Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

Abstract

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

UDP-Glucose: A Potential Signaling Molecule in Plants?

“…UDP-GlcNac, produced by UAP, is the precursor for glycoprotein and glycolipid synthesis. In Arabidopsis, UAP1 can reversibly convert GlcNAc1P and GalNAc1P to UDP-GlcNAc and UDP-GalNAc, whereas UAP2 converts GlcNAc1P, GalNAc1P and to some extent G1P into their corresponding UDP-sugars ( Yang et al, 2010 ; Decker and Kleczkowski, 2017 ). UAP2 showed higher affinity for UDP-Glc than for G1P, suggesting a role in metabolizing UDP-Glc rather than producing it Decker and Kleczkowski (2017) .…”

“…In Arabidopsis, UAP1 can reversibly convert GlcNAc1P and GalNAc1P to UDP-GlcNAc and UDP-GalNAc, whereas UAP2 converts GlcNAc1P, GalNAc1P and to some extent G1P into their corresponding UDP-sugars ( Yang et al, 2010 ; Decker and Kleczkowski, 2017 ). UAP2 showed higher affinity for UDP-Glc than for G1P, suggesting a role in metabolizing UDP-Glc rather than producing it Decker and Kleczkowski (2017) . In rice, UAP1 was initially shown to reversibly convert GlcNAc1P to UDP-GlcNAc and UDP-GalNAc to GalNAc1P, respectively ( Wang et al, 2015 ).…”

“…Phylogenetic analysis revealed that the rice UAP1 had high identity to its homolog UAP2 (88%) and to the Arabidopsis UAP1 and UAP2 (both 78%) ( Wang et al, 2015 ). The ability of the rice UAP1 to use UDP-Glc as substrate was particularly interesting ( Xiao et al, 2017 ), as the Arabidopsis UAP2 showed very low activity with G1P as substrate with high K m (3.2 mM), but showed a low K m (0.21 mM) for UDP-Glc ( Decker and Kleczkowski, 2017 ). Arabidopsis UAP2 also shows more common amino acids for UDP binding than for sugar binding.…”

“…The UDP-Glc accumulation in these mutants might also be indirectly caused by the lowered levels of UDP-GlcNAc/UDP-GalNAc required for protein glycosylation (e.g., UGPase/USPase/SuSy). It will be interesting to investigate whether Arabidopsis UAP2 mutants show similar UDP-Glc accumulation, since UAP2 shows a high affinity for UDP-Glc in vivo ( Decker and Kleczkowski, 2017 ). Further, the expression levels of UGPase , USPase , and SuSy together with their enzyme activity should be followed in rice UAP1 ( spl29 ) and Arabidopsis UAP2 mutants as compared to WT, to pinpoint the reason for UDP-Glc accumulation.…”

The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases

“…The uncompetitive mode of inhibition by cmpd #6D and #6D2 seems useful for in vivo studies, as the effect of the inhibitor will not be outcompeted (as in the case of competitive inhibitors) by accumulating substrates 14 . In a more recent study, 15 activity of Arabidopsis UDP- N -acetyl glucosamine pyrophosphorylase2 (UAGPase2) was also found to be inhibited by cmpd #6D, further underscoring common structural features at or near the active sites of distinct pyrophosphorylases, which differ in substrate specificity.…”