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

Daniel Decker,Leszek A Kleczkowski

doi:10.3389/fpls.2017.01610

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.

Highlights

UDP-sugar formation is an essential pre-requirement for any cell to produce larger and more complex carbohydrate-containing compounds
UDP-glucose pyrophosphorylase (UGPase) is considered to be more or less specific for glucose-1-P (Glc-1-P), and has by far the highest activity among the pyrophosphorylases (Kleczkowski et al, 2010; Kleczkowski and Decker, 2015). This is in contrast to UDP-sugar pyrophosphorylase (USPase), which uses a variety of sugar-1-phosphates as substrates (Kotake et al, 2004; Damerow et al, 2010; Kleczkowski et al, 2011a), and to UDP-N-acetylglucosamine pyrophosphorylase (UAGPase), which prefers N-acetylglucosamine-1-P (GlcNAc-1-P) and N-acetylgalactosamine-1-P (GalNAc-1-P) as substrates (Yang et al, 2010)
We demonstrated that an inhibitor earlier shown to affect UGPase and USPase activities (Decker et al, 2017), had similar effects on purified UAGPase2

Summary

Introduction

UDP-sugar formation is an essential pre-requirement for any cell to produce larger and more complex carbohydrate-containing compounds. UDP-glucose pyrophosphorylase (UGPase) is considered to be more or less specific for glucose-1-P (Glc-1-P), and has by far the highest activity among the pyrophosphorylases (Kleczkowski et al, 2010; Kleczkowski and Decker, 2015) This is in contrast to UDP-sugar pyrophosphorylase (USPase), which uses a variety of sugar-1-phosphates as substrates (Kotake et al, 2004; Damerow et al, 2010; Kleczkowski et al, 2011a), and to UDP-N-acetylglucosamine pyrophosphorylase (UAGPase), which prefers N-acetylglucosamine-1-P (GlcNAc-1-P) and N-acetylgalactosamine-1-P (GalNAc-1-P) as substrates (Yang et al, 2010). As the synthesis of the UDP-sugars is a fully reversible reaction, each pyrophosphorylase can be involved in the production of a given sugar-1-P from the respective UDP-sugar, and contributing to an equilibrium concentration between those metabolites

Methods

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Conclusion