7 research outputs found
Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation
ISSN:1559-744XISSN:1559-743
Thio-linked UDP-peptide conjugates as O-GlcNAc transferase inhibitors
O-GlcNAc
transferase (OGT) is an essential glycosyltransferase
that installs the O-GlcNAc post-translational modification on the
nucleocytoplasmic proteome. We report the development of S-linked
UDP–peptide conjugates as potent bisubstrate OGT inhibitors.
These compounds were assembled in a modular fashion by photoinitiated
thiol–ene conjugation of allyl-UDP and optimal acceptor peptides
in which the acceptor serine was replaced with cysteine. The conjugate
VTPVCÂ(S-propyl-UDP)ÂTA (<i>K</i><sub>i</sub> = 1.3 μM)
inhibits the OGT activity in HeLa cell lysates. Linear fusions of
this conjugate with cell penetrating peptides were explored as prototypes
of cell-penetrant OGT inhibitors. A crystal structure of human OGT
with the inhibitor revealed mimicry of the interactions seen in the
pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative
of the inhibitor works as a high affinity probe in a fluorescence
polarimetry hOGT assay
Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis
Background: Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. Results: In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. Conclusions: Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. General significance: This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen