Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

Small molecule substrates for the rapid quantification of acyl transfer activity of nylon hydrolase NylCA.

The widespread use of polyamides such as nylons has led to the accumulation of nylon waste, which is particularly resistant to decomposition due to the intrinsic stability of the amide bond. New methods are required for the true recycling of these waste materials by depolymerization. Enzymes that are capable of hydrolysing polyamides have been proposed as biocatalysts that may be suitable for this application. NylC is an enzyme that can mediate the hydrolysis of aminohexanoic acid oligomers, and to some extent, bulk polymers. However, current assays to characterize the activity of this enzyme require long reaction times and/or rely on secondary reactions to quantify hydrolysis. Herein, we have designed structurally-optimized small molecule chromogenic esters that serve as substrate analogues for monitoring NylC acyltransferase activity in a continuous manner. This assay can be performed in minutes at room temperature, and the substrate N-acetyl-GABA-pNP ester (kcat = 0.37 s-1, KM = 256 µM) shows selectivity for NylC in complex biological media. We also demonstrate that activity towards this substrate analogue correlates with amide hydrolysis, which is the primary activity of this enzyme. Furthermore, our screening of substrate analogues provides insight into the substrate specificity of NylC, which is relevant to biocatalytic applications.

High-Affinity Fluorogenic Substrate for Tissue Transglutaminase Reveals Enzymatic Hysteresis.

Transglutaminases (TGases) are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins. Transglutaminase 2 (TG2) is one isozyme in this family whose role is multifaceted. TG2 can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site. These two discrete activities are tightly regulated by both environmental stimuli and redox reactions. Ubiquitously expressed in humans, TG2 has been implicated in numerous disease pathologies that require extensive investigation. The catalytic activity of TG2 can be monitored through various mechanisms, including hydrolysis, transamidation, or cleavage of isopeptide bonds. Activity assays are required to monitor the activity of this isozyme not only for studying its transamidation reaction but also for validation of therapeutics designed to abolish this activity. Herein, we present the design, synthesis, and evaluation of a new TG2 activity substrate based on a previously optimized inhibitor scaffold. The substrate APH7 exhibits excellent affinity, selectivity, and reactivity with TG2 (KM = 3.0 µM). Furthermore, its application also allowed the discovery of unique hysteresis at play within the catalytic activity and inhibition reactivity of TG2.

A mechanistic study of thiol addition to N-acryloylpiperidine.

Nucleophilic cysteine (Cys) residues are present in many enzyme active sites and represent the target of many different irreversible enzyme inhibitors. Given its fine balance between aqueous stability and thiolate reactivity, the acrylamide group is a particularly popular warhead pharmacophore among inhibitors designed for biological and therapeutic application. The acrylamide group is well known to undergo thiol addition, but the precise mechanism of this addition reaction has not been studied in as much detail. In this work we have focussed on the reaction of N-acryloylpiperidine (AcrPip), which represents a motif found in many targeted covalent inhibitor drugs. Using a precise HPLC-based assay, we measured the second order rate constants for the reaction of AcrPip with a panel of thiols possessing different pKa values. This allowed construction of a Brønsted-type plot that reveals the relative insensitivity of the reaction to the nucleophilicity of the thiolate. By studying temperature effects, we were able to construct an Eyring plot from which the enthalpy and entropy of activation were calculated. Ionic strength and solvent kinetic isotope effects were also studied, informing on charge dispersal and proton transfer in the transition state. DFT calculations were also performed, providing information on the potential structure of the activated complex. Taken together, these data strongly support one cohesive addition mechanism that is the microscopic reverse of the E1cb elimination, and highly relevant to the intrinsic thiol selectivity of AcrPip inhibitors and their subsequent design.

A kinetic study of thiol addition to N-phenylchloroacetamide.

