The crystal structure of mismatch-specific uracil-DNA glycosylase (MUG) from Deinococcus radiodurans reveals a novel catalytic residue and broad substrate specificity

Deinococcus radiodurans is extremely resistant to the effects of ionizing radiation. The source of the radiation resistance is not known, but an expansion of specific protein families related to stress response and damage control has been observed. DNA repair enzymes are among the expanded protein families in D. radiodurans, and genes encoding five different uracil-DNA glycosylases are identified in the genome. Here we report the three-dimensional structure of the mismatch-specific uracil-DNA glycosylase (MUG) from D. radiodurans (drMUG) to a resolution of 1.75 angstroms. Structural analyses suggest that drMUG possesses a novel catalytic residue, Asp-93. Activity measurements show that drMUG has a modified and broadened substrate specificity compared with Escherichia coli MUG. The importance of Asp-93 for activity was confirmed by structural analysis and abolished activity for the mutant drMUGD93A. Two other microorganisms, Bradyrhizobium japonicum and Rhodopseudomonas palustris, possess genes that encode MUGs with the highest sequence identity to drMUG among all of the bacterial MUGs examined. A phylogenetic analysis indicates that these three MUGs form a new MUG/thymidine-DNA glycosylase subfamily, here called the MUG2 family. We suggest that the novel catalytic residue (Asp-93) has evolved to provide drMUG with broad substrate specificity to increase the DNA repair repertoire of D. radiodurans.     

Characterisation of the substrate specificity of homogeneous vaccinia virus uracil-DNA glycosylase

The decision to stop smallpox vaccination and the loss of specific immunity in a large proportion of the population could jeopardise world health due to the possibility of a natural or provoked re-emergence of smallpox. Therefore, it is mandatory to improve the current capability to prevent or treat such infections. The DNA repair protein uracil-DNA glycosylase (UNG) is one of the viral enzymes important for poxvirus pathogenesis. Consequently, the inhibition of UNG could be a rational strategy for the treatment of infections with poxviruses. In order to develop inhibitor assays for UNG, as a first step, we have characterised the recombinant vaccinia virus UNG (vUNG) and compared it with the human nuclear form (hUNG2) and catalytic fragment (hUNG) UNG. In contrast to hUNG2, vUNG is strongly inhibited in the presence of 7.5 mM MgCl₂. We have shown that highly purified vUNG is not inhibited by a specific uracil-DNA glycosylase inhibitor. Interestingly, both viral and human enzymes preferentially excise uracil when it is opposite to cytosine. The present study provides the basis for the design of specific inhibitors for vUNG.     

Dissecting the broad substrate specificity of human 3-methyladenine-DNA glycosylase

Human alkyladenine-DNA glycosylase (AAG) catalyzes the excision of a broad range of modified bases, protecting the genome from many types of alkylative and oxidative DNA damage. We have investigated how AAG discriminates against normal DNA bases, while accommodating a structurally diverse set of lesioned bases, by measuring the rates of AAG-catalyzed (k(st)) and spontaneous N-glycosidic bond hydrolysis (k(non)) for damaged and undamaged DNA oligonucleotides. The rate enhancements for excision of different bases reveal that AAG is most adept at excising the deaminated lesion hypoxanthine (k(st)/k(non) = 10(8)), suggesting that enzymatic activity may have evolved in response to this lesion. Comparisons of the rate enhancements for excision of normal and modified purine nucleobases provide evidence that AAG excludes the normal purines via steric clashes with the exocyclic amino groups of adenine and guanine. However, methylated purines are more chemically labile, and only modest rate enhancements are required for their efficient excision. Base flipping also contributes to specificity as destabilized mismatched base pairs are better substrates than stable Watson-Crick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by specifically recognizing each lesion.     

A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

Cytosine bases can be deaminated spontaneously to uracil, causing DNA damage. Uracil-DNA glycosylase (UDG), a ubiquitous uracil-excising enzyme found in bacteria and eukaryotes, is one of the enzymes that repair this kind of DNA damage. To date, no UDG-coding gene has been identified in Methanococcus jannaschii, although its entire genome was deciphered. Here, we have identified and characterized a novel UDG from M.jannaschii designated as MjUDG. It efficiently removed uracil from both single- and double-stranded DNA. MjUDG also catalyzes the excision of 8-oxoguanine from DNA. MjUDG has a helix–hairpin–helix motif and a [4Fe–4S]-binding cluster that is considered to be important for the DNA binding and catalytic activity. Although MjUDG shares these features with other structural families such as endonuclease III and mismatch-specific DNA glycosylase (MIG), unique conserved amino acids and substrate specificity distinguish MjUDG from other families. Also, a homologous member of MjUDG was identified in Aquifex aeolicus. We report that MjUDG belongs to a novel UDG family that has not been described to date.     

