TcGLIP GDSL Lipase Substrate Specificity Co-determines the Pyrethrin Composition in Tanacetum cinerariifolium

Abstract

Natural pesticides pyrethrins biosynthesized by Tanacetum cinrerariifolium are biodegradable and safer insecticides for pest insect control. TcGLIP, a GDSL lipase underpinning the ester bond formation in pyrethrins, exhibits high stereo-specificity for acyl-CoA and alcohol substrates. However, it is unknown how the enzyme recognizes the other structural features of the substrates and whether such specificity affects the product amount and composition in T. cinrerariifolium. We report here that the cysteamine moiety in (1R,3R)-chrysanthemoyl CoA and the conjugated diene moiety in (S)-pyrethrolone play key roles in the interactions with TcGLIP. CoA released from chrysanthemoyl CoA in the pyrethrin-forming reaction reduces the substrate affinity for TcGLIP by feedback inhibition. (S)-Pyrethrolone shows the highest catalytic efficiency for TcGLIP, followed by (S)-cinerolone and (S)-jasmololone, contributing, at least in part, to determine the pyrethrin compositions in T. cinerariifolium.

Creating Pyrethrin Mimetic Phosphonates as Chemical Genetics Tools Targeting the GDSL Esterase/Lipase TcGLIP to Investigate Pyrethrin Biosynthesis

“…The model revealed that the C5 alkyl chain of 5a could interact with Pro211 located inside the binding site, while the alkyl chain of 8b can escape from such steric interaction, accounting at least in part for the different structure–activity relationships for the ( S ) p ,( S ) c , and ( R ) p ,( S ) c compounds. On the other hand, the conjugated diene in the side chain of the pyrethrolonyl group underwent π–π and hydrophobic interactions with the aromatic residues (Phe56, Tyr297, and Phe320), in support of higher affinity of ( S )-pyrethrolone than ( S )-jasmololone lacking such moiety for the enzyme …”

“…Recombinant TcGLIP was prepared as previously described. ,, TcGLIP was expressed functionally as the fusion protein with maltose binding protein using the pMAL-c4E vector (New England Biolabs, USA) in the Origami B strain of Escherichia coli (Merck Millipore, USA) with 0.1 mM isopropyl-β- d -thiogalactopyranoside as an inducer. The recombinant TcGLIP was purified with amylose resin (New England Biolabs) and a Mono Q column (Cytiva, USA) and dissolved in 50 mM Tris-HCl buffer (pH 7.5).…”

“…The reaction was stopped by acetic acid and extracted with an equal volume of n -hexane. Pyrethrin I concentrations in the extract were determined in terms of UV absorption at 230 nm by HPLC using a Cadenza CD-C18 column with an 80% MeCN–20% water mixture at a flow rate of 1.0 mL min –1 as previously described …”

“…Pyrethrins are esters of mono-terpene chrysanthemic or pyrethric acid with octadecanoid pathway products, the rethrolone alcohols (pyrethrolone, cinerolone, and jasmololone), resulting in the six pyrethrin members (Figure A). − To date, both the involvement of chrysanthemyl diphosphate synthase in cyclopropane ring formation and enzymes that convert chrysanthemic acid to pyrethric acid and facilitate rethrolone biosynthesis are identified. , In addition, we discovered a GELP called “TcGLIP” that catalyzes ester bond formation during the final pyrethrin biosynthesis step . GELPs are a large family of enzymes that facilitate homeostasis in microorganisms, plants, insects, and vertebrates. − Notably, >100 GELPs are expressed in Oryza sativa and Arabidopsis thaliana, yet many functions of GELPs remain unknown. , TcGLIP is the only GELP for which the native substrates are known to function as a defense against herbivores, with the substrate structure–activity relationship studied in detail. ,, TcGLIP undergoes feedback control by coenzyme A (CoA) released in the pyrethrin I ( 1 )-forming reaction of (1 R ,3 R )-chrysanthemoyl CoA and ( S )-pyrethrolone . The TcGLIP gene is expressed dominantly in flowers and leaves but much less in roots, accounting for pyrethrin content variations in these tissues .…”

TcMYB8, a R3-MYB Transcription Factor, Positively Regulates Pyrethrin Biosynthesis in Tanacetum cinerariifolium

“…GLIP functions at the last step of pyrethrin biosynthesis [ 18 ]. Our previous research reported that the binding affinity between the TcGLIP enzyme and substrate directly affects the pyrethrin content [ 20 ]. These findings suggest that CHS and GLIP are key enzymes in the pyrethrum biosynthesis pathway.…”