Irreversible enzyme inhibitors bind covalently to their target and permanently limit its function. The redox-sensitive thiol group on the side chain of cysteine (Cys) residues is often the nucleophilic group that is targeted for reaction with the electrophilic warhead of irreversible inhibitors. While the acrylamide group is the warhead applied most frequently currently in the design of inhibitors with therapeutic potential, the chloroacetamide group offers a comparable reactivity profile. In that context, we have studied the details of the mechanism of thiol addition to N-phenylchloroacetamide (NPC). A kinetic assay was developed to accurately monitor the reaction progress between NPC and a small library of thiols with varying pKa values. From these data, a Brønsted-type plot was constructed, from which a ßnucRS- value of 0.22 ± 0.07 was derived, indicative of a relatively early transition state with respect to attack by the thiolate. The halide leaving group was also varied, for the reaction with one thiol, providing rate constants consistent with a transition state that is early with respect to leaving group departure. The effects of temperature and ionic strength were also studied, and all data are consistent with an early transition state for a concerted SN2 mechanism of addition. Molecular modelling was also performed, and these calculations confirm the concerted transition state and relative reactivity of the haloacetamides. Finally, this study allows a detailed comparison of the reactivity and reaction mechanisms of the chloroacetamide group with the benchmark acrylamides used in many irreversible inhibitor drugs.

Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype.

Transglutaminase 2 (TG2) is a multifunctional enzyme primarily responsible for crosslinking proteins. Ubiquitously expressed in humans, TG2 can act either as a transamidase by crosslinking two substrates through formation of an Nε(ɣ-glutaminyl)lysine bond or as an intracellular G-protein. These discrete roles are tightly regulated by both allosteric and environmental stimuli and are associated with dramatic changes in the conformation of the enzyme. The pleiotropic nature of TG2 and multi-faceted activities have resulted in TG2 being implicated in numerous disease pathologies including celiac disease, fibrosis, and cancer. Targeted TG2 therapies have not been selective for subcellular localization, such that currently no tools exist to selectively target extracellular over intracellular TG2. Herein, we have designed novel TG2-selective inhibitors that are not only highly potent and irreversible, but also cell impermeable, targeting only extracellular TG2. We have also further derivatized the scaffold to develop probes that are intrinsically fluorescent or bear an alkyne handle, which target both intra- and extracellular TG2, in order to facilitate cellular labelling and pull-down assays. The fluorescent probes were internalized and imaged in cellulo, and provide the first implicit experimental evidence that by comparison with their cell-impermeable analogues, it is specifically intracellular TG2, and presumably its G-protein activity, that contributes to transglutaminase-associated cancer progression.

Peptidic Inhibitors and a Fluorescent Probe for the Selective Inhibition and Labelling of Factor XIIIa Transglutaminase.

Factor XIIIa (FXIIIa) is a transglutaminase of major therapeutic interest for the development of anticoagulants due to its essential role in the blood coagulation cascade. While numerous FXIIIa inhibitors have been reported, they failed to reach clinical evaluation due to their lack of metabolic stability and low selectivity over transglutaminase 2 (TG2). Furthermore, the chemical tools available for the study of FXIIIa activity and localization are extremely limited. To combat these shortcomings, we designed, synthesised, and evaluated a library of 21 novel FXIIIa inhibitors. Electrophilic warheads, linker lengths, and hydrophobic units were varied on small molecule and peptidic scaffolds to optimize isozyme selectivity and potency. A previously reported FXIIIa inhibitor was then adapted for the design of a probe bearing a rhodamine B moiety, producing the innovative KM93 as the first known fluorescent probe designed to selectively label active FXIIIa with high efficiency (kinact/KI = 127,300 M-1 min-1) and 6.5-fold selectivity over TG2. The probe KM93 facilitated fluorescent microscopy studies within bone marrow macrophages, labelling FXIIIa with high efficiency and selectivity in cell culture. The structure-activity trends with these novel inhibitors and probes will help in the future study of the activity, inhibition, and localization of FXIIIa.

Sulforaphane covalently interacts with the transglutaminase 2 cancer maintenance protein to alter its structure and suppress its activity.

