9CTD

CtfAB F42TS45C mutant co-crystallized with acetyl-CoA

Summary for 9CTD

• Entry DOI: 10.2210/pdb9ctd/pdb
• Related: 9CQ2 9CRY 9CSC
• Descriptor: 3-oxoacid CoA-transferase, A subunit, 3-oxoacid CoA-transferase, B subunit, ACETATE ION, ... (6 entities in total)
• Functional Keywords: acetoacetate-coa transferase, thermophile, acetyl-coa, acetate binding site, transferase
• Biological source: Thermosipho melanesiensis; More
• Total number of polymer chains: 4
• Total formula weight: 95538.97

Authors

Buhrman, G.,Bing, R. (deposition date: 2024-07-24, release date: 2025-02-05, Last modification date: 2025-03-05)

Primary citation

Bing, R.G.,Buhrman, G.K.,Ford, K.C.,Straub, C.T.,Laemthong, T.,Rose, R.B.,Adams, M.W.W.,Kelly, R.M.
Structural and kinetic characterization of an acetoacetyl-Coenzyme A: acetate Coenzyme A transferase from the extreme thermophile Thermosipho melanesiensis.
Biochem.J., 482:225-240, 2025

PubMed Abstract: Family 1 Coenzyme A transferases (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 Coenzyme A (CoA) transferases with Km values for acetate and acetoacetyl-CoA of 85 mM and 135 μM, respectively. Product inhibition by acetyl-CoA was competitive with respect to acetoacetyl-CoA and non-competitive with respect to acetate. Crystal structures of wild-type 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 CoA 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 CoA, and the variable organic acid binding site is a low-affinity binding site with few specific interactions.

PubMed: 39869497
DOI: 10.1042/BCJ20240747

Experimental method

X-RAY DIFFRACTION (1.9 Å)