Transpeptidation Reaction Research Articles

Yeast protein‐derived γ‐glutamyl peptides prepared by transpeptidation reaction exhibit a pronounced taste‐enhancing effect

AbstractA high‐salt diet can induce hypertension, so salt reduction can prevent hypertension. γ‐Glutamyl peptides (GGPs) have obvious taste‐enhancing effects, while their contents in natural foods are relatively low. Yeast protein rich in Glu/Gln is a good precursor for the preparation of GGPs. In this study, yeast protein‐derived GGPs were prepared through hydrolysis and transpeptidation reactions, followed by sensory evaluation and E‐tongue analysis. Peptide sequences were identified by LC−MS/MS and screened for molecular docking. The optimal reaction conditions were hydrolysis for 4 h, enzyme concentration of 16 U/g, and transpeptidation for 4 h. GGPs could increase salt and umami intensity by 60.78% and 40.93% based on sensory evaluation, 22.52%, and 16.40% according to E‐tongue analysis. Fifteen γ‐glutamyl peptides with different peptide lengths were selected for molecular docking. Molecular docking confirmed their binding to calcium‐sensing receptors (CaSr) through hydrogen bonds and hydrophobic interaction, while interaction between CaSR receptor and γ‐glutamyl di‐, tri‐, and oligo‐peptides varied in binding energy. The stimulation received by CaSR lasted a longer time and varied in intensity. It was further proved that the flavor of mixed peptides has a layered sense and can give people a rich taste experience. Overall, yeast protein‐derived GGPs can enhance salt and umami taste, which can reduce salt usage without compromising taste.

A monoclonal antibody that recognizes a unique 13-residue epitope in the cytoplasmic tail of HLA-E

The Class I MHC molecule (MHC-I) HLA-E presents peptides that are derived from the signal sequences, either those of other MHC-I products, or of viral type I membrane glycoproteins. Monoclonal antibodies with proven specificity for HLA-E, and with no cross-reactions with other MHC-I products, have yet to be described. To obtain anti-HLA-E-specific antibodies suitable for a range of applications, we generated monoclonal antibodies against a unique feature of HLA-E: its cytoplasmic tail. We created an immunogen by performing an enzymatically catalyzed transpeptidation reaction to obtain a fusion of the cytoplasmic tail of HLA-E with a nanobody that recognizes murine Class II MHC (MHC-II) products. We obtained a mouse monoclonal antibody that recognizes a 13-residue stretch in the HLA-E cytoplasmic tail. We cloned the genes that encode this antibody in expression vectors to place an LPETG sortase recognition motif at the C-terminus of the heavy and light chains. This arrangement allows the site-specific installation of fluorophores or biotin at these C-termini. The resulting immunoglobulin preparations, labeled with 4 equivalents of a fluorescent or biotinylated payload of choice, can then be used for direct immunofluorescence or detection of the tag by fluorescence or by streptavidin-based methods. We also show that the 13-residue sequence can serve as an epitope tag, independent of the site of its placement within a protein’s sequence. The antibody can be used diagnostically to stain for HLA-E on patient tumor samples, it can be used as an antibody-epitope tag for extracellular proteins, and it enables research into the unique role of the cytoplasmic tail of HLA-E.

Взаимодействие лидерного пептида CmlAL с рибосомой E. coli, связавшей хлорамфеникол в неканоническом сайте