Type 2 transglutaminase (TG2) functions as an important cancer cell survival protein in a range of cancers including epidermal squamous cell carcinoma. TG2 exists in open and closed conformations each of which has a distinct and mutually exclusive activity. The closed conformation has GTP-binding/GTPase activity while the open conformation functions as a transamidase to catalyze protein-protein crosslinking. GTP-binding/GTPase activity is required for TG2 maintenance of the aggressive cancer phenotype. Thus, identifying agents that convert TG2 from the closed to the open GTP-binding/GTPase inactive conformation is an important cancer prevention/treatment strategy. Sulforaphane (SFN) is an important diet-derived cancer prevention agent that is known to possess a reactive isothiocyanate group and has potent anticancer activity. Using a biotin-tagged SFN analog (Biotin-ITC) and kinetic analysis we show that SFN covalently and irreversibly binds to recombinant TG2 to inhibit transamidase activity and shift TG2 to an open/extended conformation, leading to a partial inhibition of GTP binding. We also show that incubation of cancer cells or cancer cell extract with Biotin-ITC results in formation of a TG2/Biotin-ITC complex and that SFN treatment of cancer cells inhibits TG2 transamidase activity and shifts TG2 to an open/extended conformation. These findings identify TG2 as a direct SFN anticancer target in epidermal squamous cell carcinoma.

A mechanistic study of thiol addition to N-phenylacrylamide.

Cysteine (Cys) residues contain a redox-sensitive thiol and are commonly found in enzyme active sites. In recent years, the presence of a reactive thiolate group on a protein has been exploited in the development of irreversible enzyme inhibitors as therapeutic agents. Many targeted covalent inhibitors (TCIs) are designed to covalently react with a specific Cys residue on a target protein active site, irreversibly modifying the target and inhibiting its normal function. The electrophilic warhead most commonly used in this way is the acrylamide functional group. Although the acrylamide group is well known for its ability to undergo thiol-addition reactions, very few studies have been conducted to elucidate the detailed mechanism of this reaction, which inspired us to conduct a thorough kinetic investigation. First, we developed a robust kinetic assay to accurately monitor reaction progress between N-phenylacrylamide (NPA) and a small library of alkyl thiols having widely varying pKa values. This allowed us to construct a Brønsted-type plot for the thiol addition reaction, revealing a ßnucRS- value of 0.07 ± 0.04. We also studied the solvent kinetic isotope effects (SKIEs), pH dependence, and temperature dependence of the reaction, which showed that reaction has a relatively large negative ΔS‡, and a small ΔH‡. Computational studies provided a structure for the transition state that is consistent with the experimental data. All of these data are consistent with rate-limiting nucleophilic attack, followed by rapid protonation of the enolate, corresponding to the microscopic reverse of the E1revcb elimination mechanism.

A Precision Strategy to Cure Renal Cell Carcinoma by Targeting Transglutaminase 2.

In a recent report, no significance of transglutaminase 2 (TGase 2) was noted in the analyses of expression differences between normal and clear cell renal cell carcinoma (ccRCC), although we found that knock down of TGase 2 induced significant p53-mediated cell death in ccRCC. Generally, to find effective therapeutic targets, we need to identify targets that belong specifically to a cancer phenotype that can be differentiated from a normal phenotype. Here, we offer precise reasons why TGase 2 may be the first therapeutic target for ccRCC, according to several lines of evidence. TGase 2 is negatively regulated by von Hippel-Lindau tumor suppressor protein (pVHL) and positively regulated by hypoxia-inducible factor 1-α (HIF-1α in renal cell carcinoma (RCC). Therefore, most of ccRCC presents high level expression of TGase 2 because over 90% of ccRCC showed VHL inactivity through mutation and methylation. Cell death, angiogenesis and drug resistance were specifically regulated by TGase 2 through p53 depletion in ccRCC because over 90% of ccRCC express wild type p53, which is a cell death inducer as well as a HIF-1α suppressor. Although there have been no detailed studies of the physiological role of TGase 2 in multi-omics analyses of ccRCC, a life-long study of the physiological roles of TGase 2 led to the discovery of the first target as well as the first therapeutic treatment for ccRCC in the clinical field.

Transglutaminase 2 programs differentiating acute promyelocytic leukemia cells in all-trans retinoic acid treatment to inflammatory stage through NF-κB activation.

Differentiation syndrome (DS) is a life-threatening complication arising during retinoid treatment of acute promyelocytic leukemia (APL). Administration of all-trans retinoic acid leads to significant changes in gene expression, among the most induced of which is transglutaminase 2, which is not normally expressed in neutrophil granulocytes. To evaluate the pathophysiological function of transglutaminase 2 in the context of immunological function and disease outcomes, such as excessive superoxide anion, cytokine, and chemokine production in differentiated NB4 cells, we used an NB4 transglutaminase knock-out cell line and a transglutaminase inhibitor, NC9, which inhibits both transamidase- and guanosine triphosphate-binding activities, to clarify the contribution of transglutaminase to the development of potentially lethal DS during all-trans retinoic acid treatment of APL. We found that such treatment not only enhanced cell-surface expression of CD11b and CD11c but also induced high-affinity states; atypical transglutaminase 2 expression in NB4 cells activated the nuclear factor kappa (κ)-light-chain-enhancer of the activated B-cell pathway, driving pathogenic processes with an inflammatory cascade through the expression of numerous cytokines, including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), and monocyte chemoattractant protein 1. NC9 decreased the amount of transglutaminase 2, p65/RelA, and p50 in differentiated NB4 cells and their nuclei, leading to attenuated inflammatory cytokine synthesis. NC9 significantly inhibits transglutaminase 2 nuclear translocation but accelerates its proteasomal breakdown. This study demonstrates that transglutaminase 2 expression induced by all-trans retinoic acid treatment reprograms inflammatory signaling networks governed by nuclear factor κ-light-chain-enhancer of activated B-cell activation, resulting in overexpression of TNF-α and IL-1ß in differentiating APL cells, suggesting that atypically expressed transglutaminase 2 is a promising target for leukemia treatment.

Antimicrobial peptide LL-37 and its truncated forms, GI-20 and GF-17, exert spermicidal effects and microbicidal activity against Neisseria gonorrhoeae.

STUDY QUESTION: Do the truncated LL-37 peptides, GI-20 and GF-17, have spermicidal activity and microbicidal effects on the sexually transmitted infection (STI) pathogen Neisseria gonorrhoeae with equivalent potency to LL-37? SUMMARY ANSWER: GI-20 and GF-17 exhibited spermicidal effects on both mouse and human sperm as well as microbicidal action on N. gonorrhoeae with the same efficacy as LL-37. WHAT IS KNOWN ALREADY: The antimicrobial peptide LL-37 exerts microbicidal activity against various STI pathogens as well as spermicidal effects on both mouse and human sperm. STUDY DESIGN, SIZE, DURATION: Spermicidal activities of GI-20 and GF-17 were evaluated in vitro in mouse and human sperm and in vivo in mice. Finally, in vitro antimicrobial effects of LL-37, GI-20 and GF-17 on an STI pathogen, N. gonorrhoeae were determined. All experiments were repeated three times or more. In particular, sperm samples from different males were used on each experimental day. PARTICIPANTS/MATERIALS, SETTING, METHODS: The plasma membrane integrity of peptide-treated sperm was assessed by cellular exclusion of Sytox Green, a membrane impermeable fluorescent DNA dye. Successful mouse in vitro fertilization was revealed by the presence of two pronuclei in oocytes following co-incubation with capacitated untreated/peptide-pretreated sperm. Sperm plus each peptide were transcervically injected into female mice and the success of in vivo fertilization was scored by the formation of 2-4 cell embryos 42 h afterward. Reproductive tract tissues of peptide pre-exposed females were then assessed histologically for any damage. Minimal inhibitory/bactericidal concentrations of LL-37, GI-20 and GF-17 on N. gonorrhoeae were determined by a standard method. MAIN RESULTS AND THE ROLE OF CHANCE: Like LL-37, treatment of sperm with GI-20 and GF-17 resulted in dose-dependent increases in sperm plasma membrane permeabilization, reaching the maximum at 18 and 3.6 µM for human and mouse sperm, respectively (P < 0.0001, as compared with untreated sperm). Mouse sperm treated with 3.6 µM GI-20 or GF-17 did not fertilize oocytes either in vitro or in vivo. Moreover, reproductive tract tissues of female mice pre-exposed to 3.6 µM GI-20 or GF-17 remained intact with no lesions, erosions or ulcerations. At 1.8-7.2 µM, LL-37, GI-20 and GF-17 exerted bactericidal effects on N. gonorrhoeae. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration of the inhibitory effects of GI-20 and GF-17 on human in vitro and in vivo fertilization cannot be performed due to ethical issues. WIDER IMPLICATIONS OF THE FINDINGS: Like LL-37, GI-20 and GF-17 acted as spermicides and microbicides against N. gonorrhoeae, without adverse effects on female reproductive tissues. With lower synthesis costs, GI-20 and GF-17 are attractive peptides for further development into vaginal spermicides/microbicides. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Canadian Institutes of Health Research (MOP119438 and CCI82413 to N.T.) and NIH (R01 AI105147 to G.W.). There are no competing interests to declare.

Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis-MS.

To enable the detection of protein conformational isomers, their enzymatic activity and their inhibition in a single experiment, we developed a method based on kinetic capillary electrophoresis coupled on-line with UV detection and ion mobility mass spectrometry (CE-UV-IM-MS). Kinetic CE-UV separated protein conformers and monitored their interconversion dynamics in solution. Ion mobility mass spectrometry analyzed the conformer sizes, exact molecular weights, and structures of an enzyme and of its substrates, inhibitors and corresponding products. This coupled CE-UV-IM-MS system allowed the simultaneous, real-time observation of the effect of small-molecule inhibitors on both the conformational distribution and enzymatic activity of the human tissue transglutaminase TG2. By expanding mass spectrometry profiling of enzymatic reactions beyond proteins and substrates to include protein dynamics, CE-UV-IM-MS opens a new avenue for the modulation and regulation of cellular functions, drug development and protein engineering.

Mechanisms of Alkyl and Aryl Thiol Addition to N-Methylmaleimide.

A mechanistic study was undertaken to elucidate the reaction pathways for thiol addition to N-methylmaleimide in water. We used linear free energy relationships, solvent kinetic isotope effects (SKIEs), activation parameters, and ionic strength effects to probe the nature of the rate-limiting transition states. Calculations were also employed and assisted in illuminating three possible mechanistic pathways: (1) stepwise addition with rate-limiting nucleophilic attack, (2) stepwise addition with rate-limiting proton transfer, and (3) concerted addition with nucleophilic attack and proton transfer occurring concurrently. Alkyl thiolate addition exhibits ßnucRS-= 0.4, small negative Δ S‡ values, prominent ionic strength effects, and no evidence of general acid catalysis, consistent with pathway 1. Aryl thiolate addition exhibited ßnucArS- = 1.0, large negative Δ S‡ values, normal primary SKIEs, general acid catalysis, and negligible sensitivity to ionic strength, consistent with pathways 2 and 3. The experimental and computational data depict an energy surface where ground state effects, namely the energy of the alkyl/aryl thiolate, play a major role in shaping the governing pathway. Application of these findings to bioconjugation chemistry is also discussed.

Rational design of a highly reactive dicysteine peptide tag for fluorogenic protein labelling.

Rationally designed libraries of a short helical peptide sequence containing two cysteine residues were screened kinetically for their reactivity towards complementary dimaleimide fluorogens. This screening revealed variant sequences whose reactivity has been increased by an order of magnitude relative to the original sequence. The most reactive engineered sequences feature mutant residues bearing positive charges, suggesting the pKa values of the adjacent thiol groups have been significantly lowered, through electrostatic stabilization of the thiolate ionization state. pH-Rate profiles measured for several mutant sequences support this mechanism of rate enhancement. The practical utility of the enhanced reactivity of the final engineered dicysteine tag ('dC10*') was then demonstrated in the fluorogenic intracellular labelling of histone H2B in living HeLa cells.

A Green BODIPY-Based, Super-Fluorogenic, Protein-Specific Labelling Agent.

We report the development of YC23, a novel green BODIPY-based dimaleimide derivative that undergoes a fluorogenic addition reaction (FlARe) with a genetically encodable peptide tag (dC10α) that can be fused to a protein of interest (POI). We also demonstrate the application of this reaction for the fluorogenic labelling of a specific POI in bacterial lysate and in living mammalian cells.

Inhibition or ablation of transglutaminase 2 impairs astrocyte migration.

