SummaryPlant cyclotides are a large family of naturally occurring circular proteins that are produced from linear precursors containing one, two or three cyclotide domains. The mechanism of excision of the cyclotide domains and ligation of the free N-and C-termini to produce the circular peptides has not been elucidated. Here, we investigate production of the prototypic cyclotide kalata B1 from the precursor Oak1 from the African plant Oldenlandia affinis. Immunoprecipitation experiments and MALDI-TOF mass spectrometry analysis showed that O. affinis only produces mature kalata B1, whereas transgenic Arabidopsis thaliana, Nicotiana tabacum and Nicotiana benthamiana produced both linear and circular forms. Circular peptides were not produced when a highly conserved asparagine residue at the C-terminal processing site of the cyclotide domain was replaced with an alanine or an aspartate residue, or when the conserved C-terminal tripeptide motif was truncated. We propose that there are two processing pathways in planta: one to produce the mature cyclotide and the other to produce linear variants that ultimately cannot be cyclized.

Asparaginyl endopeptidases (AEPs) are proteases that have crucial roles in plant defense and seed storage protein maturation. Select plant AEPs, however, do not function as proteases but as transpeptidases (ligases) catalyzing the intra-molecular ligation of peptide termini, which leads to peptide cyclization. These ligase-type AEPs have potential biotechnological applications ranging from in vitro peptide engineering to plant molecular farming, but the structural features enabling these enzymes to catalyze peptide ligation/cyclization rather than proteolysis are currently unknown. Here, we compare the sequences, structures, and functions of diverse plant AEPs by combining molecular modeling, sequence space analysis, and functional testing in planta. We find that changes within the substrate-binding pocket and an adjacent loop, here named the “marker of ligase activity”, together play a key role for AEP ligase efficiency. Identification of these structural determinants may facilitate the discovery of more ligase-type AEPs and the engineering of AEPs with tailored catalytic properties.

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These “ligase-type” AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1 b . These AEPs work preferentially as ligases at both acidic and neutral pH and we term them “canonical AEP ligases” to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

Background: Production of insecticidal and nematocidal cyclic peptides is inefficient in transgenic plants. Results: Efficient cyclization requires cleavage of the N-terminal propeptide from the mature cyclotide domain and a C terminus containing a binding motif. Conclusion:The cyclization motif has Asn at position P1, a small amino acid at position P1Ј, and a Leu at position P2Ј. Significance: Understanding substrate requirements will help produce cyclotides in transgenic plants.

Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPIsusceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe→Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.chymotrypsin mutants | inhibitor-resistant proteinase | Lepidoptera S erine peptidases from the chymotrypsin family are a large group of enzymes and, although they have a highly conserved tertiary structural fold, they have developed a range of substrate specificities critical to many biological functions, including blood coagulation and immune responses (1, 2).Chymotrypsin and trypsin are the major digestive serine proteases of insects from the order Lepidoptera, which comprises some of the most important agricultural pests. When ingested, protease inhibitors (PI) block protease activity and increase insect mortality by restricting the availability of essential amino acids. Mechanisms of insect resistance to PIs include the upregulation of enzymes that degrade the PIs (3, 4), induction of enzymes that resist inactivation by PIs (5, 6), and overproduction of enzymes to maintain normal levels of gut proteolysis (7,8).As part of a strategy to develop novel insecticidal agents, we investigated the properties of a series of 6-kDa chymotrypsin and trypsin inhibitors (C1, C2, T1-T4) from Nicotiana alata (NaPI), which are members of the potato type II family of inhibitors (pin II). In our companion paper (9), we report that larvae from the major agricultural insect pest Helicoverpa punctigera that survive consumption of NaPI have high levels of an NaPI-resistant chymotrypsin. We discovered that a potato type I inhibitor (StPin1A) abolished the NaPI-resistant chymotrypsin activity and that the combination of these two PIs in artificial diets substantially stunted the growth of another agronomically important pest, H. armigera (9). Furthermore, field-grown transgenic cotton expressing both NaPI and StPin1A showed greater insect protection over the growing season than plants expressing a single inhibitor. In the current stud...

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