Rubber transferase

This enzyme [EC 2.5.1.20], also known as rubber allyl-transferase and rubber transferase, catalyzes the reaction of poly-dx-polyprenyl diphosphate (or, rubber particles) with isopentenyl diphosphate to produce a poly-cK-... 

Rubber is synthesized by plants via a side branch of the isoprenoid pathway by the enzyme rubber transferase (dy-prenyl transferase systematic name poly-dy-polyprenyl-diphosphate isopentenyl-diphosphate polyprenylcistransferase EC 2.5.1.20). Surprisingly, although this process has been studied for decades, due to the labile nature of the rubber transferase and the fact that it is a membrane-associated enzyme present in relatively low abundance, the identification of its protein subunits remain elusive. For some recent reviews on rubber biosynthesis, please refer to [248-251]. 

Rubber is synthesized and sequestered on cytsolic vesicles known as rubber particles. Rubber transferase is localized to the surface of the rubber particles, and biosynthesis is initiated through the binding of an allylic pyrophosphate (APP, a pyrophosphate, produced by soluble trans- rtnyl transferases) primer. Progressive additions of IPP molecules ultimately result in the formation of high molecular weight cjT-l,4-polyisoprene. The rubber transferase also requires a divalent cation, such as Mg + or Mn +, as cofactor. 

All rubber transferases exhibit similar kinetic constants and pH optima, and are able to accept a similar range of APPs as initiating substrate [263, 264], In vitro studies have shown that several compounds (DMAPP, GPP, FPP, and GGPP) can initiate rubber biosynthesis, with a faster rate of rubber biosynthesis the longer the APP (up to C15 or C20) [254, 265], Non-natural APPs were also shown to be able to function as a primer for the rubber biosynthesis [266],... 

Rubber transferase active site with bound IPP and FPP... 

Ji, W., Benedict, C.R. and Foster, M.A. (1993) Seasonal variations in rubber biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and rubber transferase activities in Parthenium argentatum in the Chihuahuan Desert. Plant Physiol, 103, 535-42. 

Natural Rubber Isopentenylphosphate E Rubber transferase cis polyisoprene Phospholipids 10-2000 48... 

The formation of c s-l,4-polyisoprene is a heterogeneous polymerization where the polymerization mainly occurs at the surface of the rubber particles. The propagating rubber transferase molecule is mainly situated at the surface of a rubber latex particle [52,53]. The sizes of rubber latex particles can vary from 10 nm to several microns [50], The outCT layer of these latex particles mainly consists of phospholipids and proteins. 

In the case of natural rubber from Hevea brasiliensis, which is almost entirely made of cis-isoprene units, it could be assumed that ci5-prenyltransferase is exclusively involved in phase 2. Unfortunately it is more complex. Authors agree on phase 1, which is traws -condensation catalyzed by a cytosolic soluble trans-prenyl transferase" Famesyl diphosphate synthase has been cloned from rubber latex.The most probable prenyldiphosphate used as a primer for phase 2 is famesyl-PP (15) or geranylgeranyl-PP (16)." " Phase 2 is catalyzed by a still not clearly identified rubber transferase system. Different proteins have been... 

The biosynthesis of rubber may be divided into three steps (1) initiation, which requires an allylic diphosphate molecule, (2) elongation, in which IPP units are added to a Z-l,4-polyisoprene chain, and (3) termination, in which the polymer is released from the rubber transferase enzyme (Cornish, 1993). In plants, the elongation of Z-l,4-polyiso-prene (natural rubber) requires a small -allylic diphosphate initiator (less than or equal to C20). Famesyl pyrophosphate (FPP) is an effective initiator of polyisoprene biosynthesis (Light et al, 1989) further, because only one molecule of FPP is needed for each molecule of rubber formed, small traces of this substance that are inadvertently present complicate biosynthetic studies. The E-allylic diphosphates are hydrophilic cytosolic compounds, whereas Z-l,4-polyisoprene is hydrophobic and compartmentalized in subcellular rubber particles. A soluble E-prenyl transferase from the latex of Hevea brasiliensis serves as a famesyl diphosphate synthase and plays no direct role in elongation of Z-l,4-polyisoprene (Cornish, 1993). Because the hydro-phobic rubber molecule is produced inside a rubber particle but is formed from hydrophilic precursors from the cytoplasm, the polymerization reaction must take place at the particle surface. 