Substrate specificity of homogeneous monkeypox virus uracil-DNA glycosylase

Weak or nonexistent smallpox immunity in today's human population raises concerns about the possibility of natural or provoked genetic modifications leading to re-emergence of variola virus and other poxviruses. Thus, the development of new antiviral strategies aimed at poxvirus infections in humans is a high priority. The DNA repair protein uracil-DNA glycosylase (UNG) is one of the viral enzymes important for poxvirus pathogenesis. Consequently, the inhibition of UNG is a rational therapeutic strategy for infections with poxviruses. Monkeypox virus, which occurs naturally in Africa, can cause a smallpox-like disease in humans. Here, the monkeypox virus UNG (mpUNG) is characterized and compared to vaccinia virus UNG (vUNG) and human UNG (hUNG). The mpUNG protein excises uracil preferentially from single-stranded DNA. Furthermore, mpUNG prefers the U.G pair over the U.A pair and does not excise oxidized bases. Both mpUNG and vUNG viral proteins are strongly inhibited by physiological concentrations of NaCl and MgCl2. Although the two viral DNA repair enzymes have similar substrate specificities, the kcat/KM values of mpUNG are higher than those of vUNG. The mpUNG protein was strongly inhibited by 5-azauracil and to a lesser extent by 4(6)-aminouracil and 5-halogenated uracil analogues, whereas uracil had no effect. To develop antiviral drugs toward mpUNG, we also validated a repair assay using the molecular beacons containing multiple uracil residues. Potential targets and strategies for combating pathogenic orthopoxviruses, including smallpox, are discussed.     

Intracellular localization and tissue specificity of human uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG) purified by various procedures from the human placenta was used to obtain immune antisera with specific antibodies, the antibodies being affinity-purified on UDG-sepharose. Two immunoreactive polypeptides were found in crude extracts of the human placenta with the help of the antibodies. Their apparent molecular masses were about 37,000 and 34,000 dalton. Only the former polypeptide was found in crude extracts of the human embryonal heart, liver and in HeLa cells. The indirect immunofluorescent staining shows both slight and intensive fluorescence of HeLa cell nuclei. The similarity of antigenic properties of the human and rat UDG was confirmed.     

Structure and specificity of the vertebrate anti-mutator uracil-DNA glycosylase SMUG1

Cytosine deamination is a major promutagenic process, generating G:U mismatches that can cause transition mutations if not repaired. Uracil is also introduced into DNA via nonmutagenic incorporation of dUTP during replication. In bacteria, uracil is excised by uracil-DNA glycosylases (UDG) related to E. coli UNG, and UNG homologs are found in mammals and viruses. Ung knockout mice display no increase in mutation frequency due to a second UDG activity, SMUG1, which is specialized for antimutational uracil excision in mammalian cells. Remarkably, SMUG1 also excises the oxidation-damage product 5-hydroxymethyluracil (HmU), but like UNG is inactive against thymine (5-methyluracil), a chemical substructure of HmU. We have solved the crystal structure of SMUG1 complexed with DNA and base-excision products. This structure indicates a more invasive interaction with dsDNA than observed with other UDGs and reveals an elegant water displacement/replacement mechanism that allows SMUG1 to exclude thymine from its active site while accepting HmU.     

