Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

Czekster, Clarissa Melo; Ludewig, Hannes; McMahon, S.A.; Naismith, J.H.

doi:10.1038/s41467-017-00862-4

Cited by 36 publications

(46 citation statements)

References 57 publications

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“…Notably, the enzyme plays its bifunctional role in the same in vitro condition resulting in an expected cyclic product when incubated with DIN-MCoTI-II-DAL. This bifunctional role of MCoAEP2 bears some similarity to that described for GmPOPB from the autumn skullcap mushroom (Galerina marginata), which can perform N-terminal cleavage and subsequent cyclization steps to produce cyclic amatoxins 48,49 . Interestingly, we observed a mis-cyclized DIN-MCoTI-II that resulted from premature cyclization of the precursor prior to release of the leader peptide.…”

Section: Discussionmentioning

confidence: 56%

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

et al. 2020

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Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a k cat /K m of 620 mM −1 s −1 , making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.

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Section: Discussionmentioning

confidence: 56%

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

et al. 2020

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“…The core sequence of the macrocyclization substrate was not experimentally observed in GmPOPB complexes (PDB codes 5N4B and 5N4D). 24…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates

et al. 2018

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Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.

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“…Residue X acid+2 , one of the conserved structural elements that is involved in the coordination of the imidazole ring of the catalytic histidine, is usually a hydrophobic or an aromatic residue located at the entrance of the active site cleft, mostly reviewed for its role in ligand binding [51][52][53][54][55][56] or the release of products of catalysis [51,57]. Consequently, the site-directed mutagenesis of residue X acid+2 in different ABH enzymes has led to various results, which are consistent with its role in ligand binding, including the compromising [52,54,55,58,59] or the enhancement [52] of catalytic activity, the alteration of transport tunnels [57,60], the modification of substrate specificity [53,55,56] and the inversion of enantioselectivity [52]. For the remaining residues that coordinate the imidazole ring of the catalytic histidine, including residue X acid+3 , we have only found a few mutational studies that have resulted in reduced activity [58,61,62], with a single study highlighting the residue's role in the stability of the enzyme and the catalytic activity rather than in ligand binding [58].…”

Section: The Conserved Structural Elements That Line the Catalytic Stmentioning

confidence: 99%

The acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements

et al. 2020

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The alpha/beta-Hydrolases (ABH) are a structural class of proteins that are found widespread in nature and includes enzymes that can catalyze various reactions in different substrates. The catalytic versatility of the ABH fold enzymes, which has been a valuable property in protein engineering applications, is based on a similar acid-base-nucleophile catalytic mechanism. In our research, we are concerned with the structure that surrounds the key units of the catalytic machinery, and we have previously found conserved structural organizations that coordinate the catalytic acid, the catalytic nucleophile and the residues of the oxyanion hole. Here, we explore the architecture that surrounds the catalytic histidine at the active sites of enzymes from 40 ABH fold families, where we have identified six conserved interactions that coordinate the catalytic histidine next to the catalytic acid and the catalytic nucleophile. Specifically, the catalytic nucleophile is coordinated next to the catalytic histidine by two weak hydrogen bonds, while the catalytic acid is directly involved in the coordination of the catalytic histidine through by two weak hydrogen bonds. The imidazole ring of the catalytic histidine is coordinated by a CH-π contact and a hydrophobic interaction. Moreover, the catalytic triad residues are connected with a residue that is located at the core of the active site of ABH fold, which is suggested to be the fourth member of a "structural catalytic tetrad". Besides their role in the stability of the catalytic mechanism, the conserved elements of the catalytic site are actively involved in ligand binding and affect other properties of the catalytic activity, such as substrate specificity, enantioselectivity, pH optimum and thermostability of ABH fold enzymes. These properties are regularly targeted in protein engineering applications, and thus, the identified conserved structural elements can serve as potential modification sites in order to develop ABH fold enzymes with altered activities.

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Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

Cited by 36 publications

References 57 publications

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates

The acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements

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