Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plants and humans

• Published in: Biochemical and biophysical research communications Vol. 591; pp. 110 - 117
• Main Authors: Das, Bhaba Krishna, Kumar, Amit, Sreekumar, Sreeshma Nellootil, Ponraj, Kannapiran, Gadave, Kaustubh, Kumar, Saravanan, Murali Achary, V. Mohan, Ray, Pratima, Reddy, Malireddy K., Arockiasamy, Arulandu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.02.2022
• Subjects: Amino Acid Sequence; Ascorbate recycling; Ascorbic Acid - metabolism; Biocatalysis; Catalytic Domain; CLIC1; CLIC3; CLIC4; Conserved Sequence; Crystallography, X-Ray; Cysteine - metabolism; Dehydroascorbate reductase; Humans; Kinetics; Models, Molecular; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Oxidation-Reduction; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Pearl millet; Pennisetum - enzymology; Dehydroascorbate reductase; CLIC4; PgDHAR; CLIC3; Pearl millet; DHAR; Ascorbate recycling; CLIC; CLIC1; HsCLIC
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2021.12.103

Ascorbate is an important cellular antioxidant that gets readily oxidized to dehydroascorbate (DHA). Recycling of DHA is therefore paramount in the maintenance of cellular homeostasis and preventing oxidative stress. Dehydroascorbate reductases (DHARs), in conjunction with glutathione (GSH), carry out this vital process in eukaryotes, among which plant DHARs have garnered considerable attention. A detailed kinetic analysis of plant DHARs relative to their human counterparts is, however, lacking. Chloride intracellular channels (HsCLICs) are close homologs of plant DHARs, recently demonstrated to share their enzymatic activity. This study reports the highest turnover rate for a plant DHAR from stress adapted Pennisetum glaucum (PgDHAR). In comparison, HsCLICs 1, 3, and 4 reduced DHA at a significantly lower rate. We further show that the catalytic cysteine from both homologs was susceptible to varying degrees of oxidation, validated by crystal structures and mass-spectrometry. Our findings may have broader implications on crop improvement using pearl millet DHAR vis-à-vis discovery of cancer therapeutics targeting Vitamin-C recycling capability of human CLICs. •Stress adapted pearl millet DHA reductase (DHAR) possess higher turnover rate.•Detailed kinetic analysis presented for a plant DHAR and human CLIC1, 3 and 4.•Crystal structures reveal different oxidation states of catalytic cysteine.•Possible implications in crop improvement and anti-cancer drug-discovery.