Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plant and human

Ascorbate, a primary antioxidant, gets readily oxidized to dehydroascorbate (DHA). Hence, recycling by dehydroascorbate reductase (DHAR) enzymes is vital for protection from cellular oxidative stress in eukaryotes. However, a detailed kinetic analysis of plant DHARs and their human orthologs; chloride intracellular channels ( Hs CLICs) is lacking. We demonstrate that DHAR from stress adapted pearl millet Pennisetum glaucum ( Pg DHAR) shows the highest turnover rate whereas Hs CLIC1, 3, and 4 reduce DHA, albeit at lower rates. We further show that the catalytic cysteine is susceptible to varying levels of oxidation, supported by crystal structures and mass-spectrometry analysis. The differences in kinetic parameters among plant and human DHA reductases corroborate with the levels of reactive oxygen species H 2 O 2 encountered in their respective intracellular environment. Our findings may have broader implications in crop improvement using pearl millet DHAR, and anti-cancer therapeutics targeting Vitamin-C recycling capability of human CLICs.