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

Abstract

Acetyl-coenzyme A (CoA) is a central metabolic intermediate that links carbon and energy metabolism across all domains of life. The interconversion 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 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) thermodynamically favors synthesizing acetate and ATP from acetyl-CoA and AMP using an AMP-forming acetyl-CoA synthetase (SaAcs1). The origin of the preference for AMP formation and the structural determinants of both the thioester-forming step and catalytic bias 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 support 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 catalytic bias in SaAcs1.