Structural Studies of 4-CBA-CoA Dehalogenase From Pseudomonas CBS-3. Matthew M. Benning¹, Debra Dunaway-Mariano² and Hazel M. Holden^{1 1}Institute for Enzyme Research, Graduate School and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705 ²Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742

4-chlorobenzoyl Coenzyme A dehalogenase has attracted interest due to its possible employment as a bioremediation agent. Because of widespread use as industrial and agricultural agents, halogenated hydrocarbons constitute a particularly formidable class of environmental pollutants. Microbial degradation of these compounds may be an alternative to conventional disposal methods which are costly and inefficient. Involved in the 4-CBA biodegradative pathway of Pseudomonas CBS-3 strain, 4-CBA-CoA dehalogenase catalyzes an aromatic substitution reaction involving the replacement of the chloride substituent with a hydroxide. Studies have indicated that the reaction proceeds through a nucleophilic substitution process involving a Meisenheimer complex.

In order to learn more about the dehalogenase, we have determined the structures of both the wild-type enzyme and several site-directed mutants in the presence the product, 4-hydroxybenzoyl-Coenzyme A. The enzyme functions as a trimer with the binding pocket formed by two separate subunits. For the most part, the pocket consists of hydrophobic residues except for Asp 145 and His 90. Asp 145 is within hydrogen bonding distance of the hydroxyl oxygen of the benzoyl ring and most likely participates in the formation of the intermediate. His 90 is also close to the benzoyl ring and may activate a water molecule participating in the hydrolysis of 4-CBA-CoA. The site-directed mutants D145A and H90Q exhibit greatly reduced activity. The structures of the native enzyme as well as these site-directed mutants will be presented.