Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity

Gerasymenko, I. M.; Sheludko, Y. V.; Navarro, Ismael; Schmidts, Volker; Steinel, Lara; Haumann, Elisabeth; Warzecha, Heribert

doi:10.1002/cbic.202100465

Cited by 6 publications

(4 citation statements)

References 39 publications

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“…Recently, Warzecha et al. engineered NDPS1 to increase its promiscuity by substituting Asn88 with His [22] . As the recombinant NDPS1 in this report was expressed from Leu56, the first Cys residue at position 54 of the NTR did not exist; thus, the inter‐subunit S−S bond could not be formed.…”

Section: Resultsmentioning

confidence: 89%

“…Recently, Warzecha et al engineered NDPS1 to increase its promiscuity by substituting Asn88 with His. [22] As the recombinant NDPS1 in this report was expressed from Leu56, the first Cys residue at position 54 of the NTR did not exist; thus, the inter-subunit SÀ S bond could not be formed. The Asn88His mutant exhibited increased amounts of irregular condensation, including a head-to-middle reaction between two DMAPP molecules, although the disulfide bond formation in the recombinant NDPS1 was not confirmed.…”

Section: Complex Structures Of Trndps1 With Ligands Revealed Heteroge...mentioning

confidence: 88%

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Structural‐Functional Correlations between Unique N‐terminal Region and C‐terminal Conserved Motif in Short‐chain cis‐Prenyltransferase from Tomato

Imaizumi,

Matsuura,

Yanai

et al. 2024

ChemBioChem

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Neryl diphosphate (C10) synthase (NDPS1), a homodimeric soluble cis‐prenyltransferase from tomato, contains four disulfide bonds, including two inter‐subunit S–S bonds in the N‐terminal region. Mutagenesis studies demonstrated that the S–S bond formation affects not only the stability of the dimer but also the catalytic efficiency of NDPS1. Structural polymorphs in the crystal structures of NDPS1 complexed with its substrate and substrate analog were identified by employing massive data collections and hierarchical clustering analysis. Heterogeneity of the C‐terminal region, including the conserved RXG motifs, was observed in addition to the polymorphs of the binding mode of the ligands. One of the RXG motifs covers the active site with an elongated random coil when the ligands are well‐ordered. Conversely, the other RXG motif was located away from the active site with a helical structure. The heterogeneous C‐terminal regions suggest alternating structural transitions of the RXG motifs that result in closed and open states of the active sites. Site‐directed mutagenesis studies demonstrated that the conserved glycine residue cannot be replaced. We propose that the putative structural transitions of the order/disorder of N‐terminal regions and the closed/open states of C‐terminal regions may cooperate and be important for the catalytic mechanism of NDPS1.

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Section: Resultsmentioning

confidence: 89%

Section: Complex Structures Of Trndps1 With Ligands Revealed Heteroge...mentioning

confidence: 88%

Structural‐Functional Correlations between Unique N‐terminal Region and C‐terminal Conserved Motif in Short‐chain cis‐Prenyltransferase from Tomato

Imaizumi,

Matsuura,

Yanai

et al. 2024

ChemBioChem

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“…The identification of the P. citri IDS gene family provides an important foundation for deciphering terpenoid biosynthesis in this species as well as other mealybugs. The candidate FPPS and FPPS-like sequences with confirmed regular and irregular activity might also present a starting point for protein engineering; mutations have been shown to increase or even spur irregular coupling activity in IDSs (Chan et al, 2017; Gerasymenko et al, 2022). Diversification of IDS sequences in Coccoidea is an intriguing genomic enigma for future investigation, especially in relation to their capacity for species-specific irregular monoterpene synthesis, a unique feature in this insect superfamily, which still remains elusive.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Identification and characterisation ofPlanococcus citri cis- andtrans-isoprenyl diphosphate synthase genes, supported by short- and long-read transcriptome data

Juteršek

Gerasymenko

Petek

et al. 2023

Preprint

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Many insect species rely on diverse terpenoids for their development and interorganismal interactions. However, little is known about terpenoid biosynthesis in insects. The monoterpenoid sex pheromones of mealybugs and scale insects (Coccoidea) are particularly enigmatic, with several species producing unique structures presumed to result from the irregular coupling activity of unidentified isopentenyl diphosphate synthases (IDSs). Enzymes capable of similar transformations have previously only been described from a few plant, bacterial and archaeal species. To investigate if insect irregular monoterpenes can be biosynthesised by similar enzymes, we performed a comprehensive search for IDS coding sequences in the genome ofPlanococcus citri, a widespread agricultural pest. We complemented the availableP. citrigenome data with newly generated short- and long-read transcriptome data. The identified candidate genes had homology to both short- and long-chain IDSs and some appeared to be paralogous, indicating gene duplications and consequent IDS gene family expansion inP. citri. We tested the activity of eleven candidate gene products, confirming in vitro regular activity for five enzymes, one of which (transIDS5) also produced the irregular prenyl diphosphates, maconelliyl and lavandulyl diphosphate. Targeted mutagenesis of selected aspartates and a lysine in the active site oftransIDS5 uncovered their importance for chain length preference and irregular coupling. This work provides an important foundation for deciphering terpenoid biosynthesis in mealybugs, as well as a potential source of enzymes for the biotechnological production of sustainable insect pest management products.

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