Structural and Kinetic Characterization of an Acetoacetyl-Coenzyme A: Acetate Coenzyme A Transferase from the Extreme Thermophile Thermosipho melanesiensis.

Abstract

CtfAB from the extremely thermophilic bacterium, Thermosipho melanesiensis, has been used for in vivo acetone production up to 70°C. This enzyme has tentatively been identified as the rate-limiting step, due to its relatively low binding affinity for acetate. However, existing kinetic and mechanistic studies on this enzyme are insufficient to evaluate this hypothesis. Here, kinetic analysis of purified recombinant T. melanesiensis CtfAB showed that it has a ping pong bi bi mechanism typical of CoA transferases with Km values for acetate and acetoacetyl-CoA of 85 mM and 135 mM, respectively. Product inhibition by acetyl-CoA was competitive with respect to acetoacetyl-CoA and non-competitive with respect to acetate. Crystal structures of wildtype and mutant T. melanesiensis CtfAB were solved in the presence of acetate and in the presence or absence of acetyl-CoA. These structures led to a proposed structural basis for the competitive and non-competitive inhibition of acetyl-CoA: acetate binds independently of acetyl-CoA in an apparent low affinity binding pocket in CtfA that is directly adjacent to a catalytic glutamate in CtfB. Similar to other CoA transferases, acetyl-CoA is bound in an apparent high affinity binding site in CtfB with most interactions occurring between the phospho-ADP of coenzyme A and CtfB residues far from the acetate binding pocket. This structural-based mechanism also explains the organic acid promiscuity of CtfAB. High affinity interactions are predominantly between the conserved phospho-ADP of coenzyme A and the variable organic acid binding site is a low affinity binding site with few specific interactions.