notes: ---

Periplasmic Screening for Artificial Metalloenzymes

Review

Ward, T.R.

Methods Enzymol. 2016, 539-556, 10.1016/bs.mie.2016.05.037

Artificial metalloenzymes represent an attractive means of combining state-of-the-art transition metal catalysis with the benefits of natural enzymes. Despite the tremendous recent progress in this field, current efforts toward the directed evolution of these hybrid biocatalysts mainly rely on the laborious, individual purification of protein variants rendering the throughput, and hence the outcome of these campaigns feeble. We have recently developed a screening platform for the directed evolution of artificial metalloenzymes based on the streptavidin–biotin technology in the periplasm of the Gram-negative bacterium Escherichia coli. This periplasmic compartmentalization strategy comprises a number of compelling advantages, in particular with respect to artificial metalloenzymes, which lead to a drastic increase in the throughput of screening campaigns and additionally are of unique value for future in vivo applications. Therefore, we highlight here the benefits of this strategy and intend to propose a generalized guideline for the development of novel transition metal-based biocatalysts by directed evolution in order to extend the natural enzymatic repertoire.


Notes: Book chapter

Reconstitution of Heme Enzymes with Artificial Metalloporphyrinoids

Review

Hayashi, T

Methods Enzymol. 2016, 439-454, 10.1016/bs.mie.2016.05.049

An important strategy used in engineering of hemoproteins to generate artificial enzymes involves replacement of heme with an artificial cofactor after removal of the native heme cofactor under acidic conditions. Replacement of heme in an enzyme with a nonnatural metalloporphyrinoid can significantly alter the reactivity of the enzyme. This chapter describes the design and synthesis of three types of artificial metalloporphyrinoid cofactors consisting of mono-, di-, and tri-anionic ligands (tetradehydrocorrin, porphycene, and corrole, respectively). In addition, practical procedures for the preparation of apo-hemoproteins, incorporation of artificial cofactors, and characterization techniques are presented. Furthermore, the representative catalytic activities of artificial enzymes generated by reconstitution of hemoproteins are summarized.


Notes: Book chapter