Kiwifruit l-galactose dehydrogenase: molecular, biochemical and physiological aspects of the enzyme
William A Laing A B , Nicky Frearson A , Sean Bulley A and Elspeth MacRae AA Gene Technologies Sector, The Horticultural and Food Research Institute of NZ Ltd PB 92169, Auckland, NZ.
B Corresponding author; email: wlaing@hortresearch.co.nz
Functional Plant Biology 31(10) 1015-1025 https://doi.org/10.1071/FP04090
Submitted: 24 May 2004 Accepted: 23 June 2004 Published: 14 October 2004
Abstract
l-Galactose dehydrogenase, an enzyme in the pathway of ascorbate biosynthesis, was purified to homogeneity from leaves of kiwifruit [Actinidia deliciosa (A.Chev.) CF Liang et AR Ferguson var. deliciosa ‘Hayward’]. The enzyme had a molecular mass of 34.2 kD, and behaved as a monomer during gel filtration. The Km(galactose) and Km(NAD) decreased as pH increased from 6.5 to 9, while the Vmax increased over this range. A number of related sugars were tested as alternative substrates or inhibitors, but these were ineffective. Ascorbate caused slow inactivation of the enzyme, possibly through metal catalysed generation of oxygen radicals. Inactivation appeared to be active-site directed as it was protected by the substrate NAD, and not by NADH or l-galactose. This is not likely to be physiologically significant. Through partially sequencing the protein, the gene was identified in the HortResearch Actinidia EST database, and the translation of the full length sequence of this cDNA showed a high homology (80% identity, 90% similarity) to the translation of an Arabidopsis gene (accession CAD10386) and to translations of other genes identified in GenBank. Levels of l-GalDH activity decreased during fruit and leaf development, and levels of mRNA showed a similar reduction. Activity varied between flower parts, with ovaries and styles showing equivalent activity to young fruitlets and sink leaves. Nucleotide sequences reported are available in the Genbank database under the accession number AY176585 (kiwifruit) and AY264803 (apple).
Keywords: ascorbic acid, biosynthesis, vitamin C.
Acknowledgments
We thank Dr Andrew Gleave (HortResearch) for providing sequencing facilities, Dr Ross Crowhurst (HortResearch), manager of the EST sequence database and bioinformatics suite, and Dr Lesley Beuning (HortResearch), who oversaw tissue collection and library construction for kiwifruit and apple ESTs. Di Barraclough conducted the SDS-PAGE analysis and Janine Cooney conducted the mass spectrometry based sequencing.
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