Functional diversification of Viola arcuata protein disulfide isomerases and their roles in cyclotide oxidative...

Cyclotides are ubiquitously expressed in the Violaceae family, where their hallmark cyclic cystine knot (CCK) structure-stabilized by three disulfide bonds-confers exceptional chemical and biological stability. In plants, disulfide bond formation and isomerization are predominantly catalyzed by protein disulfide isomerases (PDIs), yet their specific roles in cyclotide folding remain unclear. Here, we identified five PDI isoforms (VaPDI1-VaPDI5) from Viola arcuata and compared their catalytic properties with that of Oldenlandia affinis PDI (OaPDI). Oxidase and isomerase activities were quantified using the reduced and denatured RNase A refolding assay, revealing that VaPDI3, VaPDI4, and VaPDI5 possess strong isomerase activity comparable to OaPDI, whereas VaPDI1 and VaPDI2 exhibit a predominant oxidative bias. Using reduced cyclic Viar C, a member of the Möbius family of cyclotides, as a model substrate, we found that all VaPDIs markedly promoted the formation of correctly folded peptides under near-physiological redox conditions, and the folding yield was strongly dependent on the redox environment. Molecular dynamics simulations and structural modeling suggested that conserved phenylalanine and tyrosine residues in the b' domain of VaPDIs may contribute to substrate engagement during cyclotide folding. Overall, this study reveals functional divergence among V. arcuata PDI isoforms and provides insight into their possible involvement in the oxidative folding of cyclotides, based on analyses using Viar C as a model substrate.