Research in my group is focused on elucidating mechanisms leading to formation of nucleotide- sugars (especially UDP-sugars), which are direct precursors to polysaccharides (e.g. sucrose, cellulose, hemicellulose). The function of relevant enzymes is elucidated both in vivo, using transgenic plants, but also wt plants with the use of specific inhibitors, and by studying purified proteins. Structure/ function properties of key enzymes are investigated, using site-directed mutagenesis, domain deletion approaches, specific inhibitors, etc.

UDP-sugars are used in hundreds of glycosylation reactions (e.g. those forming sucrose, cell wall polysaccharides, etc.), and they represent the most important precursors for biomass production in nature. Thus, mechanisms of UDP-sugar formation, together with selected glycosyltransferases, may represent primary targets for biotechnological approaches aiming at increased biomass production for needs of agriculture, forestry and other industries. We are studying both genes and proteins responsible for UDP-sugar production, including those for UDP-Glc pyrophosphorylase (UGPase), sucrose synthase and, recently, UDP-sugar pyrophosphorylase (USPase) and UDP-N-acetylglucosamine pyrophosphorylase (UAGPase).

Some roles of UDP-sugars in plants (Decker and Kleczkowski, 2019)

We are using transgenic plants with impaired expression of a given protein to elucidate its physiological role. Parameters studied include growth/development of transgenics, metabolite contents, physiological properties, co-expressed gene networks, etc. At the protein level, purified plant pyrophosphorylases are investigated with respect to molecular determinants of their substrate specificity and regulation, including effects of oligomerization and post-translational modifications. The enzymes differ in specificity for sugar and nucleotide portions of their substrates, and may be involved in distinct metabolic pathways.

Substrate specificity of UDP-sugar producing enzymes (Decker and Kleczkowski, 2019)

Properties of the enzymes are modified via site-directed mutagenesis and domain swapping. We have also identified several inhibitors of pyrophosphorylases, using a chemical library developed at the Department of Chemistry, Umeå University. Inhibitors are used to study the role of the target protein in selected physiological/ developmental processes in plant tissues/ cells (reverse chemical-genetics approach) and, possibly, as potential drugs against human protozoan pathogens (e.g. Leishmania, Trypanosoma). Since sugars are essential for UDP-sugar synthesis, we also investigate sugar-mediated regulation of plant gene expression, using both cell culture, pollen germination test, and whole plants.

Structures of plant UDP-sugar producing pyrophosphorylases (Kleczkowski et al., 2011)

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