Elucidation of the Regulatory and Structural Mechanisms of GDP-Mannose Pyrophosphorylase (GMP) and Its Interaction with Kjc-1/Kjc-2 Enzymes in Ascorbic Acid Biosynthesis

• Grant number: 24/21019-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2025
• End date: January 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Grazieli Moreira Cordeiro
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

Ascorbic acid (AsA), commonly known as vitamin C, is a multifunctional molecule with crucial roles in cellular redox control in both plants and animals. Among plants, the Amazonian species Myrciaria dubia (camu-camu) is notable for its exceptionally high AsA content, ranging from 1 to 3 g per 100 g of pulp, making it a promising source for nutraceutical and biotechnological applications. In plants, AsA biosynthesis occurs predominantly via the Smirnoff-Wheeler pathway, which converts glucose-6-phosphate into ascorbic acid through multiple enzymatic steps. The enzyme GDP-mannose pyrophosphorylase (GMP) plays a critical role in this process by catalyzing the formation of GDP-mannose from GTP and mannose-1-phosphate, an essential precursor for L-galactose production, which is subsequently converted into AsA. Thus, GMP activity and regulation directly impact the metabolic flux through this pathway and, consequently, AsA levels in the plant. Although the Smirnoff-Wheeler pathway is well-characterized, the regulatory and structural mechanisms governing GMP activity, particularly the roles of Kjc-1 and Kjc-2 proteins as activators of GMP, remain poorly understood. This project aims to elucidate these regulatory and structural mechanisms of GMP and the Kjc-1 and Kjc-2 proteins, providing insights into the control of AsA biosynthesis. To achieve this, the gene sequences of the enzymes were optimized, synthesized, and cloned into pET28a+ and pET-Duet expression vectors. Recombinant protein expression will be performed in the Rosetta BL21 (DE3) strain, induced with 0.3 mM IPTG, followed by purification through affinity and size-exclusion chromatography. Biochemical characterization will be conducted using specific substrates under controlled conditions of pH, temperature, and buffer. Structural analysis of the interactions between GMP, Kjc-1, and Kjc-2 will be carried out using bioinformatics tools, X-ray crystallography, and cryo-electron microscopy (cryo-EM), enabling a detailed understanding of GMP activation by Kjc-1 and Kjc-2 and the regulatory control of the AsA biosynthetic pathway in plants.