Takani, Y. | bioRxiv Science

bioRxiv2021

Reconstitution of prenyltransferase activity on nanodiscs by components of the rubber synthesis machinery of the Para rubber tree and guayule

Prenyltransferases mediate the biosynthesis of various types of polyisoprene compound in living organisms. Natural rubber (NR) of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity, with the proteins HRT1, HRT2, and HRBP having been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Cell-free synthesis of HRT1, HRT2, and HRBP in the presence of asolectin nanodiscs revealed that all three proteins were membrane associated. A complex of HRT1 and HRBP formed as a result of co-expression of the two proteins in the presence of nanodiscs manifested marked polyisoprene synthesis activity, whereas neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP exhibited such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (CPT1-3) manifested prenyltransferease activity only if co-expressed with the homolog of HRBP (CBP). Our results thus indicate that the core prenyltransferase of the rubber biosynthetic machinery of both the Para rubber tree and guayule is formed by the assembly of heterologous subunits (HRT1 and HRBP in the former species).

plant biology↗

bioRxiv2020

Catalytic Promiscuity of Rice 2-Oxoglutarate/Fe(II)-Dependent Dioxygenases Supports Xenobiotic Metabolism

The rice 2-oxoglutarate/Fe(II)-dependent dioxygenase HIS1 mediates the catalytic inactivation of five distinct {beta}-triketone herbicides (bTHs). During a search for potential inhibitors of HIS1, we found that it mediates the hydroxylation of trinexapac-ethyl (TE) in the presence of Fe2+ and 2-oxoglutarate. TE is a plant growth regulator that blocks gibberellin biosynthesis, and we observed that its addition to culture medium induced growth retardation of rice seedlings in a concentration-dependent manner. Similar treatment with hydroxylated TE revealed that hydroxylation greatly attenuated the inhibitory effect of TE on plant growth. Forced expression of HIS1 in a rice his1 mutant also reduced its sensitivity to TE compared with that of the nontransformant. These results indicated that HIS1 metabolizes TE and thereby markedly reduces its ability to slow plant growth. Furthermore, testing of five HIS1-related proteins (HSLs) of rice revealed that OsHSL2 and OsHSL4 also metabolize TE in vitro. HSLs from wheat and barley also showed such activity. In contrast, OsHSL1, which shares the highest amino acid sequence identity with HIS1 and metabolizes the bTH tefuryltrione, did not manifest TE-metabolizing activity. Site-directed mutagenesis of OsHSL1 informed by structural models showed that substitution of three amino acids with the corresponding residues of HIS1 conferred TE-metabolizing activity similar to that of HIS1. Our results thus reveal a catalytic promiscuity of HIS1 and its related enzymes that supports xenobiotic metabolism in plants. One-sentence summaryThe rice 2-oxoglutarate/Fe(II)-dependent dioxygenase HIS1 and related enzymes show broad substrate specificity and mediate metabolism of the growth regulator trinexapac-ethyl as well as of herbicides.

plant biology↗