Regulation of UDP-glucose pyrophosphorylase isozyme UGP5 associated with cold-sweetening resistance in potatoes.
The regulation of UDP-Glc pyrophosphorylase (UGPase) isozyme, UGP5, was investigated in potato tuber. The cDNA for UGP5 was cloned into the bacterial expression vector pET21d and recombinant (RC) enzyme was expressed in E. coli (BL21 star cells). The RC-UGP5 isozyme was purified to near homogeneity using salt precipitation, hydrophobic interaction, and anion-exchange column chromatography. Kinetic analysis revealed that in the synthesis direction, K(m) values for Glc-1-P (0.83 mM) and UTP (0.22 mM) were similar to those observed previously with the mother tuber (MT)-UGP5. In the pyrophosphorolysis direction, the K(m) values for UDP-Glc (0.68 mM) and PPi (0.56 mM) were slightly higher than those observed previously. Maximum reaction velocities (V(max)) for RC-UGP5 were also elevated. Since the molecular mass, charge, and amino acid sequence of the MT- and RC-UGP5 isozymes were identical, it was assumed that altered kinetic constants may be due to an improper folding of RC-UGP5 polypeptide. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and proteomic analysis demonstrated that the UGP5 isozyme was a single polypeptide with a calculated molecular mass of 51.8kDa consisting of 477 amino acids. Native PAGE and kinetic analysis revealed that this polypeptide was monomeric in nature. Immunoblotting with specific antibodies and LC-MS/MS data indicated that UGP5 did not require any post-translational modification (e.g., phosphorylation, O-glycosylation, oligomerization/de-oligomerization, or the presence of the regulatory 14-3-3 proteins) for its regulation. Additionally, the two closely associated isozymes UGP5 and UGP6 in the cv. Snowden are likely the result of allelic differences of UGPase at a single locus.
Gupta
Sanjay K
SK
Department of Horticultural Science, University of Minnesota, 305 Alderman Hall, 1970 Folwell Avenue, St. Paul, MN 55108, USA.
Sowokinos
Joseph R
JR
Hahn
In-Su
IS
D048948:14-3-3 Proteins / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003080:Cold Temperature; D006031:Glycosylation; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D040901:Proteomics; D011994:Recombinant Proteins / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology*; D013549:Sweetening Agents / Q000378:metabolism*; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D048948:14-3-3 Proteins; D007527:Isoenzymes; D010455:Peptides; D011994:Recombinant Proteins; D013549:Sweetening Agents; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase