Temporal analysis of natural rubber transferases reveals intrinsic distinctions for in vitro synthesis in two rubber-producing species

Authors: CORNISH, KATRINA - The Ohio State University; DACOSTA, BERNARDO - Former ARS Employee; McMahan, Colleen

Submitted to: Current Topics in Biochemical Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/4/2020
Publication Date: 10/1/2020
Citation: Cornish, K., Dacosta, B., McMahan, C.M. 2020. Temporal analysis of natural rubber transferases reveals intrinsic distinctions for in vitro synthesis in two rubber-producing species. Current Topics in Biochemical Research. 21:45-58.

Interpretive Summary: Development of rubber-producing crops will benefit from improved natural rubber yield in plants such as guayule (Parthenium argentatum). Guayule is currently under development in the SW USA as a source of natural rubber, resins, and biomass. Knowledge of the fundamental biology and biochemistry of natural rubber synthesis in plants will lead to practical approaches to improve yield. In this investigation, rubber particles were isolated from plants such that they could still make rubber on the bench (in vitro). Concentrations of the initiator molecules and the monomer molecules were varied, and rubber production measured over time. Some of the surprising discoveries were that guayule can synthesis long polymer chains very rapidly (under 15 minutes), and that the maximum rubber polymer size (molecular weight) can exceed 50 million grams/mole, even though the product usually found in nature is about 1 million grams/mole.

Technical Abstract: The temporal relationship of rubber molecule initiation, polymerization and termination, as affected by limited, optimal, and non-limiting initiator concentrations, and in optimal and or non-limiting isopentenyl pyrophosphate (IPP) monomer concentrations, was investigated in vitro using enzymatically active rubber particles purified from Hevea brasiliensis and Parthenium argentatum. Polymer initiation occurred at the beginning of the experiments and reinitiation in excess farnesyl pyrophosphate (FPP) occurred more quickly in P. argentatum than H. brasiliensis. The number of FPP binding sites (six per rubber transferase complex, RT-ase), and thus the number of RT-ase complexes, was similar in both P. argentatum and H. brasiliensis per gram of rubber particles. Since the RT-ase complexes are bound to the rubber particle surface, the difference in mean rubber particle size, and the abundance of small particles in H. brasiliensis washed rubber particles (WRP) implies at least double the surface density of active RT-ases in H. brasiliensis WRP than in P. argentatum WRP in these samples. The rate of rubber biosynthesis was approximately linear with time in H. brasiliensis, but in P. argentatum IPP incorporation slowed between 1.5 and 4 h, under some conditions, and then generally accelerated between 4 and 8 h to even higher rates. Under most conditions, it took between 1.5 h and 4 h to produce mature rubber polymers. The chain transfer reaction of both RT-ase’s was accelerated by excess initiator resulting in much lower molecular weight polymers than typically extracted from living plants. However, in both species, rubber molecules kept growing when synthesized in limited initiator concentrations even at non-saturating monomer concentrations. The P. argentatum RT-ase was able to make higher molecular weight rubber than the H. brasiliensis RT-ase, and under extremely limited FPP (0.001 µM), rubber molecular weights above 50 Mg/mol were achieved. However, in addition to primary biochemical substrate concentration effects on rubber molecular weight and, thus termination, spatial constraints appear to be a factor in the as yet undefined rubber polymer termination mechanism.