Butelase-1 is a peptide asparaginyl ligase (PAL) that efficiently catalyzes peptide bond formation after Asn/Asp (Asx), whereas its homologue butelase-2 is an asparaginyl endopeptidase (AEP) that ...catalyzes the reverse reaction, hydrolyzing Asx-peptide bonds. Since PALs and AEPs share essentially similar overall structures, we surmised that the S2 and S1′ substrate-binding pockets immediately flanking the catalytic S1 site constitute the major ligase activity determinants (LADs) that control the catalytic directionality of these enzymes. Here, we report the successful conversion of butelase-2 into butelase-1-like ligases based on the LAD hypothesis. We prepared 23 LAD mutants by mutating residues of the S2 pocket (LAD1) and/or the S1′ pocket (LAD2) of butelase-2 into homologous residues in PALs. These LAD mutants markedly diminished protease activity and increased ligase activity. In contrast, substituting 12 non-LAD residues to the corresponding residues in butelase-1 did not change their protease profiles. At physiological pH, mutations targeting both LADs resulted in shifting the catalytic directionality from 95% hydrolysis to >95% peptide ligation. This results in the engineering of efficient recombinant peptide ligases that were demonstrated to be useful for macrocyclization and site-specific labeling of bioactive peptides and proteins. Five high-resolution crystal structures of butelase-2 and its engineered mutants reveal subtle changes proximal to the catalytic S1 site that account for the reversal in enzymatic activity. Computational simulations of enzyme–substrate complexes suggest how the S-acyl intermediate is positioned at the S2 site and the substrate orientated, controlling accessibility of either water or incoming nucleophiles from the prime-side (S1′ site) of the catalytic center. Together, these features determine the catalytic directionality between hydrolysis and ligation in AEPs and PALs. Overall, this work validates the LAD hypothesis as a central guide for making peptide ligases from their corresponding widely available protease counterparts, to produce precision tools for exquisite site-specific conjugation and the biomanufacturing of biologics.
Butelase-1 is an efficient ligase from Clitoria ternatea with wide applications in the food and biopharmaceutical fields. This research aimed to achieve high-efficiency expression of butelase-1 and ...explore its application in food-derived angiotensin I-converting enzyme (ACE) inhibitory peptides. The recombinant butelase-1 zymogen was prepared at a yield of 100 mg/L in Escherichia coli and successfully activated at pH 4.5, resulting in a 6973.8 U/L yield of activated butelase-1 with a specific activity of 348.69 U/mg and a catalytic efficiency of 9956 M–1 s–1. Activated butelase-1 exhibited considerable resistance to Tween-20, Triton X-100, and methanol. The “traceless” cyclization of ACE inhibitory peptides was realized using activated butelase-1, which resulted in higher stability and ACE inhibitory activity than those of the linear peptides. Our work proposed an efficient method for the preparation of butelase-1 and provided a promising model for its application in food fields.
Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In ...particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.
Background: Sortase A (SrtA) is a transpeptidase capable of catalyzing the formation of amide bonds.
Results: SrtA was used to backbone-cyclize disulfide-rich peptides, including kalata B1, α-conotoxin Vc1.1, and SFTI-1.
Conclusion: SrtA-mediated cyclization is applicable to small disulfide-rich peptides.
Significance: SrtA-mediated cyclization is an alternative to native chemical ligation for the cyclization of small peptides of therapeutic interest.
The sheer size and vast chemical space (i.e., diverse repertoire and spatial distribution of functional groups) underlie peptides' ability to engage in specific interactions with targets of various ...structures. However, the inherent flexibility of the peptide chain negatively affects binding affinity and metabolic stability, thereby severely limiting the use of peptides as medicines. Imposing conformational constraints to the peptide chain offers to solve these problems but typically requires laborious structure optimization. Alternatively, libraries of constrained peptides with randomized modules can be screened for specific functions. Here, we present the properties of conformationally constrained peptides and review rigidification chemistries/strategies, as well as synthetic and enzymatic methods of producing macrocyclic peptides. Furthermore, we discuss the in vitro molecular evolution methods for the development of constrained peptides with pre-defined functions. Finally, we briefly present applications of selected constrained peptides to illustrate their exceptional properties as drug candidates, molecular recognition probes, and minimalist catalysts.
Flexizymes, highly flexible tRNA aminoacylation ribozymes, have enabled charging of virtually any amino acid (including non-proteogenic ones) onto tRNA molecules. Coupling to a custom-made in vitro ...translation system, namely the flexible in vitro translation (FIT) system, has unveiled the remarkable tolerance of the ribosome toward molecules, remote from what nature has selected to carry out its elaborate functions. Among the very diverse molecules and chemistries that have been ribosomally incorporated, a plethora of entities capable of mediating intramolecular cyclization have revolutionized the design and discovery of macrocyclic peptides. These macrocyclization reactions (which can be spontaneous, chemical, or enzymatic) have all served as tools for the discovery of peptides with natural-like structures and properties. Coupling of the FIT system and mRNA display techniques, known as the random non-standard peptide integrated discovery (RaPID) system, has in turn allowed for the simultaneous screening of trillions of macrocyclic peptides against challenging biological targets. The macrocyclization methodologies are chosen depending on the structural and functional characteristics of the desired molecule. Thus, they can emanate from the peptide's N-terminus or its side chains, attributing flexibility or rigidity, or even result in the installation of fluorescent probes.
Cyclic oligomers of hexamethylene furanoate and hexamethylene terephthalate were obtained from 1,6-hexanediol and the corresponding methyl esters using Candida antarctica lipase B (CALB) enzyme ...catalyst. HPLC, MALDI-TOF MS, and NMR analyses showed that mixtures composed from cyclic dimer up to heptamer were obtained almost quantitatively. Subsequently, these cycles were polymerized by ring opening polymerization (ROP) mediated by CALB to obtain semicrystalline polymers. In addition, we demonstrated that the polymers obtained from the ROP process could be transformed into cyclic oligomers in high yields using enzymatic cyclodepolymerization, thereby recycling the polymer in a circular biosynthetic path.
Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions ...of (+)-germacrene A (7) with relatively minor changes in k cat and k cat/K m. By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg2+-binding aspartate-rich domain, 80DDLFD, resulted in reduction in the k cat/K m for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a k cat/K m which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product β-caryophyllene (9) (9%). Finally, the F77Y mutant had a k cat/K m which was reduced by 20-fold compared to that of the wild-type synthase.
The crude enzyme (PH-1) isolated from Pseudostellaria heterophylla by our group has catalyzed enzymatic cyclization of linear peptide NH2-Gly1-Gly2-Leu-Pro-Pro-Pro-Ile-Phe-COOH (4) into cyclopeptide ...heterophyllin B (HB) from plant for the first time. To ensure this reaction, some analytical methods including TLC, HPLC, MS, NMR, and 3C labeling were used to prove that the reaction of substrate 4 sharing residue of NH-Phe-Gly-CO was successful.
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