Factor Xa active site substrate specificity with substrate phage display and computational molecular modeling

Structural origin of substrate-enzyme recognition remains incompletely understood. In the model enzyme system of serine protease, canonical anti-parallel beta-structure substrate-enzyme complex is the predominant hypothesis for the substrate-enzyme interaction at the atomic level. We used factor Xa (fXa), a key serine protease of the coagulation system, as a model enzyme to test the canonical conformation hypothesis. More than 160 fXa-cleavable substrate phage variants were experimentally selected from three designed substrate phage display libraries. These substrate phage variants were sequenced and their specificities to the model enzyme were quantified with quantitative enzyme-linked immunosorbent assay for substrate phage-enzyme reaction kinetics. At least three substrate-enzyme recognition modes emerged from the experimental data as necessary to account for the sequence-dependent specificity of the model enzyme. Computational molecular models were constructed, with both energetics and pharmacophore criteria, for the substrate-enzyme complexes of several of the representative substrate peptide sequences. In contrast to the canonical conformation hypothesis, the binding modes of the substrates to the model enzyme varied according to the substrate peptide sequence, indicating that an ensemble of binding modes underlay the observed specificity of the model serine protease.     

Soluble aldose sugar dehydrogenase from Escherichia coli: a highly exposed active site conferring broad substrate specificity

A water-soluble aldose sugar dehydrogenase (Asd) has been purified for the first time from Escherichia coli. The enzyme is able to act upon a broad range of aldose sugars, encompassing hexoses, pentoses, disaccharides, and trisaccharides, and is able to oxidize glucose to gluconolactone with subsequent hydrolysis to gluconic acid. The enzyme shows the ability to bind pyrroloquinoline quinone (PQQ) in the presence of Ca2+ in a manner that is proportional to its catalytic activity. The x-ray structure has been determined in the apo-form and as the PQQ-bound active holoenzyme. The beta-propeller fold of this protein is conserved between E. coli Asd and Acinetobacter calcoaceticus soluble glucose dehydrogenase (sGdh), with major structural differences lying in loop and surface-exposed regions. Many of the residues involved in binding the cofactor are conserved between the two enzymes, but significant differences exist in residues likely to contact substrates. PQQ is bound in a large cleft in the protein surface and is uniquely solvent-accessible compared with other PQQ enzymes. The exposed and charged nature of the active site and the activity profile of this enzyme indicate possible factors that underlie a low affinity for glucose but generic broad substrate specificity for aldose sugars. These structural and catalytic properties of the enzymes have led us to propose that E. coli Asd provides a prototype structure for a new subgroup of PQQ-dependent soluble dehydrogenases that is distinct from the A. calcoaceticus sGdh subgroup.     

Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis

The role of the conserved histidine-187 located in the leucine intercalation loop of Escherichia coli uracil-DNA glycosylase (Ung) was investigated. Using site-directed mutagenesis, an Ung H187D mutant protein was created, overproduced, purified to apparent homogeneity, and characterized in comparison to wild-type Ung. The properties of Ung H187D differed from Ung with respect to specific activity, substrate specificity, DNA binding, pH optimum, and inhibition by uracil analogues. Ung H187D exhibited a 55000-fold lower specific activity and a shift in pH optimum from pH 8.0 to 7.0. Under reaction conditions optimal for wild-type Ung (pH 8.0), the substrate preference of Ung H187D on defined single- and double-stranded oligonucleotides (25-mers) containing a site-specific uracil target was U/G-25-mer > U-25-mer > U/A-25-mer. However, Ung H187D processed these same DNA substrates at comparable rates at pH 7.0 and the activity was stimulated approximately 3-fold relative to the U-25-mer substrate. Ung H187D was less susceptible than Ung to inhibition by uracil, 6-amino uracil, and 5-fluorouracil. Using UV-catalyzed protein/DNA cross-linking to measure DNA binding affinity, the efficiency of Ung H187D binding to thymine-, uracil-, and apyrimidinic-site-containing DNA was (dT20) = (dT19-U) >/= (dT19-AP). Comparative analysis of the biochemical properties and the X-ray crystallographic structures of Ung and Ung H187D [Putnam, C. D., Shroyer, M. J. N., Lundquist, A. J., Mol, C. D., Arvai, A. S., Mosbaugh, D. W., and Tainer, J. A. (1999) J. Mol. Biol. 287, 331-346] provided insight regarding the role of His-187 in the catalytic mechanism of glycosylic bond cleavage. A novel mechanism is proposed wherein the developing negative charge on the uracil ring and concomitant polarization of the N1-C1' bond is sustained by resonance effects and hydrogen bonding involving the imidazole side chain of His-187.     

