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

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.

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

Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity. The proteins HRT1, HRT2, and HRBP have 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. Co-expression of HRT1 and HRBP in the presence of nanodiscs yielded marked polyisoprene synthesis activity. By contrast, neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP manifested such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (PaCPT1-3) manifested prenyltransferase activity only when co-expressed with PaCBP, the homolog of HRBP. Our results thus indicate that two heterologous subunits form the core prenyltransferase of the rubber biosynthetic machinery. A recently developed structure modeling program predicted the structure of such heterodimer complexes including HRT1/HRBP and PaCPT2/PaCBP. HRT and PaCPT proteins were also found to possess affinity for a lipid membrane in the absence of HRBP or PaCBP, and structure modeling implicated an amphipathic α-helical domain of HRT1 and PaCPT2 in membrane binding of these proteins.

Structure-based engineering of a short-chain cis-prenyltransferase to biosynthesize nonnatural all-cis-polyisoprenoids: molecular mechanisms for primer substrate recognition and ultimate product chain-length determination.

Most cis-prenyltransferases (cPTs) use all-trans-oligoprenyl diphosphate, such as (E,E)-farnesyl diphosphate (FPP, C15 ), but scarcely accept dimethylallyl diphosphate (DMAPP, C5 ), as an allylic diphosphate primer in consecutive cis-condensations of isopentenyl diphosphate. Consequently, naturally occurring cis-1,4-polyisoprenoids contain a few trans-isoprene units at their ω-end. However, some Solanum plants have distinct cPTs that primarily use DMAPP as a primer to synthesize all-cis-oligoprenyl diphosphates, such as neryl diphosphate (NPP, C10 ). However, the mechanism underlying the allylic substrate preference of cPTs remains unclear. In this study, we determined the crystal structure of NDPS1, an NPP synthase from tomato, and investigated critical residues for primer substrate preference through structural comparisons of cPTs. Highly conserved Gly and Trp in the primer substrate-binding region of cPTs were discovered to be substituted for Ile/Leu and Phe, respectively, in DMAPP-preferring cPTs. An I106G mutant of NDPS1 exhibited a low preference for DMAPP, but a higher preference for FPP. However, an I106G/F276W mutant preferred not only DMAPP but also all-trans-oligoprenyl diphosphates, with 15-fold higher catalytic efficiency than WT. Surprisingly, the mutant synthesized longer polyisoprenoids (~C50 ). Furthermore, one of the helix domains that constitute the hydrophobic cleft for accommodating elongating prenyl chains was also demonstrated to be critical in primer substrate preference. An NDPS1 I106G/F276W mutant with a chimeric helix domain swapped with that of a medium-chain cPT synthesizing C50-60 polyisoprenoids showed over 94-fold increase in catalytic efficiency for all primer substrates tested, resulting in longer products (~C70 ). These NDPS1 mutants could be used in the enzymatic synthesis of nonnatural all-cis-polyisoprenoids.

Purification and characterization of small and large rubber particles from Hevea brasiliensis.

Natural rubber (NR) is synthesized by the rubber transferase (RTase) on rubber particles (RPs) in latex. Due to the heterogeneity of the RPs in latex, it is difficult to precisely characterize the RTase activity. In this study, we separated the RPs of Hevea brasiliensis with different particle size distributions, via stepwise centrifugations. Analyses of protein compositions and size distributions of NR in the RPs suggest that RPs in Hevea latex can be categorized into two distinct subclasses, the larger RPs (termed 1kRP, 2kRP, and 8kRP) and the smaller RPs (termed 20kRP and 50kRP). Precise enzymatic assays using the RPs revealed that 50kRP showed the highest RTase activity, whereas the larger RPs, which had been regarded to have quite low activity, also exhibited a comparable activity to the smaller RPs. Immunological detections of cis-prenyltransferases in the RPs showed that the abundance of these enzymes correlates with the extent of RTase activity.

Platform for "Chemical Metabolic Switching" to Increase Sesquiterpene Content in Plants.

The biosynthetic pathway of cytosolic isoprenoids bifurcates after farnesyl diphosphate into sesquiterpene and triterpene pathways. "Metabolic switching" has been used to increase sesquiterpene content in plants by suppressing the competitive triterpene pathway using transgenic technology. To develop "metabolic switching" without using transgenic technology, we developed a model system of "chemical metabolic switching" using inhibitors of the competitive pathway. Arabidopsis plants that overexpress the amorpha-4,11-diene synthase gene were treated with squalestatin, a squalene synthase inhibitor, or terbinafine, a squalene epoxidase inhibitor. We then analyzed total sterol content as major triterpenes and amorpha-4,11-diene in the plant. Plants treated with squalestatin showed decreased total sterol content and increased amorpha-4,11-diene content. In contrast, plants treated with terbinafine showed decreased total sterol content, but amorpha-4,11-diene accumulation was quite low. These results suggest that inhibition of the enzyme just below the branch point is more effective than inhibition of enzymes far from the branch point for "chemical metabolic switching". In addition, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of the cytosolic isoprenoid biosynthetic pathway, was upregulated in plants treated with squalestatin, suggesting that feedback regulation of 3-hydroxy-3-methylglutaryl-CoA reductase may contribute to amorpha-4,11-diene production. Here we demonstrated the effectiveness of "chemical metabolic switching" in plants.

Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis.

Natural rubber (NR) is stored in latex as rubber particles (RPs), rubber molecules surrounded by a lipid monolayer. Rubber transferase (RTase), the enzyme responsible for NR biosynthesis, is believed to be a member of the cis-prenyltransferase (cPT) family. However, none of the recombinant cPTs have shown RTase activity independently. We show that HRT1, a cPT from Heveabrasiliensis, exhibits distinct RTase activity in vitro only when it is introduced on detergent-washed HeveaRPs (WRPs) by a cell-free translation-coupled system. Using this system, a heterologous cPT from Lactucasativa also exhibited RTase activity, indicating proper introduction of cPT on RP is the key to reconstitute active RTase. RP proteomics and interaction network analyses revealed the formation of the protein complex consisting of HRT1, rubber elongation factor (REF) and HRT1-REF BRIDGING PROTEIN. The RTase activity enhancement observed for the complex assembled on WRPs indicates the HRT1-containing complex functions as the NR biosynthetic machinery.