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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Cloning and Expression Analysis of a UDP-Galactose/Glucose Pyrophosphorylase from Melon Fruit Provides Evidence for the Major Metabolic Pathway of Galactose Metabolism in Raffinose Oligosaccharide Metabolizing Plants¹

Department of Vegetable Research, Agricultural Research Organization, Volcani Center, Bet Dagan, 50250, Israel

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by -galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Arthur A. Schaffer (vcaris{at}volcani.agri.gov.il).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.083634

^* Corresponding author; e-mail vcaris{at}volcani.agri.gov.il; fax 972–3–9669642.

Received May 15, 2006; accepted June 30, 2006.