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Biochemical Characterization of L‐idonate Dehydrogenase from Vitis vinifera
Author(s) -
Steiner Stephen A.,
Selby Anna,
Moore Kristen,
McKinley Katie
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.581.1
Subject(s) - nad+ kinase , gluconic acid , cofactor , enzyme , vitis vinifera , dehydrogenase , chemistry , biochemistry , tartaric acid , ascorbic acid , enzyme assay , biology , food science , horticulture , citric acid
The enzyme L‐idonate dehydrogenase (L‐IdDH) is an important enzyme in the pathway for the biosynthesis of tartaric acid from ascorbic acid in grapes. IdDH is an NAD + /NADH‐dependent enzyme which catalyzes the conversion of L‐idonate to 5‐keto‐D‐gluconic acid. For this study, the enzyme was produced in BL21 cells which have been transformed by the pET14b plasmid containing the IdDH gene produced from a Vitis vinifera cDNA library. The active enzyme was isolated by TALON affinity chromatography. Initial characterization of the enzyme in a reaction using 5‐keto‐D‐gluconate and NADH as substrates showed a pH optimum of 6.0. The reaction proceeding the opposite direction, with the conversion of L‐idonate to 5‐keto‐D‐gluconate using NAD + as a cosubstrate, has optimal activity at a significantly higher pH, near 8.0. Kinetics studies have indicated that the Km for 5‐keto‐D‐gluconate is 4 mM in the presence of a saturating concentration of NADH. This work is supported by a grant from the Indiana Academy of Science.