Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.

Arabidopsis Rab-E GTPases exhibit a novel interaction with a plasma-membrane phosphatidylinositol-4-phosphate 5-kinase

Rab GTPases of the Arabidopsis Rab-E subclass are related to mammalian Rab8 and are implicated in membrane trafficking from the Golgi to the plasma membrane. Using a yeast two-hybrid assay, Arabidopsis phosphatidylinositol-4-phosphate 5-kinase 2 (PtdIns(4)P 5-kinase 2; also known as PIP5K2), was shown to interact with all five members of the Rab-E subclass but not with other Rab subclasses residing at the Golgi or trans-Golgi network. Interactions in yeast and in vitro were strongest with RAB-E1d[Q74L] and weakest with the RAB-E1d[S29N] suggesting that PIP5K2 interacts with the GTP-bound form. PIP5K2 exhibited kinase activity towards phosphatidylinositol phosphates with a free 5-hydroxyl group, consistent with PtdIns(4)P 5-kinase activity and this activity was stimulated by Rab binding. Rab-E proteins interacted with PIP5K2 via its membrane occupancy and recognition nexus (MORN) domain which is missing from animal and fungal PtdIns(4)P 5-kinases. In plant cells, GFP:PIP5K2 accumulated at the plasma membrane and caused YFP:RAB-E1d to relocate there from its usual position at the Golgi. GFP:PIP5K2 was rapidly turned over by proteasomal activity in planta, and overexpression of YFP:PIP5K2 caused pleiotropic growth abnormalities in transgenic Arabidopsis. We propose that plant cells exhibit a novel interaction in which PIP5K2 binds GTP-bound Rab-E proteins, which may stimulate temporally or spatially localized PtdIns(4,5)P(2) production at the plasma membrane.

In Vitro UDP-Sugar:Undecaprenyl-Phosphate Sugar-1-Phosphate Transferase Assay and Product Detection by Thin Layer Chromatography

In vitro assays are invaluable for the biochemical characterization of UDP-sugar:undecaprenyl-phosphate sugar-1-phosphate transferases. These assays typically involve the use of a radiolabeled substrate and subsequent extraction of the product, which resides in a lipid environment. Here, we describe the preparation of bacterial membranes containing these enzymes, a standard in vitro transferase assay with solvents containing chloroform and methanol, and two methods to measure product formation: scintillation counting and thin layer chromatography.

Structural-Functional Studies of Burkholderia cenocepacia D-Glycero-beta-D-manno-heptose 7-Phosphate Kinase (HldA) and Characterization of Inhibitors with Antibiotic Adjuvant and Antivirulence Properties

As an essential constituent of the outer membrane of Gram-negative bacteria, lipopolysaccharide contributes significantly to virulence and antibiotic resistance. The lipopolysaccharide biosynthetic pathway therefore serves as a promising therapeutic target for antivirulence drugs and antibiotic adjuvants. Here we report the structural-functional studies of D-glycero-beta-D-manno-heptose 7-phosphate kinase (HldA), an absolutely conserved enzyme in this pathway, from Burkholderia cenocepacia. HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270. Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites. Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.