Expanding the Versatility of Microbial Transglutaminase Using α‐Effect Nucleophiles as Noncanonical Substrates

Abstract

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology,inparticular for the attachment of alkylamines to glutamine-containing peptides and proteins.H ere,w e expand the substrate repertoire to include hydrazines,h ydrazides,a nd alkoxyamines,resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG.F urthermore,w ed emonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues.The distinct hydrazide handles can be further coupled with carbonyls,i ncluding ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability.T he extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme,providing anew approach to facilitate novel applications. Scheme 1. Transamidation reaction catalyzed by microbial transglutaminase (mTG) using an amine or an a-effect nucleophile a) hydrazine, b) hydrazide, or c) alkoxyaminea sthe acyl acceptor substrate.

A tailored phosphoaspartate probe unravels CprR as a response regulator in Pseudomonas aeruginosa interkingdom signaling

“…The sequence motif enrichment of this refined methodology is more pronounced compared to the previous study demonstrating the general utility of the approach in acyl phosphate trapping. 14 As we cannot a priori exclude the capture of other electrophilic modifications such as ADP-ribosylation, 29 glutamate methylation 30 and transamidation 31 we extended our analysis to all amino acids as possible modification sites. To our surprise we obtained significant labeling at Glu, Asn and Gln which cumulated in about 6000 additional sites, foremost on Gln, in both proteomes (Fig.…”

Site-selective modification strategies in antibody–drug conjugates

“…19D). 295 Modification of genetically installed glutamine residues in the light or heavy chain was demonstrated with these nucleophiles with minimal modification of native glutamines observed. Furthermore, conjugation with unsubstituted hydrazine and dihydrazines facilitated introduction of bioorthogonally reactive aldehydes or ketones to produce further functionalised bioconjugates.…”

Enzymatic Methods for the Site-Specific Radiolabeling of Targeting Proteins

“…Indeed, even if mTG reacts with acyl acceptor substrates with very different chemical structures, ligands containing aromatic moieties show a higher reactivity while the presence of negatively charged groups is detrimental [75]. Recently, it has been demonstrated that mTG can catalyze the reaction also with noncanonical acyl acceptor substrates containing hydrazines, hydrazides, and alkoxyamines in place of the primary ammine (Figure 2B) [76]. These substrates lead to the formation of analogous of the isopeptide bond and they also allow the introduction of reactive groups into the sidechain of Gln residues for subsequent conjugation reactions.…”