Mechanism and Metal Specificity of Zinc-Dependent Deacetylases.

Metal-dependent deacetylases catalyze a variety of essential reactions in nature, and it is estimated that over 10% of all human proteins require zinc for activity. However, most metalloamidases can be activated by a number of divalent metal ions. The metal functions as a catalytic cofactor in numerous classes of hydrolytic reactions by coordinating and polarizing a nucleophilic water and coordinating substrate. One metal-dependent deacetylase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), catalyzes the committed step in Lipid A biosynthesis. Lipid A is the major lipid component of the outer membrane in Gram-negative bacteria, and is essential for cell viability. Consequently, inhibitors of Lipid A biosynthesis are targets for the development of antibacterials. Two chemical mechanisms consistent with the current data have been proposed: a glutamate that functions as both a general acid and general base with a conserved histidine functioning as an electrostatic catalyst, or a general acid/general base pair mechanism. Mutagenesis and expressed protein ligation experiments were performed to distinguish between these mechanisms. LpxC was activated by a general acid analogue and inactive with a positively charged analogue, indicating the general acid/general base pair mechanism is most likely. The techniques developed here solve a persistent mechanistic question and have the potential to be applied to similar mechanistic questions in many other enzymes. Additionally, the metal specificity of LpxC was explored. In vitro, Fe2+LpxC is more active than ZnLpxC. In addition, LpxC purified anaerobically from E. coli contains mainly bound Fe, although the bound Fe/Zn ratio varies with metal availability. Thermodynamic and kinetic studies of LpxC metal binding show that these results can be explained by thermodynamics without metal chaperones, as is observed in other metalloproteins. Another zinc-dependent deacetylase, histone deacetylase 8 (HDAC8), has a similar metal dependence. HDAC8 is more active with Fe2+ than Zn2+, and exhibits Fe2+-like activity in vivo, suggesting that many “Zn”-enzymes may in fact use either Fe or Zn cofactors in vivo. These results are important for understanding how best to inhibit the many clinically useful metalloprotein drug targets, as well as for understanding a potentially important new aspect of metal ion homeostasis and metalloprotein regulation.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77870/1/sgattis_1.pd