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Investigation of the Substrate Specificity of L‐Idonate Dehydrogenase By Site‐directed Mutagenesis
Author(s) -
Steiner Steve,
Terpening Allison,
McCurdy Chelsea
Publication year - 2017
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.31.1_supplement.764.4
Subject(s) - biochemistry , dehydrogenase , enzyme , nad+ kinase , chemistry , site directed mutagenesis , substrate (aquarium) , active site , mutagenesis , stereochemistry , amino acid , ascorbic acid , mutant , biology , gene , ecology , food science
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. L‐IdDH is an NAD + /NADH‐dependent enzyme which catalyzes the conversion of L‐idonate to 5‐keto‐D‐gluconate. Analysis of the amino acid sequence indicates that L‐IdDH from Vitis vinifera is a member of the family of medium chain dehydrogenases/reductases and is 79% identical with the putative sorbitol dehydrogenase (SDH) from Arabidopsis thaliana . Despite the high degree of homology, each enzyme is specific for its substrate with neither enzyme showing activity toward the substrate of the other. One of the notable differences in the amino acid sequences of L‐IdDH compared to SDH is at position 42 where His is located in L‐IdDH in contrast to Tyr in SDH. It is possible that the cationic His in L‐IdDH is essential to the binding of the anionic substrates L‐idonate and 5‐keto‐D‐gluconate. For this study, site‐directed mutagenesis was performed to convert 42H to 42R and to 42Y in L‐IdDH. Assay results indicate that mutation of His (positive charge) to Arg (positive charge) causes a significant reduction in activity toward 5‐keto‐D‐gluconate and a mutation to Tyr (uncharged) at this position completely eliminates activity. These finding illustrate the critical role of 42H in the activity and substrate specificity of L‐IdDH. Support or Funding Information This work is supported by a grant from the Indiana Academy of Science.