Characterization of the dullard protein by the HAD Bridging Project served as an early model project for examination of single domain C0 enzymes which have no cap domain (generally known to direct specificity), and thus present a special challenge to development of the EFI’s large-scale strategies. Intriguingly this work, together with our work on enzymes involved in lipid A biosynthesis show that "capless" C0 HADSF members can be extremely specific and efficient.
Human lipin1 catalyzes the highly regulated conversion of phosphatidic acids to diacylglycerides. Lipin's cellular location, protein partners, and biological function are directed by phosphorylation-dephosphorylation events catalyzed by the phosphoserine phosphatase dullard. To define the determinants of dullard substrate recognition and catalysis, and hence, lipin regulation, steady-state kinetic analysis was performed on phosphoserine-bearing nonapeptides based on the phosphorylation sites of lipin. The results demonstrate that dullard shows specificity for the peptide corresponding to the insulin-dependent phosphorylation site (Ser106) of lipin with a k(cat)/K(m) of 2.9 × 10(4) M(-1) s(-1). These results are consistent with a coil-loop structure for the insulin-dependent phosphorylation site on human lipin1 and make unlikely the requirement for an adaptor protein to confer activity such as that proposed for the yeast homologue.
Figure 2. Ribbon diagrams of the homology model of dullard (left) and X-ray crystal structures of Mg(II)-dependent phosphatase 1 (center) and the phosphatase domain of polynucleotide kinase/phosphatase (right). The conserved Rossmann fold of the HADSF is colored blue, and inserted segments are colored yellow, with the catalytic nucleophile colored red to mark the active site.
Figure 3. Dullard model (red, stringently conserved) shown as a coil (left) and in space-filling representation (center and right) with peptide (cyan sticks) from Scp1 overlaid (right) by superimposing the Scp1 9-mer peptide complex structure (PDB entry 2GHQ) onto the dullard model and removing the Scp1 protein.
2011 HAD Superfamily Publication
Reprinted with permission from Biochemistry.
© 2011 American Chemical Society.