Crystal structure and insights into the oligomeric state of UDP-glucose pyrophosphorylase from sugarcane

<div><p>UDP-glucose pyrophosphorylase (UGPase) is found in all organisms and catalyses the formation of UDP-glucose. In sugarcane, UDP-glucose is a branch-point in the carbon channelling into other carbohydrates, such as sucrose and cellulose, which are the major factors for sugarcane productivity. In most plants, UGPase has been described to be enzymatically active in the monomeric form, while in human and yeast, homo-octamers represent the active form of the protein. Here, we present the crystal structure of UGPase from sugarcane (ScUGPase-1) at resolution of 2.0 Å. The crystals of ScUGPase-1 reveal the presence of two molecules in the asymmetric unit and the multi-angle light scattering analysis shows that ScUGPase-1 forms a mixture of species ranging from monomers to larger oligomers in solution, suggesting similarities with the orthologs from yeast and human.</p></div

Structural comparison of ScUGPase-1 and AtUGPase.

<p><b>(A)</b> Superposition of monomers of ScUGPase-1 (blue) and AtUGPase (green) (PDB code: 2ICY; RMSD value of 0.552 Å for 369 Cα atoms). UDP-glucose (yellow sticks) is shown in the active site. The arrow indicates β19 and β20 of ScUGPase-1; they are replaced by a unique β-strand in the AtUGPase structure. <b>(B)</b> Comparison of the active site of apo-ScUGPase-1 and AtUGPase containing UDP-glucose. Residues of AtUGPase involved in the stabilization of UDP-glucose are shown as green sticks and residues of ScUGPase-1 likely important for ligand-binding are shown in blue. The active site of the apo-AtUGPase (PDB code: 1Z90) is also included (purple sticks), showing the same arrangement as for AtUGPase containing UDP-glucose.</p

Size exclusion chromatography and MALS analysis.

<p><b>(A)</b> Size exclusion chromatogram of ScUGPase-1. Peaks between 40 and 60 mL correspond presumably to higher oligomeric forms of ScUGPase-1, whereas the peak at 75 mL corresponds to the monomeric form. <b>(B)</b> Coomassie-stained gel under reducing conditions showing the purity of ScUGPase-1. <b>(C)</b> SEC-MALS analysis of ScUGPase-1. Blue and green lines indicate the trace from the refractive index detector during SEC for the octamer and monomer, respectively. Black lines on each peak correspond to the averaged molecular weight (Mw; y axis) distribution across the peak.</p

Crystal structure of ScUGPase-1.

<p><b>(A)</b> Cartoon representation of the two molecules of ScUGPase-1 present in the asymmetric unit. Monomer A is coloured in blue and monomer B in orange. <b>(B)</b> The ScUGPase-1 monomer and its domains. The N-terminal domain is shown in purple, catalytic domain in blue and the C-terminal domain in wheat colour. The RFKS⁴¹⁹IPSI motif is shown in red.</p

Multiple sequence alignment of UGPase orthologs.

<p>Proteins were aligned by MUSCLE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193667#pone.0193667.ref021" target="_blank">21</a>] and the alignment optimized in Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193667#pone.0193667.ref022" target="_blank">22</a>]. The aligned sequences from top to bottom with their accession numbers are: ScUGPase-1 from <i>Saccharum</i> spp—sugarcane (A0A075E2Q1); AtUGPase from <i>Arabidopsis thaliana</i> (Q9M9P3); StUGPase from <i>Solanum tuberosum–</i>potato (P19595); SbUGPase from <i>Sorghum bicolor</i> (C5XSC5); HvUGPase from <i>Hordeum vulgare</i>–barley (Q43772); OsUGPase from <i>Oryza sativa–</i>rice (Q93X08); ZmUGPase from <i>Zea mays–</i>maize (B6T4R3); bUGPase from bovine–<i>Bos taurus</i> (Q07130); mUGPase from mouse–<i>Mus musculus</i> (Q91ZJ5-2); hUGPase from human–<i>Homo sapiens</i> (Q16851-2) and yUGPase from yeast–<i>Saccharomyces cerevisiae</i> (P32861). Region coloured in green corresponds to the residues in the C-terminal region involved in the end-to-end interactions in the yeast and human orthologs. Black asterisks (<b>*</b>) indicate the residues important for ligand binding, and red asterisks (<b>*</b>) indicate residues involved in the dimer interface in the crystal of ScUGPase-1. Elements of secondary structure are shown based on the crystal structure of ScUGPase-1. β-sheets and α-helices are shown in green and red, respectively.</p