Proceedings of the 47th Annual Meeting of the Brazilian Biophysical Society

Myrciaria dubia (Kunth) McVaugh, known as “camu-camu” or “aracá d’agua”, is a fruit native to the Amazon, characterized by its high content of L-Ascorbic Acid (Vitamin C or AsA). Due to this property, the enzymes that participate in the biosynthesis of AsA could have potential biotechnological applications for the artificial production of AsA. However, the structural biology of the enzymes involved has been poorly explored. Here, we describe the biophysical, functional, and structural properties for three enzymes of the D-mannose/L-galactose pathway (Smirnoff & Wheeler). The enzymes are purified by affinity chromatography and molecular exclusion. GDP-D-mannose-3´,5´-epimerase (MdGME), which catalyzes a double epimerization of GDP-mannose to produce GDP-L-galactose and GDP-L-gulose, proved to be a dimer in solution and its crystallographic structure was solved, in both its apo (2.5 Å, bound to NAD+) and holo (1.25 Å bound to NAD+/substrate/product) forms. L-galactose dehydrogenase (MdGDH) and L-galactono-1,4-lactone dehydrogenase (MdGalDH) proved to be monomeric in solution. MdGDH showed a catalytic activity with a Km of 0.128 mM and an optimal pH of 7. We show that inhibition by AsA in MdGDH is due to pH changes and is not necessarily due to a competitive inhibition at its active site as reported for the homologous enzyme of spinafre (SoGDH). The crystallographic structure of SoGDH was solved at 1.4 and 1.75Å in its apo and holo (NAD+-bound) forms, respectively. Finally, MdGalDH showed a catalytic activity with a Km of 0.044 mM and an optimal pH of 8 and was inhibited by its own substrate. The crystallographic structure was solved to 2.1 Å. The present work contributed to a broader understanding of the structure-function relationships of enzymes involved in the synthesis of vitamin C.