Biochemical and structural characterization of ancestral L-galactono-1,4-lactone dehydrogenase

Global warming and environmental damage caused by the use of fossil fuels have prompted the search for more sustainable production methods. In this context, industrial biocatalysis plays a pivotal role in developing green chemistry solutions and biorefinery processes for producing valuable products. This study focuses on flavin-dependent carbohydrate oxidoreductases, specifically from the aldonolactone oxidoreductase family, crucial for the final step of L-ascorbic acid biosynthesis in eukaryotes. By investigating the evolution of ancient plant species, we resurrected through Ancestral sequence reconstruction a variant of the L-galactono-1,4-lactone dehydrogenase (vGalDH). This research aims to decipher Biochemical and structural characterization of ancestral L-galactono-1,4-lactone dehydrogenase specifically investigating plant GalDH expressed as recombinant proteins. Using ASR, we seek to gain insights into the catalytic mechanisms and evolutionary history of these enzymes. Recognizing the efficacy of ASR for studying challenging enzymes, we employed this approach to explore the evolutionary history and functionality of vGalDH. Through substrate screening, vGalDH demonstrated significant activity on several lactone sugars, with a pronounced preference for L-galactono-1,4-lactone. Mutagenesis studies, particularly the introduction of an asparagine at position 413, improved the enzyme's affinity towards L-gulono-1,4-lactone while retaining high catalytic efficiency for L-galactono-1,4-lactone. The enzyme exhibited robust thermostability with a melting temperature around 87 ºC at pH 8. Crystallization trials were successful, and several protein crystals were grown. These findings underscore the evolutionary adaptation and catalytic versatility of vGalDH, highlighting its potential applications in biotechnological industries focused on sustainable production practices.