Biosynthesis of UDP‐glucuronic acid in developing soybean embryos: possible role of UDP‐sugar pyrophosphorylase

Abstract

During soybean [Glycine max (L.) Merrill] embryo development, cell wall polysaccharides (CWPs) derived from UDP-glucuronic acid (UDP-GlcA) (uronic acids, arabinose, xylose) exhibited a linear increase during the period of 25-45 days after flowering (daf). At embryo maturity, CWPs derived from UDP-GlcA accounted for 39% of total CWPs. To ascertain the relative importance of the nucleotide sugar oxidation (NSO) and the myo-inositol oxidation (MIO) pathways to UDP-GlcA biosynthesis, UDP-glucose (UDPGlc) dehydrogenase (UDP-Glc DH, EC 1.1.1.22) and UDP-glucuronic acid pyrophosphorylase (UDP-GlcA PPase, EC 2.7.7.44) activities, respectively, were measured in desalted extracts of developing embryos. UDP-Glc DH and UDP-GlcA PPase activities, expressed on a per seed basis, increased 3.5-and 3.9-fold, respectively, during the period of 25-45 daf. However, UDP-GlcA PPase activity was 35-50-fold greater than UDP-Glc DH activity. The soybean UDP-sugar pyrophosphorylase gene (USP1), a homolog of pea USP, and a candidate gene for UDP-GlcA PPase, was cloned and the recombinant enzyme characterized. Recombinant soybean USP1 (71 kDa) exhibited high activity with glucuronic acid 1-phosphate (GlcA-1-P), glucose 1-phosphate (Glc-1-P) and galactose 1-phosphate (Gal-1-P), but low activity with mannose 1-phosphate (Man-1-P), N-acetylglucosamine 1-phosphate and Glc-6-P. Determination of kinetic constants indicated that USP1 has a higher affinity for GlcA-1-P (K m ¼ 0.14 AE 0.02 mM) than for Glc-1-P (K m ¼ 0.23 AE 0.02 mM). Semiquantitative RT-PCR was used to measure transcript levels of the UDPglucose DH (UGD) and USP gene families in developing soybean embryos. Transcript levels, normalized to the 18S rRNA controls, were greater for UGD than USP throughout embryo development. The possibility that USP serves as UDP-GlcA PPase, the terminal enzyme of the MIO pathway, is discussed.

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

“…In those experiments, USPases were either purified from plant extracts (pea) or overexpressed in Escherichia coli and purified as recombinant proteins (for Arabidopsis, soybean [Glycine max], Leishmania, and Trypanosoma enzymes). In most cases, the activities with Glc-1-P, Gal-1-P, and GlcA-1-P were higher than with Xyl-1-P and Ara-1-P. On the other hand, the enzyme had low (below 7%) or no activity with GalA-1-P, Man-1-P, N-acetyl-GlcA-1-P, Fuc-1-P, inositol-1-P, and Glc-6-P (Kotake et al, 2004(Kotake et al, , 2007Litterer et al, 2006aLitterer et al, , 2006bDamerow et al, 2010). For all USPases, the K m values for UTP were low (0.03-0.19 mM), regardless of the nature of the second substrate, whereas K m values for sugar-1-phosphate were in the range of 0.13 to 2.54 mM (Supplemental Table S1).…”

“…3). Studies with purified soybean and Arabidopsis USPases using products and alternative substrates/products as possible inhibitors have revealed relatively small inhibition (Litterer et al, 2006a(Litterer et al, , 2006bSchnurr et al, 2006;Gronwald et al, 2008).…”

“…It is unknown whether this activity belongs to USPase or reflects the involvement of other Figure 3. Relative UTP-dependent activities with different sugar-1-phosphates of purified USPases from pea (Kotake et al, 2004), Arabidopsis A (Litterer et al, 2006b), Arabidopsis B (Kotake et al, 2007), soybean (Litterer et al, 2006a), Leishmania , and T. cruzi . Only activities with substrates showing over 7% activity in comparison with the most active substrate are shown.…”

“…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.…”

“…These proteins share low or very low identity at the amino acid level (below 20%; Kleczkowski et al, 2010). Phylogenetic analyses revealed that USPases, UGPases (either A or B type), and UAGPases should all be categorized into distinct groups Litterer et al, 2006aLitterer et al, , 2006bOkazaki et al, 2009;Kleczkowski et al, 2010).…”