Search

Abstract The uridine diphosphate glycosyltransferase (UGT) plays the central role in glycosylation of small molecules by transferring sugars to various acceptors including bioactive natural products in plants. UGT89C1 from Arabidopsis thaliana is a novel UGT, a rhamnosyltransferase, specifically recognizes UDP- l -rhamnose as donor. To provide an insight into the sugar specificity for UDP- l -rhamnose and interactions between UGT89C1 and its substrates, the UGT89C1 was expressed in Escherichia coli and purified toward biochemical and structural studies. Enzyme activity assay was performed, and the recombinant UGT89C1 recognized UDP- l -rhamnose and rhamnosylated kaempferol. Crystals of At UGT89C1 were obtained, they diffracted to 2.7 Å resolution and belonged to space group I 4 1. At UGT89C1 was also co-crystallized with UDP. Interestingly, two crystal forms were obtained in the same crystallization condition, including the previous I 4 1 crystal form, and the new crystal form that diffracted to 3.0 Å resolution and belonged to space group P 2 1. Highlights • Expression in Escherichia coli and purification of the recombinant UGT89C1 have been established. • The recombinant UGT89C1 specifically recognized UDP- l -rhamnose and rhamnosylated kaempferol. • UGT89C1 was crystallized without and with UDP, and two crystal forms were obtained with UDP. • The X-ray crystallographic analysis of UGT89C1 was performed and discussed. [ABSTRACT FROM AUTHOR]

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca 2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca 2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria. [ABSTRACT FROM AUTHOR]