Enzyme-catalyzed protein crosslinking

Abstract

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.

Fig. 1

Schematic illustration of enzyme-catalyzed covalent modifications of proteins in vivo; for a more comprehensive overview we refer to Walsh (2006) and Walsh et al. (2005). The cartoon depicted in the center was created with the program PyMOL and shows the structure of ubiquitin (PDB ID: 1ubq) (Schrodinger 2010)

Fig. 2

Simplified schematic illustration of the enzymatic cascade leading to ubiquitination of target proteins in eukaryotic cells (Spasser and Brik 2012)

Fig. 3

Protein crosslinking through transamidation reactions catalyzed by transglutaminase and sortase A. The reactive thioester intermediates generated at the active site of the enzymes are framed

Fig. 4

Crosslinking of proteins mediated through oxidation by oxidoreductases. The reactive species generated by the enzymes are framed. The illustration of the laccase- and peroxidase-catalyzed oxidation reactions is simplified and does not show the stoichiometry of substrates and products (laccase oxidizes four substrate molecules per molecule of oxygen, whereas peroxidase oxidizes only two substrate molecules per molecule of hydrogen peroxide)