Abstract
Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.
Highlights
Substrate-dependent ordering of flexible active site loops can transmute substrate binding affinity into faster enzymatic reactions (Jencks, 1975; Malabanan et al, 2010)
The comparatively immobile external oxyanion hole component Gly388 N was chosen to be the reference point for four interatomic distances that capture 230s loop motion, active site constriction, and the in/out conformations of Arg228, which occur twice per half-reaction and are typically reciprocal to the movements of Asn229. This parametrization groups closed conformations and open conformations and allows ready identification of intermediate states such as those associated with covalent enzyme adducts
In addition to the important role the “viselike grip” exerted in the immediate vicinity of the acyl-Coenzyme A (CoA) thioester has on catalysis (White and Jencks, 1976), we suggest that acid bacteria use succinyl-CoA:acetate CoA-transferase (AarC), and by extension other class I CoA-transferase active sites, positively recognizes free CoA to prevent the final stage of active site closure
Summary
Substrate-dependent ordering of flexible active site loops can transmute substrate (ligand) binding affinity into faster enzymatic reactions (Jencks, 1975; Malabanan et al, 2010). When loop motions move critical functional groups, it is unclear if protein motions bias the enzyme-substrate complex conformational ensemble toward a reactive configuration or assemble the catalytic. A classic example of this phenomenon is provided by the CoAtransferases, which activate metabolism of diverse carboxylate substrates by introducing the versatile and reactive CoA thioester (Moore and Jencks, 1982; Amyes and Richard, 1992; Yang and Drueckhammer, 2003), often at the expense of acetyl-CoA (AcCoA). Strong selection for key roles in acetic acid resistance and central metabolism appear to have optimized the structural and functional properties of AarC, making it an excellent representative of the class I CoA-transferase superfamily
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