With the use of docking, equilibrium and biased molecular dynamics simulations, the structure of the 70S E. coli A/A,P/P-ribosome complex with chloramphenicol, an antibiotic, bound in a non-canonical site near the peptidyl transferase center, and ClmAL leader peptide, placed in the nascent peptide exit tunnel, has been obtained. In this structure, chloramphenicol is retained in the ribosome due to wedging of its nitrophenyl residue into the cavity between the Ψ2504 and U2506 residues of 23S rRNA and formation of hydrophobic contacts with them, as well as hydrogen bonds with the G2505 and G2061 residues of 23S rRNA. The ClmAL leader peptide forms many stable hydrogen bonds with the G2061, m2A2503, U2609, and C2610 residues of 23S rRNA. Molecular dynamics simulations of this ternary complex has shown that the mechanism of antibiotic action is to induce divergence of the peptidyl transferase reaction of the substrates relative to each other at a distance that excludes the transpeptidation reaction. This divergence of the peptidyl transferase reaction substrates is stabilized by interactions between the side chain amino group of the lysine residue in the A site, on the one hand, and the C2063 base and chloramphenicol, on the other hand. In this case, the α-amino group of the lysine residue forms a hydrogen bond with the carbonyl group of the Ala-7 CmlAL residue. Together with specific interactions of the CmlAL peptide residues with the rRNA residues of the nascent peptide exit tunnel, this explains the arrest of translation in the presence of chloramphenicol at this particular sequence and, as a consequence, the presence of this sequence in the genes encoding the CmlA transporter protein, which is responsible for ribosomal resistance to this antibiotic.

Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography.

Gram-positive bacteria display pili whose protein components (pilins) are covalently crosslinked by pilus-specific sortase enzymes. These cysteine transpeptidase enzymes catalyze a transpeptidation reaction that joins the pilins together via lysine isopeptide bonds. The crosslinking reaction that builds the SpaA pilus in Corynebacterium diphtheriae is mediated by the SrtA sortase (CdSrtA) and has been reconstituted in vitro. Here, we present a protocol that can be used to measure the kinetics of CdSrtA-catalyzed crosslinking using high-performance liquid chromatography (HPLC). In principle, this biochemical procedure can be used to measure the in vitro crosslinking activity of any pilus-specific sortase.

Differential Study of Antimicrobial Activity of Vancomycin and Teicoplanin (Targocid) against Strains of Staphylococcus aureus and Streptococci sp.

The glycopeptide antibiotics vancomycin and teicoplanin (targocid) are widely used in the treatment of infections caused by gram positive bacteria. Vancomyc inhibits both transglycosylation and transpeptidation reaction during peptidoglycan assembly, targocid inhibits peptidoglycan polymerization, resulting in inhibition of bacterial cell wall synthesis and cell death. In this study twenty strains of streptococcus pyogenes and streptococcus pneumoniae and twenty five strains of staphylococcus aureus were collected from different infections. MI Cs and MBCs of vancomycin and targocid were determined against the bacterial strains, the MICs of vancomycin for the streptococcus strains were 4-8 µg /ml compared with 8-16 µg/ml for targocid and MI Cs of targocid for staphylococcus aureus were 4-16 µg/ml MBC for the same strains were 8-32 µg/ml. the conclusion of this study was that targocid was less active than vancomycin against staphylococci but equal or more active against streptococci specifically it presents excellent in vitro activity against streptococcus pneumonia strains.

Combined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling.

Staphylococcus aureus sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is reported. Substrate specificities of three sortases have been investigated allowing identification of an orthogonal pair of enzymes accepting LPEToG and LPESoG depsipeptides, which have been applied to dual N-terminal labeling of a model protein mutant containing a second, latent N-terminal glycine residue. The method provides an efficient orthogonal site-specific labeling technique that further expands the biochemical protein labeling toolkit.

From Batch to Continuous Flow Bioprocessing: Use of an Immobilized γ-Glutamyl Transferase from B. subtilis for the Synthesis of Biologically Active Peptide Derivatives.

γ-Glutamyl-peptides are frequently endowed with biological activities. In this work, “kokumi peptides” such as γ-glutamyl-methionine (1) and γ-glutamyl-(S)-allyl-cysteine (2), as well as the neuroprotective γ-glutamyl-taurine (3) and the antioxidant ophthalmic acid (4), were synthesized through an enzymatic transpeptidation reaction catalyzed by the γ-glutamyl transferase from Bacillus subtilis (BsGGT) using glutamine as the γ-glutamyl donor. BsGGT was covalently immobilized on glyoxyl-agarose resulting in high protein immobilization yield and activity recovery (>95%). Compounds 1–4 were obtained in moderate yields (19–40%, 5–10 g/L) with a variable purity depending on the presence of the main byproduct (γ-glutamyl-glutamine, 0–16%). To achieve process intensification and better control of side reactions, the synthesis of 2 was moved from batch to continuous flow. The specific productivity was 1.5 times higher than that in batch synthesis (13.7 μmol/min*g), but it was not accompanied by a paralleled improvement of the impurity profile.

