Location: Plant Physiology and Genetics Research
Project Number: 2020-21000-014-019-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 15, 2025
End Date: Oct 31, 2027
Objective:
Domestic rubber production is of significant national economic and security importance as specified in the new National Farm Security Action Plan. Understanding the mechanism of rubber biosynthesis by rubber transferase (RTase), the enzyme complex that polymerizes natural rubber, is essential to inform genetic engineering approaches designed to improve the yield of rubber-producing crops. The RTase complexes of Ficus elastica (ficus, a model system) and Parthenium argentatum (guayule, the leading alternative rubber crop in the US) each appear to contain two large subunits (scaffold proteins) and six copies of each of two small subunits directly involved in rubber synthesis. This composition was inferred from enzymatic and protein chemistry studies. These two small subunits are polypeptides, called "toddler" (29 - 36 amino-acid residues) and "baby" (18 amino acid residues for ficus). Modeling studies predict that the three-dimensional structures of these small subunits are symmetric hexamers. However, these predicted models for ficus and for guayule are distinct from each other. Our goal is to produce, reconstitute, and determine three-dimensional structures of these small subunit complexes of RTase from ficus and guayule using recombinant protein production using strains developed in the NSF-ERC-TARDISS project. We will carry out analytical gel filtration, circular dichroism spectroscopy, NMR spectroscopy, and X-ray crystallography, and in the presence of substrates, such as isopentenyl pyrophosphate (IPP, the elongation substrate), farnesyl pyrophosphate (FPP, a polymer initiator) and magnesium and manganese cations, essential activators of polymerization. These experimental structural studies are not part of the funded TARDISS program. They will provide valuable insights into the mechanisms of rubber biosynthesis and the evolution of rubber biosynthesis systems.
Approach:
The project is motivated by the following fundamental research questions:
- What are the quaternary structures of the Ficus elastica and Parthenium argentatum (guayule) rubber transferase (RTase) toddler and baby subunits?
- How do the 3D structures interact with the substrates from which rubber is made?
- What are the substrate-binding mechanisms?