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 Plants

Abstract

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by a-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.

Metabolism of soluble sugars in developing melon fruit: a global transcriptional view of the metabolic transition to sucrose accumulation

“…Carbohydrate metabolism in cucurbit fruit has been studied and the pathway has been elucidated ( Fig. 1, based on Carmi et al 2003;Dai et al 2006;Gao et al , 2004Gross and Pharr 1982;Keller and Pharr 1996;Schaffer et al 1996). The major photosynthates, sucrose and the raffinose oligosaccharides (RFOs), raffinose and stachyose, are translocated from source leaf to the melon fruit sink (Chrost and Schmitz 1997;Mitchell et al 1992;Schaffer et al 1996) and the near absence of raffinose and stachyose in the melon fruit flesh points to the rapid metabolism of these translocated sugars in the fruit (Chrost and Schmitz 1997;Hubbard et al 1989).…”

“…1) begins with the hydrolysis of the translocated RFOs, stachyose and raffinose by the enzymes a-galactosidase, of which there are at least three in the developing melon fruit . The released gal is phosphorylated by a galactokinase and the gal-1P product subsequently transformed to glc-1P by the concerted actions of a novel UDP-gal/glc pyrophosphorylase (UGGP), UDP-glc-4 0 -epimerase and UDP-glc pyrophosphorylase (UGP) (Dai et al 2006). The resultant glc-1P can undergo further transformations to glc-6P and fru-6P via the phosphoglucomutase (PGM) and phosphoglucoisomerase (PGI) reactions, each of which occur as both cytoplasmic and plastidic enzymes.…”

UDP-Sugar Pyrophosphorylase: A New Old Mechanism for Sugar Activation

“…Those carbohydrates are the main carbon-transporting compounds in the phloem of the Cucurbitaceae family of plants (e.g. melon [Cucumis melo], cucumber [Cucumis sativus]; Dai et al, 2006).…”

“…Recent work with purified USPases (Kotake et al, 2004(Kotake et al, , 2007Litterer et al, 2006aLitterer et al, , 2006bDai et al, 2006;Gronwald et al, 2008;Damerow et al, 2010;, studies on transgenic plants with altered USPase contents (Schnurr et al, 2006;Kotake et al, 2007), and resolution of the crystal structure of protozoan USPase (Dickmanns et al, 2011) have reignited the interest in USPase and provided new clues to the possible roles of this multisubstrate-utilizing enzyme.…”

Unraveling the Leloir Pathway of Bifidobacterium bifidum: Significance of the Uridylyltransferases

“…Because of this promiscuity, a new EC number (EC 2.7.7.10) was proposed to distinguish it from UTP-glucose-1-phosphate uridylyltransferase (EC 2.7.7.9; GalU), which predominantly acts on G1P in prokaryotes (26,27). However, due to the activity on different sugar 1-phosphates, the absence of similarity with GalU-type enzymes, and a high level of identity with promiscuous eukaryotic uridylyltransferases, we suggest that UgpA should be classified as an UTP-monosaccharide-1-phosphate uridylyltransferase (EC 2.7.7.64, synonym of UDP-sugar pyrophosphorylase [USP]) (28)(29)(30). Moreover, a UDP-sugar pyrophosphorylase from B. longum (BLUSP) was recently cloned, showing 82% amino acid identity with UgpA, and was used for the efficient synthesis of a variety of UDP-sugars (31).…”