Identification and Characterisation of the Enzymes Involved in the Biosynthetic Pathway of Tartaric Acid in Vitis vinifera

Author: Crista Ann Burbidge

Burbidge, Crista Ann, 2012 Identification and Characterisation of the Enzymes Involved in the Biosynthetic Pathway of Tartaric Acid in Vitis vinifera, Flinders University, School of Biological Sciences

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Abstract

Vitis vinifera cv Shiraz is a member of Vitaceae, one of three higher plant families in which tartaric acid (TA) accumulates to significant levels. The accumulation of TA in V. vinifera berry tissue is crucial for commercial wine making, preventing discolouration and spoilage due to bacterial contamination. In V. vinifera, TA is biosynthesised via two pathways, the primary-ascorbate and secondary-glucose precursor pathways. Little is known regarding the enzymes responsible for the biosynthesis of TA with L-idonate dehydrogenase (L-idonate-5-dehydrogenase) the only enzyme identified in the primary-ascorbate precursor pathway. The results presented in this study describe a bioinformatic approach to the identification of putative candidates for a 2-keto-D-gluconate reductase (possessing 2-keto-L-gulonate reductase activity) and gluconate 5-dehydrogenase suggested as being responsible for the catalysis of the second steps of the primary-ascorbate and secondary-glucose precursor pathways respectively. In vitro biochemical characterisation of recombinantly expressed proteins in conjunction with in vivo molecular analysis was performed to support the candidates inclusion in the respective pathways. Comparative analysis of the V. vinifera genome with enzymes identified as catalysing identical reactions in bacteria identified three candidate 2-keto-D-gluconate reductases, TC61548, TC59682 and TC55752 and three candidate gluconate 5-dehydrogenases, TC52437, TC58004 and TC55097 (Gene Indices: Grape database). All candidate genes were identified in immature berry cDNA except TC52437, therefore TC52437 was not pursued further. No active recombinant protein was obtained for candidates TC61548, TC58004 and TC55097. Kinetic analyses were performed on purified samples of the recombinant TC59682 and TC55752 protein. Optimal activity of recombinant TC59682 was observed at 35 degrees C, pH 7.5. Activity studies indicated the primary substrate of TC59682 to be 2-keto-L-gulonate (2KGA) with a Km of 4.67mM. The Km for NADH was also determined as 0.77mM. A two-step assay utilising the highly-specific L-idonate dehydrogenase indicates L-idonate is a product of this reaction. The reversibility of TC59682 was confirmed against L-idonate in the presence of NAD+, at a rate 37-fold lower than the forward direction under identical conditions. Activity was also observed against ascorbate, the pathway precursor, 8.9-fold lower than that observed against 2KGA. Activity increased against all tested substrates in the presence of coenzyme NADP(H) as compared to NAD(H). Candidate TC55752 showed activity against 2KGA and ascorbate, 7.13-fold lower and 1.82 fold higher than TC59682 in the presence of coenzyme NADPH respectively. QRT-PCR analysis of the candidate genes expression was conducted in developing V. vinifera cv Shiraz berries over the 2007-2008 season. The expression pattern of TC59682 strongly coincided with the biosynthesis of TA over development. TC55752's expression pattern does not indicate involvement in TA biosynthesis. Expression patterns of TC61548, TC58004 and TC55097 suggest these candidates are not involved in TA biosynthesis Activity of extracted soluble enzymes from a subset of the sampled Shiraz berries showed an increase in activity against 2KGA over development suggesting, as recently shown with L-idonate dehydrogenase, the presence of the enzyme late in berry development. The results presented in this study suggest TC59682 has a role in the primary TA biosynthetic pathway. Based upon primary substrate activity TC59682 will be annotated as a 2-keto-L-gulonate reductase.

Keywords: Tartrate,Organic Acid,Vitis vinifera,Shiraz,Grape Berry Development,2-keto-L-gulonate reductase
Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2012
School: School of Biological Sciences
Supervisor: Kathleen Soole