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine.

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.

Structural and Biophysical Analysis of the Phytochelatin-Synthase-Like Enzyme from Nostoc sp. Shows That Its Protease Activity is Sensitive to the Redox State of the Substrate.

Phytochelatins (PCs) are nonribosomal thiol-rich oligopeptides synthetized from glutathione (GSH) in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by PC synthases (PCSs). Ubiquitous in plant and present in some invertebrates, PCSs are involved in metal detoxification and homeostasis. The PCS-like enzyme from the cyanobacterium Nostoc sp. (NsPCS) is considered to be an evolutionary precursor enzyme of genuine PCSs because it shows sufficient sequence similarity for homology to the catalytic domain of the eukaryotic PCSs and shares the peptidase activity consisting in the deglycination of GSH. In this work, we investigate the catalytic mechanism of NsPCS by combining structural, spectroscopic, thermodynamic, and theoretical techniques. We report several crystal structures of NsPCS capturing different states of the catalyzed chemical reaction: (i) the structure of the wild-type enzyme (wt-NsPCS); (ii) the high-resolution structure of the γ-glutamyl-cysteine acyl-enzyme intermediate (acyl-NsPCS); and (iii) the structure of an inactive variant of NsPCS, with the catalytic cysteine mutated into serine (C70S-NsPCS). We characterize NsPCS as a relatively slow enzyme whose activity is sensitive to the redox state of the substrate. Namely, NsPCS is active with reduced glutathione (GSH), but is inhibited by oxidized glutathione (GSSG) because the cleavage product is not released from the enzyme. Our biophysical analysis led us to suggest that the biological function of NsPCS is being a part of a redox sensing system. In addition, we propose a mechanism how PCS-like enzymes may have evolved toward genuine PCS enzymes.

Enzymatic C‐to‐C Protein Ligation

AbstractTranspeptidase‐catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N‐to‐C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l‐/d‐ or α‐/β‐amino acid junctions, we find C‐terminal Leu‐ethylenediamine (Leu‐Eda) motifs to be bona fide mimetics of native N‐terminal Gly‐Leu sequences. Appending a C‐terminal Leu‐Eda to synthetic peptides or, via an intein‐splicing approach, to recombinant proteins enables direct transpeptidase‐catalyzed C‐to‐C ligations. This work significantly expands the synthetic scope of enzyme‐catalyzed protein transpeptidation reactions.

Enzymatic C-to-C Protein Ligation.

Transpeptidase‐catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N‐to‐C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l‐/d‐ or α‐/β‐amino acid junctions, we find C‐terminal Leu‐ethylenediamine (Leu‐Eda) motifs to be bona fide mimetics of native N‐terminal Gly‐Leu sequences. Appending a C‐terminal Leu‐Eda to synthetic peptides or, via an intein‐splicing approach, to recombinant proteins enables direct transpeptidase‐catalyzed C‐to‐C ligations. This work significantly expands the synthetic scope of enzyme‐catalyzed protein transpeptidation reactions.

