Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket

Abstract

Acetyl-CoA synthetase (ACS) is a member of a large superfamily of enzymes that display diverse substrate specificities, with a common mechanism of catalyzing the formation of a thioester bond between Coenzyme A and a carboxylic acid, while hydrolyzing ATP to AMP and pyrophosphate. As an activated form of acetate, acetyl-CoA is a key metabolic intermediate that links many metabolic processes, including the TCA cycle, amino acid metabolism, fatty acid metabolism and biosynthetic processes that generate many polyketides and some terpenes. We explored the structural basis of the specificity of ACS for only activating acetate, whereas other members of this superfamily utilize a broad range of other carboxylate substrates. By computationally modeling the structure of the Arabidopsis ACS and the Pseudomonas chlororaphis isobutyryl-CoA synthetase using the experimentally determined tertiary structures of homologous ACS enzymes as templates, we identified residues that potentially comprise the carboxylate binding pocket. These predictions were systematically tested by mutagenesis of four specific residues. The resulting rationally redesigned carboxylate binding pocket modified the size and chemo-physical properties of the carboxylate binding pocket. This redesign successfully switched a highly specific enzyme from using only acetate, to be equally specific for using longer linear (up to hexanoate) or branched chain (methylvalerate) carboxylate substrates. The significance of this achievement is that it sets a precedent for understanding the structure−function relationship of an enzyme without the need for an experimentally determined tertiary structure of that target enzyme, and rationally generates new biocatalysts for metabolic engineering of a broad range of metabolic processes.

Failure to Maintain Acetate Homeostasis by Acetate-Activating Enzymes Impacts Plant Development

“…The Arabidopsis (Arabidopsis thaliana) genome encodes a large number of acyl-activating enzymes (the AAE superfamily) that activate carboxylates through the intermediate, acyl-AMP (EC 6.2.1;Shockey et al, 2003;Shockey and Browse, 2011). Two of these, ACS and AC-ETATE NON-UTILIZING1 (ACN1; Hooks et al, 2004;Turner et al, 2005), are distantly related members of this superfamily, and both preferentially activate acetate to generate acetyl-CoA (Turner, et al, 2005;Lin and Oliver, 2008;Sofeo et al, 2019). ACS is plastid-localized (Kuhn et al, 1981), whereas ACN1 is localized in peroxisomes (Turner, et al, 2005).…”

Engineering precursor pools for increasing production of odd-chain fatty acids inYarrowia lipolytica

“…Its overexpression exhibited a strong negative effect on OCFA accumulation for any substrate compositions in our study, indicating the reaction catalyzed by Acs has a stronger preference for acetate than propionate similar to Acs1p in S. cerevisiae [Van den Gerg et al 1996] , thus resulting in a higher amount of ECFAs than OCFAs. The result suggested the further engineering of Acs such as altering substrate specificity by mutagenesis of carboxylate binding pocket [Sofeo et al 2019] or exchanging native Acs2p with heterologous propionyl-CoA synthetase [Hitschler et al 2020] might be helpful to increase propionyl-CoA/acetyl-CoA ratio.…”

O-Acetyl-L-homoserine production enhanced by pathway strengthening and acetate supplementation in Corynebacterium glutamicum

“…There are many biosynthetic pathways of acetyl-CoA based on different substrates, such as pyruvic acid, acetic acid, and fatty acids [25,26]. Pyruvate forms acetyl-CoA through decarboxylation using the pyruvate dehydrogenase complex (PDH) [27]; acetate forms acetyl-CoA through the reversible Pta-Ack pathway or the irreversible ACS pathway [28][29][30]; fatty acids form acetyl-CoA through β-oxidation [31]. In contrast to glucose, acetate can be converted to acetyl-CoA without carbon loss.…”