Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana.
Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-D-glucose/UDP-D-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-d-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30-40 degrees C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD+, whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display kcatUDP-Gal values between 23 and 128 s(-1) and KmUDP-Gal values between 0.1 and 0.3 mm. This results in enzymatic efficiencies ranging between 97 and 890 mm(-1) s(-1) for UGE4 = UGE1 < UGE3 < UGE5 < UGE2. The KmUDP-Glc values, derived from the Haldane relationship, were 0.76 mm for UGE1, 0.56 mm for UGE4, and between 0.13 and 0.23 mm for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereasUGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4rhd1 phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.
Barber
Christine
C
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom.
Rösti
Johannes
J
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom.
Rawat
Arun
A
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom.
Findlay
Kim
K
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom.
Roberts
Keith
K
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom.
Seifert
Georg J
GJ
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom. Electronic address: georg.seifert@bbsrc.ac.uk.
D017360:Arabidopsis / Q000201:enzymology*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D018506:Gene Expression Regulation, Plant; D006056:Golgi Apparatus / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D010940:Plant Proteins / Q000737:chemistry; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014534:UDPglucose 4-Epimerase / Q000737:chemistry*
D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014534:UDPglucose 4-Epimerase