Covalent coupling of Spike\u2019s receptor binding domain to a multimeric carrier produces a high immune response against SARS-CoV-2

The receptor binding domain (RBD) of the Spike protein from SARS-CoV-2 is a promising candidate to develop effective COVID-19 vaccines since it can induce potent neutralizing antibodies. We have previously reported the highly efficient production of RBD in Pichia pastoris, which is structurally similar to the same protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer with the purpose of increasing its immunogenicity. We produced multimeric particles by a transpeptidation reaction between RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric 170 kDa protein. Such particles were used to vaccinate mice with two doses 30 days apart. When the particles ratio of RBD to BLS units was high (6–7 RBD molecules per BLS decamer in average), the humoral immune response was significantly higher than that elicited by RBD alone or by RBD-BLS particles with a lower RBD to BLS ratio (1–2 RBD molecules per BLS decamer). Remarkably, multimeric particles with a high number of RBD copies elicited a high titer of neutralizing IgGs. These results indicate that multimeric particles composed of RBD covalent coupled to BLS possess an advantageous architecture for antigen presentation to the immune system, and therefore enhancing RBD immunogenicity. Thus, multimeric RBD-BLS particles are promising candidates for a protein-based vaccine.

Whole genome and RNA sequencing of oral commensal bacterium Streptococcus anginosus subsp. anginosus with vancomycin tolerance.

"Antibiotic tolerance" promotes the rapid subsequent evolution of "antibiotic resistance," however, it is often overlooked because it is difficult to distinguish between tolerant and susceptible organisms. A commensal bacterium S. anginosus subsp. anginosus strain KHUD_S1, isolated from dental biofilm was found to exhibit a high MBC/MIC ratio of 32 against vancomycin. We observed KHUD_S1 cells exposed to vancomycin did not grow but maintained viability. Transmission electron microscope showed KHUD_S1 cells possessed a dense, thick capsule and maintained the cell wall integrity upon vancomycin exposure. To infer the underlying mechanisms of the vancomycin tolerance in KHUD_S1, we performed whole genome sequencing and RNA sequencing. The KHUD_S1 genome carried three genes encoding branching enzymes that can affect peptidoglycan structure through interpeptide bridge formation. Global gene expression profiling revealed that the vancomycin-induced downregulation of carbohydrate and inorganic ion transport/metabolism as well as translation is less prominent in KHUD_S1 than in the vancomycin susceptible strain KHUD_S3. Based on the transcriptional levels of genes related to peptidoglycan synthesis, KHUD_S1 was determined to have a 3D peptidoglycan architecture distinct from KHUD_S3. It was found that, under vancomycin exposure, the peptidoglycan was remodeled through changes in the interpeptide bridge and transpeptidation reactions. Collectively, these features of S. anginosus KHUD_S1, including a dense capsule and differential gene expression in peptidoglycan synthesis, may contribute to vancomycin tolerance. Our results showing the occurrence of vancomycin tolerance amongst oral commensal bacteria highlight the need for considering future strategies for screening of antibiotic tolerance as an effort to reduce antibiotic resistance.

Asparaginyl Endopeptidase-Mediated Protein C-Terminal Hydrazinolysis for the Synthesis of Bioconjugates.

Asparaginyl endopeptidases (AEPs) are cysteinyl enzymes naturally catalyzing the hydrolysis and transpeptidation reactions at Asx-Xaa bonds. These reactions go by a common acyl-enzyme thioester intermediate, which is either attacked by water (for a protease-AEP) or by a peptidic amine nucleophile (for a ligase-AEP) to form the respective hydrolysis or aminolysis product. Herein, we show that hydrazine and hydroxylamine, two α-effect nucleophiles, are capable of resolving the thioester intermediate to yield peptide and protein products containing a C-terminal hydrazide and hydroxamic acid functionality, respectively. The hydrazinolysis reaction exhibits very high efficiency and can be completed in minutes at a low enzyme-to-substrate ratio. We further show the utility of the so-formed asparaginyl hydrazide in native chemical ligation and hydrazone conjugation. Using an EGFR-targeting affibody as a model protein, we have showcased our methodology in the preparation of a number of protein ligation or conjugation products, which are decorated with various functional moieties. The ZEGFR affibody-doxorubicin conjugate shows high selective binding and cytotoxicity toward the EGFR-positive A431 cells. Our results demonstrate the advantages of AEP-mediated protein hydrazinolysis as a simple and straightforward strategy for the precision manufacturing of protein bioconjugates.

