The cyclotides are the largest family of naturally occurring circular proteins. The mechanism by which the termini of these gene-encoded proteins are linked seamlessly with a peptide bond to form a circular backbone is unknown. Here we report cyclotide-encoding cDNA sequences from the plant Viola odorata and compare them with those from an evolutionarily distinct species, Oldenlandia affinis. Individual members of this multigene family encode one to three mature cyclotide domains. These domains are preceded by N-terminal repeat regions (NTRs) that are conserved within a plant species but not between species. We have structurally characterized peptides corresponding to these NTRs and show that, despite them having no sequence homology, they form a structurally conserved ␣-helical motif. This structural conservation suggests a vital role for the NTR in the in vivo folding, processing, or detoxification of cyclotide domains from the precursor protein.

We have isolated a protein-disulfide isomerase (PDI) from Oldenlandia affinis (OaPDI), a coffee family (Rubiaceae) plant that accumulates knotted circular proteins called cyclotides. The novel plant PDI appears to be involved in the biosynthesis of cyclotides, since it co-expresses and interacts with the cyclotide precursor protein Oak1. OaPDI exhibits similar isomerase activity but greater chaperone activity than human PDI. Since domain c of OaPDI is predicted to have a neutral pI, we conclude that this domain does not have to be acidic in nature for PDI to be a functional chaperone. Its redox potential of ؊157 ؎ 4 mV supports a role as a functional oxidoreductase in the plant. The mechanism of enzyme-assisted folding of plant cyclotides was investigated by comparing the folding of kalata B1 derivatives in the presence and absence of OaPDI. OaPDI dramatically enhanced the correct oxidative folding of kalata B1 at physiological pH. A detailed investigation of folding intermediates suggested that disulfide isomerization is an important role of the new plant PDI and is an essential step in the production of insecticidal cyclotides.Protein-disulfide isomerase (PDI 3 ; EC 5.3.4.1) is an oxidoreductase enzyme that belongs to the thioredoxin superfamily (1). It has a major role in oxidative folding of polypeptides in the endoplasmic reticulum (ER) of eukaryotic cells and functions as an ER chaperone (2). The exact mechanism of action of PDI is not clear, but it is believed to bind polypeptides through hydrophobic interactions and forms (oxidizes), breaks (reduces), and/or shuffles (isomerizes) disulfide bonds in substrate molecules via a dithiol-disulfide exchange between its active-site CXXC motif and the substrate polypeptide (3).Cyclotides are small disulfide-rich peptides found in plants of the coffee (Rubiaceae) and violet (Violaceae) families (4). They are typically about 30 amino acids in length and have the unique structural features of a cyclic backbone and a knotted arrangement of three-disulfide bonds, referred to as the cyclic cystine knot motif (5). Their compact cyclic cystine knot motif makes them exceptionally resistant to thermal, chemical, or enzymatic degradation (6). Cyclotides exhibit a range of biological activities, including anti-bacterial, cytotoxic, and anti-human immunodeficiency virus activities (7), but their natural function is as plant defense molecules (8, 9). Kalata B1, from the Rubiaceae species Oldenlandia affinis, was the first cyclotide discovered (10), although its macrocyclic structure was not delineated until 1995 (11). So far, the sequences of nearly 100 cyclotides have been reported, and it has been suggested that they may surpass the well known plant defensins in number and diversity (12, 13). Their unique structural framework, range of bioactivities, and sequence diversity make them interesting targets for pharmaceutical applications (14).Cyclotides have a characteristic surface-exposed patch of hydrophobic residues that accounts for their late elution on reverse-phase HPLC ...

Despite the use of chemical fungicides, fungal diseases have a major impact on the yield and quality of plant produce globally and hence there is a need for new approaches for disease control. Several groups have examined the potential use of antifungal plant defensins for plant protection and have produced transgenic plants expressing plant defensins with enhanced resistance to fungal disease. However, before they can be developed commercially, transgenic plants must pass a series of strict regulations to ensure that they are safe for human and animal consumption as well as the environment. One of the requirements is rapid digestion of the transgene protein in the gastrointestinal tract to minimize the risk of any potential allergic response. Here, we examine the digestibility of two plant defensins, NaD1 from Nicotiana alata and SBI6 from soybean, which have potent antifungal activity against major cereal pathogens. The native defensins were not digestible in simulated gastrointestinal fluid assays. Several modifications to the sequences enhanced the digestibility of the two small proteins without severely impacting their antifungal activity. However, these modified proteins did not accumulate as well as the native proteins when transiently expressed in planta, suggesting that the proteaseresistant structure of plant defensins facilitates their stability in planta.