FIGURE 9.

The proposed mechanism for ObcA. Upon the binding of oxaloacetate, the side chain hydroxyl group of Tyr³²² could be deprotonated by the C1 carboxylate of oxaloacetate, which is within 3.0 Å. The resulting anionic form of Tyr³²² likely serves as a general base and abstracts a proton from the C3 carbon of oxaloacetate in a regiospecific manner, producing an enolate anion intermediate of oxaloacetate. The anion intermediate can be stabilized via interactions with a metal ion bound to the active site, followed by a direct nucleophilic attack on the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral C6-CoA adduct as a product. In an immediate vicinity of the oxygen atom on a tetrahedral carbon, Arg⁴⁶⁹ possibly protonates the oxygen atom. The hydrogen bond network from Arg⁴⁶⁹ to the water molecule via Asp⁴⁷⁴ could regenerate Arg⁴⁶⁹; in particular, Asp⁴⁷⁴ is a solvent-exposed residue, and the nearby water molecule is located in the binding pocket for acetyl-CoA. Note that mutation of either Tyr³²², Arg⁴⁶⁹, or Asp⁴⁷⁴ greatly affects the enzyme activity (Fig. 7d).