Implications of alternative substrate binding modes for catalysis by uracil-DNA glycosylase: an apparent discrepancy resolved

A theoretical study showed that the base excision repair enzyme uracil-DNA glycosylase (UDG) exploits electrostatic interactions with backbone phosphate groups in the substrate for catalysis. Although experiments performed to test the calculated results confirmed the predicted importance of the -2, -1, and +1 phosphate groups, there was an apparent disagreement with regard to the +2 phosphate group. The calculations indicated that it made an important contribution, while experimentally, the effect of its deletion or neutralization was small. The +2 phosphate group interacts directly with an active site histidine (H148 in humans) in the crystal structure of UDG in complex with double-stranded (ds) DNA. We previously calculated that H148 has a strong anticatalytic effect due to its protonation, and here we use alchemical free energy simulations to estimate its site-specific pKa. We find that it is positively charged over the entire experimental pH range (4-10), so its deprotonation cannot compensate for deletion or neutralization of the +2 phosphate group. The free energy simulations are facilitated by an efficient charge-scaling procedure that allows quantitative correction for the implicit treatment of solvent far from the active site; improvements are made to that method to account carefully for differences in the truncation of electrostatic interactions in the contributing molecular-mechanical and continuum-electrostatic approaches. Additional simulations are used to demonstrate that the +2 phosphate group is fully solvent exposed in complexes with single-stranded DNA substrates like those used in the experiments. In contrast, it is well-structured and buried in the dsDNA complex used in the original simulations. Differences in solvent shielding thus account for the apparent lack of an effect observed experimentally upon neutralization or deletion of this group.     

A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α(4), homodimeric: α(2), and heterotetrameric: (αβ)(2)] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε(2)) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε(2) endonuclease cleaves both canonical and non-canonical bulge-helix-bulge motifs, similar to that of (αβ)(2) endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)(2) endonuclease. Thus, the discovery of ε(2) endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.     

A kinetic analysis of substrate recognition by uracil-DNA glycosylase from herpes simplex virus type 1

Uracil-DNA glycosylase (UDG) is responsible for the removal of uracil from DNA. It has previously been demonstrated that UDG exhibits some sequence dependence in its activity, although this has not been well characterised. This study has investigated the sequence-dependent activity of UDG from herpes simplex virus type-1 (HSV-1). A more detailed analysis has been possible by using both kinetic and binding assays with a variety of different oligonucleotide substrates. The target uracil has been placed in substrates with either A-T-rich or G-C-rich flanking sequences and analyses have been performed on both the single- and double-stranded forms of each substrate. In the latter the uracil has been placed in both a U·A base pair and a U·G mismatch. It is observed that the sequences flanking the target uracil have a greater effect on UDG activity than the partner base of the uracil. Furthermore, the sequence context effects extend to single-stranded DNA. Systematic examination of the kinetics and binding of UDG with these different substrates has enabled us to examine the origin of the sequence preferences. We conclude that the damage recognition step in the HSV-1 UDG reaction pathway is modulated by local DNA sequence.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site.

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.     

A PTEN-like phosphatase with a novel substrate specificity

We show that a novel PTEN-like phosphatase (PLIP) exhibits a unique preference for phosphatidylinositol 5-phosphate (PI(5)P) as a substrate in vitro. PI(5)P is the least characterized member of the phosphoinositide (PI) family of lipid signaling molecules. Recent studies suggest a role for PI(5)P in a variety of cellular events, such as tumor suppression, and in response to bacterial invasion. Determining the means by which PI(5)P levels are regulated is therefore key to understanding these cellular processes. PLIP is highly enriched in testis tissue and, similar to other PI phosphatases, exhibits poor activity against several proteinaceous substrates. Despite a recent report suggesting a role for PI(5)P in the regulation of Akt, the overexpression of wild-type or catalytically inactive PLIP in Chinese hamster ovary-insulin receptor cells or a dsRNA-mediated knockdown of PLIP mRNA levels in Drosophila S2 cells does not alter Akt activity or phosphorylation. The unique in vitro catalytic activity and detailed biochemical and kinetic analyses reported here will be of great value in our continued efforts to identify in vivo substrate(s) for this highly conserved phosphatase.     

Synthesis and molecular modeling of novel HSV1 uracil-DNA glycosylase inhibitors.

In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. Starting with the optimized model of binding between 6-(4'-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.     

