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.

One of the main and most astonishing characteristics of peptides comprised of beta-amino acids with proteinogenic side chains is their extraordinarily high stability towards enzymatic degradation. So far, only certain microbial enzymes have been shown to cleave N-terminal beta(3)-homoamino acid residues from peptides. In this work, the L-aminopeptidase-D-amidase/esterase (DmpA) from Ochrobactrum anthropi LMG7991 is compared to two closely related beta-peptidyl aminopeptidases (BapA), which originate from Sphingosinicella strains, and to microsomal leucine aminopeptidase (LAP) as a reference. All four enzymes are aminopeptidases cleaving N-terminal amino acids from small peptides. Degradation experiments reveal that DmpA and both BapA enzymes exhibit unique, but clearly distinct substrate specificities and preferences. DmpA also cleaves beta- and mixed alpha,beta-peptides and amides, but a short side chain of the N-terminal beta-amino acid residue seems to be a prerequisite, since only peptides carrying N-terminal betahGly and beta(3)hAla are hydrolyzed with good efficiencies. Both beta-peptidyl aminopeptidases cleave beta-amino acids from a variety of beta-peptides and mixed alpha,beta-peptides, but they do not accept alpha-amino acids in the N-terminal position. Astonishingly, DmpA exhibited much higher catalytical rates for the mixed dipeptide carnosine (H-betahGly-His-OH) than for any other substrate described until now.

We previously discovered that BapA, a bacterial β‐peptidyl aminopeptidase, is able to hydrolyze two otherwise metabolically inert β‐peptides [Geueke B, Namoto K, Seebach D & Kohler H‐PE (2005) J Bacteriol187, 5910–5917]. Here, we describe the purification and characterization of two distinct bacterial β‐peptidyl aminopeptidases that originated from different environmental isolates. Both bapA genes encode a preprotein with a signal sequence and were flanked by ORFs that code for enzymes with similar predicted functions. To form the active enzymes, which had an (αβ)4 quaternary structure, the preproteins needed to be cleaved into two subunits. The two β‐peptidyl aminopeptidases had 86% amino acid sequence identity, hydrolyzed a variety of β‐peptides and mixed β/α‐peptides, and exhibited unique substrate specificities. The prerequisite for peptides being accepted as substrates was the presence of a β‐amino acid at the N‐terminus; peptide substrates with an N‐terminal α‐amino acid were not hydrolyzed at all. Both enzymes cleaved the peptide bond between the N‐terminal β‐amino acid and the amino acid at the second position of tripeptidic substrates of the general structure H‐βhXaa‐Ile‐βhTyr‐OH according to the following preferences with regard to the side chain of the N‐terminal β‐amino acid: aliphatic and aromatic > OH‐containing > hydrogen, basic and polar. Experiments with the tripeptides H‐d‐βhVal‐Ile‐βhTyr‐OH and H‐βhVal‐Ile‐βhTyr‐OH demonstrated that the two BapA enzymes preferred the peptide with the l‐configuration of the N‐terminal β‐homovaline residue as a substrate.

Different enzyme variants of sortase A fromStaphylococcus aureuswere found to have distinct catalytic properties with regard to site-directed protein fusion and competing intermolecular crosslinking reactions.