6 publications

6 publications

An Artificial Di-Iron Oxo-Orotein with Phenol Oxidase Activity

DeGrado, W.F.; Lombardi, A.

Nat. Chem. Biol. 2009, 5, 882-884, 10.1038/nchembio.257

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Alcohol oxidation
Max TON: >50
ee: ---
PDB: 2KIK
Notes: kcat/KM ≈ 1380 M-1*min-1

Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Amine oxidation
Max TON: ---
ee: ---
PDB: 2KIK
Notes: kcat/KM ≈ 83 M-1*min-1

Artificial Diiron Enzymes with a De Novo Designed Four-Helix Bundle Structure

Review

DeGrado, W.F.; Lombardi, A.

Eur. J. Inorg. Chem. 2015, 2015, 3371-3390, 10.1002/ejic.201500470

A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal‐binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four‐helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.


Notes: ---

Artificial Heme Enzymes for the Construction of Gold-Based Biomaterials

Lombardi, A.; Nastri, F.

Int. J. Mol. Sci. 2018, 19, 2896, 10.3390/ijms19102896

Many efforts are continuously devoted to the construction of hybrid biomaterials for specific applications, by immobilizing enzymes on different types of surfaces and/or nanomaterials. In addition, advances in computational, molecular and structural biology have led to a variety of strategies for designing and engineering artificial enzymes with defined catalytic properties. Here, we report the conjugation of an artificial heme enzyme (MIMO) with lipoic acid (LA) as a building block for the development of gold-based biomaterials. We show that the artificial MIMO@LA can be successfully conjugated to gold nanoparticles or immobilized onto gold electrode surfaces, displaying quasi-reversible redox properties and peroxidase activity. The results of this work open interesting perspectives toward the development of new totally-synthetic catalytic biomaterials for application in biotechnology and biomedicine, expanding the range of the biomolecular component aside from traditional native enzymes.


Metal: Fe
Ligand type: Amino acid; Porphyrin
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Immobilization of the ArM on gold surfaces via a lipoic acid anchor.

Design and Engineering of Artificial Oxygen-Activating Metalloenzymes

Review

Lombardi, A.; Lu, Y.

Chem. Soc. Rev. 2016, 45, 5020-5054, 10.1039/C5CS00923E

Many efforts are being made in the design and engineering of metalloenzymes with catalytic properties fulfilling the needs of practical applications. Progress in this field has recently been accelerated by advances in computational, molecular and structural biology. This review article focuses on the recent examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design, miniaturization processes and protein redesign. Considerable progress in these diverse design approaches has produced many metal-containing biocatalysts able to adopt the functions of native enzymes or even novel functions beyond those found in Nature.


Notes: ---

Diiron-Containing Metalloproteins: Developing Functional Models

Review

DeGrado, W.F.; Lombardi, A.

C. R. Chim. 2007, 10, 703-720, 10.1016/j.crci.2007.03.010

A major objective in protein science is the design of enzymes with novel catalytic activities that are tailored to specific applications. Such enzymes may have great potential in biocatalysis and biosensor technology, such as in degradation of pollutants and biomass, and in drug and food processing. To reach this objective, investigations into the basic biochemical functioning of metalloproteins are still required. In this perspective, metalloprotein design provides a powerful approach first to contribute to a more comprehensive understanding of the way metalloproteins function in biology, with the ultimate goal of developing novel biocatalysts and sensing devices. Metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. We have approached the challenge of reproducing metalloprotein active sites by using a miniaturization process. We centered our attention on iron-containing proteins, and we developed models for heme proteins and diiron–oxo proteins. In this paper we summarize the results we obtained on the design, structural, and functional properties of DFs, a family of artificial diiron proteins.


Notes: ---

Oxidation Catalysis by Iron and Manganese Porphyrins within Enzyme-Like Cages

Review

Lombardi, A.; Maglio, O.; Nastri, F.

Biopolymers 2018, 109, e23107, 10.1002/bip.23107

Inspired by natural heme‐proteins, scientists have attempted for decades to design efficient and selective metalloporphyrin‐based oxidation catalysts. Starting from the pioneering work on small molecule mimics in the late 1970s, we have assisted to a tremendous progress in designing cages of different nature and complexity, able to accommodate metalloporphyrins. With the intent of tuning and controlling their reactivity, more and more sophisticated and diverse environments are continuously exploited. In this review, we will survey the current state of art in oxidation catalysis using iron‐ and manganese‐porphyrins housed within designed or engineered protein cages. We will also examine the innovative metal‐organic framework (MOF) systems, exploited to achieving an enzyme‐like environment around the metalloporphyrin cofactor.


Notes: ---