Rubber biosynthesis is an imusual enzymological reaction in that the rubber transferase can accept any one of a number of APPs as the initiating molecule (38,39,45,50). However, the identity of the initiating molecule affects the IPP condensation reaction rate as the rubber molecule polymerizes (Fig. 3). In nonlimiting... 

Fig. 3. Dependence of IPP incorporation rate by rubber transferase on IPP concentration in 20 ixM allylic pyrophosphate initiator Q DMAPP, A GPP, FPP, O GGPP. Data from Ref 54.

Kinetic analysis of the dependence of IPP incorporation on APP concentration (Fig. 4, Table 1) has indicated that multiple binding constants are involved in the reaction, and led to the interpretation that the rubber transferase active site contains a nonspecific hydrophobic binding region which traverses the particle monolayer membrane and with which the growing rubber molecule interacts... 

Rubber biosynthesis in guayule is imder strong environmental control, the rubber transferase being induced at the onset of winter (Fig. 6) (40,58-67). The substrate synthesis pathway also appears to be induced at this time (65). In the wintertime, the gua5uile plant is essentially dormant and can afford to use... 

Fig. 6. Rubber transferase activity in rubber particles purified from two lines of field-grown, five-year-old guayule throughout one year. The activity for line 11591 (open circles) and line 593 (filled bars) was determined in 80 ixM IPP in rubber particles purified from whole branches. The maximum and minimum daily temperatures also are plotted (light lines), as well as moving averages of both temperature sets (dark lines, data smoothed over 21 days). Data from Ref 58.

The subsequent chain elongation step was confirmed to proceed via an addition of IPP to polyisoprenyl diphosphate to form isoprene units in cis configuration. Rubber polymerization is catalysed by rubber transferase (EC 2.5.1.20), a cw-prenyltransferase, and requires divalent cations such as Mg " or Mn as a cofactor. The proposed mechanism of rubber biosynthesis is shown in Figure 2.1. 

The rubber biosynthesis was found to take place on the rubber surface of rubber particles by the incorporation of IPP into a terminal allylic diphosphate group of rubber molecules. It was assumed that the growing hydrocarbon chain of rubber diffuses into the rubber particles, while the hydrophilic diphosphate end group remains in the serum phase where it can react with IPP bound to the active site of the rubber transferase, as shown in Figure 2.2. It has been confirmed that the rubber transferase is tightly bound to the Hevea rubber particles even after purification by washing. 

The mechanism of rubber formation was studied by in vitro rubber biosynthesis, by the rubber transferase tightly bound to rubber particles, enzymatically active rubber particles. At least two mechanisms have been postulated for the conversion of IPP to the in vitro synthesized rubber. First is the initiation of new rubber molecules on the surface of the rubber particles and the chain extension of pre-existing rubber molecules, the rubber molecules from the rubber particles used for the rubber biosynthesis. In case of de novo rubber biosynthesis, the new rubber molecules are initiated by the added APP initiators and/or the APP initiators synthesized by the soluble proteins, i.e. IPP isomerase and trowi -prenyltransferase remained in the system. The second is... 

The building block (monomer) of NR is also IPP (Figure 4.6). At the physiological pH = 7.4 IPP is a stable dianion with potassium as counterion. The bios5mthesis of NR is catalyzed by the rubber transferase enzyme, cis-prenyl transferase. 

Cornish, K., Backhaus, R. A. Rubber transferase activity in rubber particles of guayule. Phytochemistry 1990,29 (12), 3809-3813. 

In plants, cellulose is synthesized by rosette terminal complexes (RTCs). The RTCs are hexameric protein structures containing the cellulose synthase enzyme. This enzyme synthesizes the individual cellulose chains. Each RTC is located at the interface of the cell membrane, similarly to the membrane-bound rubber transferase. Each RCT will polymerize a polysaccharide chain. This sounds a little bit complicated, but we are going to break down the synthesis into the... 

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