Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

Abstract

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.

Cyclotide discovery in Gentianales revisited—identification and characterization of cyclic cystine‐knot peptides and their phylogenetic distribution in Rubiaceae plants

“…To further evaluate the importance of specific amino acid residues at the processing sites of Rubiaceae cyclotide precursor proteins, we performed an alignment of 25 precursors, including those from Oldenlandia affinis , Chassalia chartacea , Hedyotis biflora , and those newly identified in Carapichea ipecacuanha (Figures C and D). As recently shown for Oldenlandia affinis a C‐terminal asparagine within the cyclotide domain as well as an adjacent small amino acid residue followed by leucine/isoleucine is crucial for correct bioprocessing, in particular cyclization . Analysis of the residues preceding the proto‐N‐terminus of the cyclotide domain revealed a conserved asparagine/aspartic acid in the precursor sequences of Carapichea ipecacuanha .…”

“…However, Oldenlandia affinis and Hedyotis biflora contain a highly conserved lysine residue at this position and only precursors for Oak 7, 8, and 9 contain different residues, namely a threonine (Oak 8) or a glycine (Oak 7 and 9) (Figure D). Overall this variation supports the hypothesis that the residue immediate preceding the conserved N‐terminal residues, e.g., glycine and leucine/isoleucine is not crucial for cyclization as has been suggested previously . On the other hand, the first residues of the C‐tail are presumably vital for cyclization and are usually conserved across species .…”

Joseph Rudinger memorial lecture: Discovery and applications of cyclotides

“…Cyclotides have been extensively reviewed over recent years by ourselves [2][3][4][5][6][7][8][9][10][11] and other groups [12][13][14][15][16][17][18][19][20], so I have tried to make this article complementary to these technical articles by using a more conversational style and by including background stories not already published in other reviews.…”

Macrocyclization by asparaginyl endopeptidases

“…The authors demonstrated that the macrocyclization was dependent on a C‐terminal Asn‐Gly‐Leu AEP recognition motif and removal of the Leu prevented cyclization. This supports previous findings investigating kalata B1 cyclization showing the importance of the C‐terminal tail residues for cyclization through mutagenesis (Gillon et al ., ; Conlan et al ., ).…”