Identification and Activity Characterization of γ-Glutamyltransferase from Bovine Milk.

It is well known that bovine milk contains γ-glutamyltransferase (GGT) activity. To verify the identity of the GGT and further to characterize the generation of γ-glutamyl peptides, identification of GGT from bovine milk and quantification of kokumi peptides and free amino acids were performed. GGT was purified from skim milk and identified as the bovine protein (G3N2D8), and it reveals that it is composed of two subunits. Sequence alignment with human GGT and molecular mass determination showed that the bovine GGT was glycosylated and contained an N-terminal transmembrane part. Further activity characterization was performed in comparison with GGT from Bacillus amyloliquefaciens in terms of the ability to generate γ-glutamyl peptides from casein hydrolysates. During the transpeptidation reaction catalyzed by both GGT, γ-glutamyl peptides significantly (P < 0.05) increased after γ-glutamylation; addition of glutamine contributed to the generation of γ-glutamyl peptides, suggesting that glutamine could act as a γ-glutamyl donor. This study reveals that the GGT of skim milk membranes is a glycosylated membrane protein that can generate γ-glutamyl peptides.

An overall framework for the E. coli γ-glutamyltransferase-catalyzed transpeptidation reactions

γ-Glutamyl derivatives of proteinogenic or modified amino acids raise considerable interest as flavor enhancers or biologically active compounds. However, their supply, on a large scale and at reasonable costs, remains challenging. Enzymatic synthesis has been recognized as a possible affordable alternative with respect to both isolation procedures from natural sources, burdened by low-yield and by the requirement of massive amount of starting material, and chemical synthesis, inconvenient because of the need of protection/deprotection steps. The E. coli γ-glutamyltransferase (Ec-GGT) has already been proposed as a biocatalyst for the synthesis of various γ-glutamyl derivatives. However, enzymatic syntheses using this enzyme usually provide the desired products in limited yield. Hydrolysis and autotranspeptidation of the donor substrate have been identified as the side reactions affecting the final yield of the catalytic process. In addition, experimental conditions need to be specifically adjusted for each acceptor substrate. Substrate specificity and the fine characterization of the activities exerted by the enzyme over time has so far escaped rationalization. In this work, reactions catalyzed by Ec-GGT between the γ-glutamyl donor glutamine and several representative acceptor amino acids have been finely analyzed with the identification of single reaction products over time. This approach allowed to rationalize the effect of donor/acceptor molar ratio on the outcome of the transpeptidation reaction and on the distribution of the different byproducts, inferring a general scheme for Ec-GGT-catalyzed reactions. The propensity to react of the different acceptor substrates is in agreement with recent findings obtained using model substrates and further supported by x-ray crystallography and will contribute to characterize the still elusive acceptor binding site of the enzyme.

Sequence variation in the β7–β8 loop of bacterial class A sortase enzymes alters substrate selectivity

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice to catalyze SML reactions. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this β7–β8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by β7–β8 loop residues that appear to be broadly conserved or converged upon in class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.

Enzymatic Synthesis of γ-Glutamylvalylglycine Using Bacterial γ-Glutamyltranspeptidase.

γ-Glutamylvalylglycine is known as a potent "kokumi" substance that increases the thickness, continuity, and mouthfulness of the taste of food. In this study, γ-glutamylvalylglycine was enzymatically synthesized from glutamine and valylglycine through the transpeptidation reaction of bacterial γ-glutamyltranspeptidase. The reaction conditions decided for the production of γ-glutamylvalylglycine are 20 mM glutamine, 100 mM valylglycine, 30 mU/mL γ-glutamyltranspeptidase, and 0.856 M (5%) NaCl at pH 8 and 37 °C, and 18.6 mM γ-glutamylvalylglycine was synthesized. Metal cations, Na+ and Mg2+, improved the transpeptidation reaction of γ-glutamyltranspeptidase, and the production of γ-glutamylvalylglycine increased. Moreover, by feeding a suitable amount of glutamine every 5 h, the maximum production of γ-glutamylvalylglycine increased 1.7 times after 20 h of incubation.

