Literature summary for 2.3.2.13 extracted from

Spidel, J.L.; Vaessen, B.; Albone, E.F.; Cheng, X.; Verdi, A.; Kline, J.B.
Site-specific conjugation to native and engineered lysines in human immunoglobulins by microbial transglutaminase (2017), Bioconjug. Chem., 28, 2471-2484 .

Search for more literature for 2.3.2.13

Application

Application	Comment	Organism
biotechnology	in production of homogeneous antibody-drug conjugates, the enzyme is useful for site-specific conjugation of glutamine-based acyl donor substrates and drugs to native and engineered lysines in human immunoglobulins by microbial transglutaminase, overview	Streptomyces mobaraensis

Protein Variants

Protein Variants	Comment	Organism
additional information	utility of single lysine substitutions and the C-terminal Lys447 for engineering efficient acyl acceptor sites suitable for site-specific conjugation to a range of glutamine-based acyl donor substrates. Because recombinant mAbs lack the C-terminal Lys447 due to cleavage by carboxypeptidase B in the production cell host, it is analyzed if blocking the cleavage of Lys447 by the addition of a C-terminal amino acid can result in transamidation of Lys447 by a variety of acyl donor substrates. MTG efficiently transamidates Lys447 in the presence of any nonacidic, nonproline amino acid residue at position 448. Scanning mutagenesis of the hinge region in a Fab' fragment reveals sites of transamidation that are not reactive in the context of the full-length mAb. A positive-control peptide with two known lysine acyl acceptor sites (GGSTKHKIPGGS) is genetically fused to the C-terminus of mAb1 HC or LC (HC-KTag or LC-KTag, respectively) and analyzed for transamidation. The addition of the KTag to the HC C-terminus blocks removal of Lys447, thereby allowing MTG to utilize Lys447 as an acyl acceptor site. Mutational analysis of binding sites	Streptomyces mobaraensis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
protein glutamine + alkylamine	Streptomyces mobaraensis	-	protein N5-alkylglutamine + NH3	-	?

Organism

Organism	UniProt	Comment	Textmining
Streptomyces mobaraensis	P81453	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	site-specific conjugation to native and engineered lysines in human immunoglobulins by microbial transglutaminase, overview. ESI-MS analysis of antibodies incubated with an acyl donor substrate and enzyme MTG, performed by incubation with ZQG-biotin and MTG at 37°C overnight followed by digestion with IdeS to generate Fab'2 and Fc fragments. A positive-control peptide with two known lysine acyl acceptor sites (GGSTKHKIPGGS) is genetically fused to the C-terminus of mAb1 HC or LC (HC-KTag or LC-KTag, respectively) and analyzed for transamidation. The addition of the KTag to the HC C-terminus blocks removal of Lys447, thereby allowing MTG to utilize Lys447 as an acyl acceptor site. Effect of single C-terminal amino acids on transamidation of HC Lys447, and analysis of transamidation of single lysine substitutions in gamma, iota, kappa, and lambda constant regions, overview. Optimal transamidation of an LC C-terminal lysine requires a spacer between Cys214 and the lysine. Analysis of transamidation of select single lysine substitutions, conducted by incubating samples with ZQG-biotin and MTG at 37°C overnight. Mutational analysis of binding sites	Streptomyces mobaraensis	?	-	-
protein glutamine + alkylamine	-	Streptomyces mobaraensis	protein N5-alkylglutamine + NH3	-	?

Synonyms

Synonyms	Comment	Organism
microbial transglutaminase	-	Streptomyces mobaraensis

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Release 2023.1 (January 2023)