Structural studies on the regulation of ascorbic acid (Vitamin C) production: the interaction between the regulatory protein PAS/LOV and the enzyme GDP-L-Galactose phosphorylase (GGP)

• Grant number: 24/15657-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2024
• End date: September 30, 2025
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Richard Charles Garratt
• Grantee: Beatriz Lecce Manzo
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

Ascorbic acid is essential for health, especially due to its antioxidant action. For some animals, such as primates, plants are the main source of ascorbic acid due to the lack of one of the enzymes in the biosynthesis pathway. Thus, Myciaria dubia (Kunth) McVaugh, a native Amazonian plant, stands out as it contains 0.96 to 2.99 grams of ascorbic acid per 100 grams of pulp. The metabolic pathway for ascorbic acid biosynthesis in plants, known as the Smirnoff-Wheeler pathway, is the most well-characterized in cellular and physiological terms. However, structural studies are still needed to understand the molecular mechanisms of ascorbic acid regulation and synthesis. Among the six enzymes involved in this pathway, GDP-L-Galactose phosphorylase (GGP) is proposed as the main control point. In vivo and in vitro studies have demonstrated the interaction between this enzyme and the regulatory protein known as PAS/LOV. This work, therefore, aims to study and characterize PAS/LOV and its complex with GGP. To achieve these objectives, the GGP and PAS/LOV proteins were cloned into the pET28+ and pETDuet plasmids, respectively, and the GGP protein will be subsequently subcloned into the pETDuet vector for co-expression of the proteins. Once purified, GGP enzymatic activity will be analyzed in the presence and absence of PAS/LOV. Finally, biophysical and structural characterizations will be performed using SEC-MALS, circular dichroism, microscale thermophoresis, and crystallization assays.