Structural Determinants of Catalytic Directionality in an AMP-Forming Acetyl-CoA Synthetase from Syntrophus aciditrophicus

Authors

Yaghoubi, S., Dinh, D. M., Thomas, L. M., Wofford, N. Q., McInerney, M. J., Follmer, A. H., Karr, E. A.

Abstract

Acetyl-coenzyme A (CoA) is a central metabolic intermediate that links carbon and energy metabolism across all domains of life. The conversion of acetate and acetyl-CoA is carried out by three enzyme pathways: acetate kinase/phosphotransacetylase, ADP-forming acetyl-CoA synthetase, and AMP-forming acetyl-CoA synthetase (Acs). Acs enzymes serve critical physiological roles across diverse organisms generally by catalyzing a reversible two-step reaction forming acetyl-CoA and AMP from acetate and ATP. Isolated from the wastewater reclamation facility in Norman, Oklahoma, Syntrophus aciditrophicus strain SB (Sa) relies on an AMP-forming acetyl-CoA synthetase (SaAcs1) that favors synthesizing acetate and ATP from acetyl-CoA and AMP, in contrast to all previously characterized Acs enzymes. The origin of this preference and the structural determinants of both the thioester-forming step and catalytic directionality remain poorly understood. Here, we report a 2.2 [A] crystal structure of full-length SaAcs1 in the adenylation conformation with acetyl-AMP bound in the active site. Structural comparison to the extensively characterized Acs enzymes from Salmonella enterica (SeAcs) and Cryptococcus neoformans (CnAcs) revealed a displaced CoA-binding loop in SaAcs1. Enzymatic assays confirmed that SaAcs1 preferentially catalyzes the ATP-forming reaction. Site-directed mutagenesis demonstrated that reversion of two residues, G196 and T197, at the beginning of the CoA-binding loop to the consensus sequence repositions the loop and shifts catalytic preference toward the AMP-forming direction. Together, these results establish the CoA-binding loop and G196 and T197 as the primary structural determinants of directional preference in SaAcs1.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 08 Jul 2026.

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