Enzymatic Synthesis of Putative Mycobacterial L,D‐Transpeptidase Substrates

The Mycobacterium genus is diverse, with members ranging from harmless water or soil dwellers to the causative agent of the deadly disease tuberculosis. Bacterial cell wall biosynthetic enzymes are therapeutic targets, and mycobacterial cell walls are uniquely complex. The peptidoglycan (PG) layer of mycobacterial cell walls is largely and uniquely 3-3 crosslinked, and L,D-transpeptidase (Ldt) enzymes catalyze this transpeptidation reaction. Our lab has previously demonstrated that there are six mycobacterial Ldt classes, and the reason for this apparent redundancy is not well understood. The Mycobacterium tuberculosis (Mtb) class 2 Ldt, LdtMt2, is the most well studied, whereas the physiological roles of the remaining paralogs are largely unknown. The PG tetrapeptide L-Ala-D-isoGln-mesoDAPNH2-D-Ala is a known substrate for four of the five Mtb Ldts. While all mycobacterial species have at least five Ldt paralogs, only the Mtb enzymes LdtMt1, LdtMt2, LdtMt4, and LdtMt5 enzymes have confirmed 3-3 crosslinking activity. LdtMt3 does not crosslink this tetrapeptide substrate, and the transpeptidation capability of class 6 Ldts – of which there is no homolog in Mtb – have not been investigated. Orthologous Ldts are presumed to have the same activities, although this has not been biochemically confirmed, presumably because of limited access to putative substrates. Tetrapeptide (and many pentapeptide) PG substrates are not commercially available; accessing them is challenging because they contain unusual amino acids (D-isoGlu, D-isoGln, D-Ala, meso-diaminopimelic acid (mesoDAP), and amidated mesoDAP), and they are linked via the γ-carboxyl group of D-isoGln. Thus, even solid-support peptide synthesis is a challenge due to the requirement of non-commercial protected amino acids. LdtMt2 requires a fully amidated tetrapeptide substrate, yet 50% of the 3-3 crosslinked PG isolated from Mtb lacks amidation at the mesoDAP ε-carboxyl moiety of the donor stem peptide, suggesting at least one Mtb Ldt prefers a non-amidated donor substrate (or that amino groups are removed in subsequent biosynthetic steps). Further, some mycobacterial species, including Mycobacterium smegmatis, can utilize tetrapeptides with L-Lys in the third position. Thus, while the role of LdtMt2 in Mtb is known, the functions of the remaining paralogs and orthologous Ldts remain to be determined, highlighting the need for substrate specificity studies on these enzymes toward understanding their physiological roles. Here, we present our preliminary work in cloning and overexpressing PG biosynthetic enzymes toward characterizing the substrate preferences of mycobacterial Ldt enzymes. We are actively pursuing enzymatic synthesis of putative PG substrates and will be evaluating purified tetra- and pentapeptide substrates against the six classes of mycobacterial Ldt enzymes. The results from this study are expected to inform the community as to the physiological roles of the under studied Ldt paralogs.

Plant asparaginyl endopeptidases and their structural determinants of function.

Asparaginyl endopeptidases (AEPs) are versatile enzymes that in biological systems are involved in producing three different catalytic outcomes for proteins, namely (i) routine cleavage by bond hydrolysis, (ii) peptide maturation, including macrocyclisation by a cleavage-coupled intramolecular transpeptidation and (iii) circular permutation involving separate cleavage and transpeptidation reactions resulting in a major reshuffling of protein sequence. AEPs differ in their preference for cleavage or transpeptidation reactions, catalytic efficiency, and preference for asparagine or aspartate target residues. We look at structural analyses of various AEPs that have laid the groundwork for identifying important determinants of AEP function in recent years, with much of the research impetus arising from the potential biotechnological and pharmaceutical applications.