Exploiting cofactor-dependent oxidoreductases by enzyme discovery, engineering and reconstruction

Abstract

Enzymes, also known as biocatalysts, are proteins which catalyze chemical reactions. Enzymes typically use functional groups from the side chains of their amino acid residues to perform catalysis. However, the functional groups derived from the 20 canonical amino acids cannot support the diversity of catalysis required by nature.Therefore, some enzymes utilize non-proteinaceous molecules and/or metal ions to broaden their catalytic scope. These protein ligands are called cofactors. Some of these cofactors have been suggested to originate as remnants of ancient ribozymes, whose active core was retained during evolution. Among all enzymes, oxidoreductases account for a considerable proportion (about one third) and most of them require dedicated redox cofactors. They participate in many crucial steps in metabolism and cover a huge range of reaction types. Except for a crucial role in nature, research in the last decades has revealed that these enzymes have enormous potential in industrial application for the production of many high-valued products.General introduction to cofactors and cofactor-dependent redox enzymes 9 Ⅰ. CofactorsCofactors: definitions Cofactors are non-protein molecules or metal ions that are required for the proper functioning of certain enzymes. In general, cofactors are classified into two main groups: inorganic and organic cofactors. Sometimes cofactors are also referred to as coenzymes or prosthetic groups. Because of the ambiguity in terminology 1 in literature, here in this thesis, we adopt the definition described by Lehninger (Principles of Biochemistry, 8 th edition) 2 . Therefore, cofactors are defined as either inorganic ions, or organic/metalloorganic molecules that are required for enzyme activity; coenzymes contain all cofactors excluding the inorganic ones; and prosthetic groups are either metal ions or organic non-protein molecules that tightly bound to a protein and essential to its activity. Inorganic cofactors include metal ions such as iron and copper. These ions often act as electron carriers or participate in redox reactions during enzymatic reactions 3 . Organic cofactors, or coenzymes, are more complex organic molecules that can be further divided into two groups: vitamin derived or not. For example, some of the best-known cofactors, pyridoxal phosphate, coenzyme A, flavins and nicotinamides, are derivatives of vitamin B 5 , B 6 , B 2 and B 3 respectively. Organic cofactors often act as carriers of electrons or functional groups 3 (Table 1). Moreover, cofactors can also provide structural stability to the enzyme 4 . 34 22.