Astrocytes play numerous complex roles that support and facilitate the function of neurons. Further, when there is an injury to the central nervous system (CNS) they can both facilitate or ameliorate functional recovery depending on the location and severity of the injury. When a CNS injury is relatively severe a glial scar is formed, which is primarily composed of astrocytes. The glial scar can be both beneficial, by limiting inflammation, and detrimental, by preventing neuronal projections, to functional recovery. Thus, understanding the processes and proteins that regulate astrocyte migration in response to injury is still of fundamental importance. One protein that is likely involved in astrocyte migration is transglutaminase 2 (TG2); a multifunctional protein expressed ubiquitously throughout the brain. Its functions include transamidation and GTPase activity, among others, and previous studies have implicated TG2 as a regulator of migration. Therefore, we examined the role of TG2 in primary astrocyte migration subsequent to injury. Using wild type or TG2-/- astrocytes, we manipulated the different functions and conformation of TG2 with novel irreversible inhibitors or mutant versions of the protein. Results showed that both inhibition and ablation of TG2 in primary astrocytes significantly inhibit migration. Additionally, we show that the deficiency in migration caused by deletion of TG2 can only be rescued with the native protein and not with mutants. Finally, the addition of TGFß rescued the migration deficiency independent of TG2. Taken together, our study shows that transamidation and GTP/GDP-binding are necessary for inhibiting astrocyte migration and it is TGFß independent.

Ring Substituent Effects on the Thiol Addition and Hydrolysis Reactions of N-Arylmaleimides.

Maleimide groups are used extensively in bioconjugation reactions, but limited kinetic information is available regarding their thiol addition and hydrolysis reactions. We prepared a series of fluorogenic coumarin maleimide derivatives that differ by the substituent on their maleimide C═C bond. Fluorescence-based kinetic studies of the reaction with ß-mercaptoethanol (BME) yielded the second-order rate constants (k2), while pH-rate studies from pH 7 to 9 gave base-catalyzed hydrolysis rate constants (kOH). Linear free-energy relationships were studied through the correlation of log k2 and log kOH to both electronic (σ(+)) and steric (Es(norm)) parameters of the C═C substituent. These correlations revealed the thiol addition reaction is primarily sensitive to the electronic effects, while steric effects dominate the hydrolysis reaction. These mechanistic studies provide the basis for the design of novel bioconjugation reactants or fluorogenic labeling agents.

Acyl transfer mechanisms of tissue transglutaminase.

Tissue transglutaminase (TG2) is a calcium-dependent enzyme that catalyses several acyl transfer reactions. The most biologically relevant of these involve protein-bound Gln residues as an acyl-donor substrate, and either water or a primary amine as an acyl-acceptor substrate. The former leads to deamidation of Gln to Glu, whereas the latter leads to transamidation, typically resulting in protein cross-linking when the amine substrate is a protein-bound Lys residue. In this review, we present an overview of over fifty years of mechanistic studies that have led to our current understanding of TG2-mediated hydrolysis and transamidation.

Microbial transglutaminase displays broad acyl-acceptor substrate specificity.

The great importance of amide bonds in industrial synthesis has encouraged the search for efficient catalysts of amide bond formation. Microbial transglutaminase (MTG) is heavily utilized in crosslinking proteins in the food and textile industries, where the side chain of a glutamine reacts with the side chain of a lysine, forming a secondary amide bond. Long alkylamines carrying diverse chemical entities can substitute for lysine as acyl-acceptor substrates, to link molecules of interest onto peptides or proteins. Here, we explore short and chemically varied acyl-acceptor substrates, to better understand the nature of nonnatural substrates that are tolerated by MTG, with the aim of diversifying biocatalytic applications of MTG. We show, for the first time, that very short-chain alkyl-based amino acids such as glycine can serve as acceptor substrates. The esterified α-amino acids Thr, Ser, Cys, and Trp--but not Ile--also showed reactivity. Extending the search to nonnatural compounds, a ring near the amine group--particularly if aromatic--was beneficial for reactivity, although ring substituents reduced reactivity. Overall, amines attached to a less hindered carbon increased reactivity. Importantly, very small amines carrying either the electron-rich azide or the alkyne groups required for click chemistry were highly reactive as acyl-acceptor substrates, providing a robust route to minimally modified, "clickable" peptides. These results demonstrate that MTG is tolerant to a variety of chemically varied natural and nonnatural acyl-acceptor substrates, which broadens the scope for modification of Gln-containing peptides and proteins.