On the substrate specificity of the rice strigolactone biosynthesis enzyme DWARF27

Abstract

The β-carotene isomerase OsDWARF27 is stereo- and double bond-specific. It converts bicyclic carotenoids with at least one unsubstituted β-ionone ring. OsDWARF27 may contribute to the formation of α-carotene-based strigolactone-like compounds. Strigolactones (SLs) are synthesized from all-trans-β-carotene via a pathway involving the β-carotene isomerase DWARF27, the carotenoid cleavage dioxygenases 7 and 8 (CCD7, CCD8), and cytochrome P450 enzymes from the 711 clade (MAX1 in Arabidopsis). The rice enzyme DWARF27 was shown to catalyze the reversible isomerization of all-trans- into 9-cis-β-carotene in vitro. β-carotene occurs in different cis-isomeric forms, and plants accumulate other carotenoids, which may be substrates of DWARF27. Here, we investigated the stereo and substrate specificity of the rice enzyme DWARF27 in carotenoid-accumulating E. coli strains and in in vitro assays performed with heterologously expressed and purified enzyme. Our results suggest that OsDWARF27 is strictly double bond-specific, solely targeting the C9-C10 double bond. OsDWARF27 did not introduce a 9-cis-double bond in 13-cis- or 15-cis-β-carotene. Substrates isomerized by OsDWARF27 are bicyclic carotenoids, including β-, α-carotene and β,β-cryptoxanthin, that contain at least one unsubstituted β-ionone ring. Accordingly, OsDWARF27 did not produce the abscisic acid precursors 9-cis-violaxanthin or -neoxanthin from the corresponding all-trans-isomers, excluding a direct role in the formation of this carotenoid derived hormone. The conversion of all-trans-α-carotene yielded two different isomers, including 9'-cis-α-carotene that might be the precursor of strigolactones with an ε-ionone ring, such as the recently identified heliolactone.

Enzymatic study on AtCCD4 and AtCCD7 and their potential to form acyclic regulatory metabolites

“…CCD7 enzymes are highly stereospecific, cleaving only 9- cis -configured substrates, such as 9- cis -β-carotene (Bruno et al , 2014), which is produced from the all- trans species by the isomerase DWARF27 (Alder et al , 2012; Bruno and Al-Babili, 2016). We therefore tested AtCCD7 as a candidate for the cleavage of cis -configured carotene desaturation intermediates.…”

“…In contrast, CCD7 and CCD8 are devoted to the biosynthesis of SLs (Gomez-Roldan et al , 2008; Umehara et al , 2008; Al-Babili and Bouwmeester, 2015). Here, CCD7 catalyzes the stereospecific cleavage of the trans -configured C9'–C10' double bond in 9- cis -β-carotene produced by the all- trans /9- cis -β-carotene isomerase DWARF27 (Alder et al , 2012; Bruno and Al-Babili, 2016), yielding β-ionone and 9- cis -β-apo-10'-carotenal (Alder et al , 2012; Bruno et al , 2014). The latter is then converted by CCD8, which catalyzes combination of repeated oxygenation and intramolecular rearrangements, into carlactone (Alder et al , 2012).…”

Insights into the formation of carlactone from in‐depth analysis of theCCD8‐catalyzed reactions

“…S2) into 13 C-labeled 3-methyl-5-(2,6,6-trimethylcyclohex-1-enyl)penta-2,4-dienenitrile via an in situ Horner-Wadsworth-Emmons (HWE) reaction. After deprotonation of 1-13 C acetonitrile with one equivalent of lithium diisopropylamide (LDA), the anion formed was reacted with diethyl chlorophosphate to give 13 C-labeled diethyl cyanomethylphosphonate, which was immediately deprotonated by a second equivalent of LDA [20]. The nitrile obtained as a mixture of 2-trans-and 2-cisisomers, was reduced with dibal-H to yield 13 C-labeled 3-methyl-5-(2,6,6-trimethylcyclohex-1-enyl)penta-2,4-dienal.…”

“…(9) represents mesomeric forms of the secondary product (an acidic species) that contribute to the stabilization of its negative charge (estimated pK a % 4) [37]. Finally, the pure 13 C-labeled 2-trans-, 4-trans-, 6-trans-, 8-trans-, 10-trans-, 12-cis-isomer (1) was obtained by a standard HWE elongation with diethyl cyanomethyl-phosphonate and NaH followed by a dibal-H reduction and subsequent silica gel purification and crystallization. S15.…”

“…This leads, inter alia, to the formation of physiologically important compounds, like the opsin chromophore retinal and the phytohormone abscisic acid [8,10]. In the next step, CCD7 catalyzes the stereospecific cleavage of 9-cis-b-carotene into 9-cis-b-apo-10 0 -carotenal and the volatile b-ionone [11,13]. In the next step, CCD7 catalyzes the stereospecific cleavage of 9-cis-b-carotene into 9-cis-b-apo-10 0 -carotenal and the volatile b-ionone [11,13].…”