Functional changes in a novel uracil-DNA glycosylase determined by mutational analyses

Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in bacteria and eukaryotes, which removes uracil residues from DNA strands. Methanococcus jannaschii UDG (MjUDG), a novel monofunctional glycosylase, contains a helix-hairpin-helix (HhH) motif and a Gly/Pro rich loop (GPD region), which is important for catalytic activity; it shares these features with other glycosylases, such as endonuclease III. First, to examine the role of two conserved amino acid residues (Asp150 and Tyr152) in the HhH-GPD region of MjUDG, mutant MjUDG proteins were constructed, in which Asp150 was replaced with either Glu or Trp (D150E and D150W), and Tyr152 was replaced with either Glu or Asn (Y152E and Y152N). Mutant D150W completely lacked DNA glycosylase activity, whereas D150E displayed reduced activity of about 70% of the wild type value. However, the mutants Y152E and Y152N retained unchanged levels of UDG activity. We also replaced Glu132 in the HhH motif with a lysine residue equivalent to Lys120 in endonuclease III. This mutation converted the enzyme into a bifunctional glycosylase/AP lyase capable of both removing uracil at a glycosylic bond and cleaving the phosphodiester backbone at an AP site. Mutant E132K catalyzes a β-elimination reaction at the AP site via uracil excision and forms a Schiff base intermediate in the form of a protein-DNA complex. This text was submitted by the authors in English.     

Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG), which is a critical enzyme in DNA base-excision repair that recognizes and removes uracil from DNA, is specifically and irreversably inhibited by the thermostable uracil-DNA glycosylase inhibitor protein (Ugi). A paradox for the highly specific Ugi inhibition of UDG is how Ugi can successfully mimic DNA backbone interactions for UDG without resulting in significant cross-reactivity with numerous other enzymes that possess DNA backbone binding affinity. High-resolution X-ray crystal structures of Ugi both free and in complex with wild-type and the functionally defective His187Asp mutant Escherichia coli UDGs reveal the detailed molecular basis for duplex DNA backbone mimicry by Ugi. The overall shape and charge distribution of Ugi most closely resembles a midpoint in a trajectory between B-form DNA and the kinked DNA observed in UDG:DNA product complexes. Thus, Ugi targets the mechanism of uracil flipping by UDG and appears to be a transition-state mimic for UDG-flipping of uracil nucleotides from DNA. Essentially all the exquisite shape, electrostatic and hydrophobic complementarity for the high-affinity UDG-Ugi interaction is pre-existing, except for a key flip of the Ugi Gln19 carbonyl group and Glu20 side-chain, which is triggered by the formation of the complex. Conformational changes between unbound Ugi and Ugi complexed with UDG involve the beta-zipper structural motif, which we have named for the reversible pairing observed between intramolecular beta-strands. A similar beta-zipper is observed in the conversion between the open and closed forms of UDG. The combination of extremely high levels of pre-existing structural complementarity to DNA binding features specific to UDG with key local conformational changes in Ugi resolves the UDG-Ugi paradox and suggests a potentially general structural solution to the formation of very high affinity DNA enzyme-inhibitor complexes that avoid cross- reactivity.     

Contrasting effects of mutating active site residues, aspartic acid 64 and histidine 187 of Escherichia coli uracil-DNA glycosylase on uracil excision and interaction with an inhibitor protein

Uracil, a promutagenic base, arises in DNA by spontaneous deamination of cytosine or by the malfunctioning of DNA polymerases. To maintain the genomic integrity, cells possess a highly conserved base excision repair enzyme, uracil-DNA glycosylase (UDG). UDGs have a notably high turnover number and strict specificity for uracil in DNA. UDGs are inhibited by a small proteinaceous inhibitor, Ugi, which acts as a transition state substrate mimic. Crystal structure studies have identified the residues crucial in catalysis, and in their interaction with Ugi. Here, we report on the mutational analyses of D64 (D64H and D64N) and H187 (H187C, H187L and H187R) in the active site pocket of Escherichia coli UDG. The mutants were compromised in uracil excision by approximately 200-25,000 fold when compared to the native protein. In contrast, our analysis of the in vivo formed UDG-Ugi complexes on urea gels shows that D64 and H187 contribute minimally to the interaction of the two proteins. Thus, our findings provide further evidence to the primary function of D64 and H187 in catalysis.     

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.