ACS3

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Acetate is a very abundant short-chain fatty acid in nature, and it is used in many metabolic processes in eukaryotes and prokaryotes. In prokaryotic cells, three different metabolic pathways have evolved to convert this short-chain fatty acid into acetyl-coenzyme A (Ac-CoA). In these pathways, prokaryotes can use acetate as carbon and energy. Among them, one way is to use acetate kinase (AckA, EC 2.7.2.1) /phosphotransacetylase (Pta EC 2.3.1.8) enzymes to activate acetate into Ac-CoA through acetate-phosphate (Ac-P). The second pathway is composed of Ac-CoA synthase (ADP forming), and the third pathway is composed of Ac-CoA synthetase (AMP forming). Acetyl-CoA synthase (ADP forming, EC 6.2.1.1.3) catalyzes the reversible reaction of acetate + ATP      ADP + Pi + Ac-CoA.

Figure 1. Pathways for the conversion of acetate to ac-CoA (Starai, V. J.; Escalante-Semerena, J. C. 2004)

The AMP-forming acetyl-CoA synthetase (Acs) enzyme is conserved in nature

AMP-forming enzymes catalyze the synthesis of products through acyl-AMP intermediates and participate in many anabolic processes. Due to the importance of this enzyme, many enzymologists, molecular biologists, cell physiologists, structural biologists have begun to study this enzyme in depth. Acs orthologs are found in bacteria, some thermophilic and extremely thermophilic archaea, and eukaryotes, including fungi, plants, and humans. Bioinformatics studies have shown that all Acs orthologs have the same ancestry. There are many conserved regions in the entire Acs protein, and they are roughly the same size (~70 kDa). This phenomenon of size conservation indicates that the existing Acs may be the smallest structure required to catalyze the two half-reactions (Figure 1B), and there is no need for a larger enzyme in nature.

Figure 2. Structure of S. enterica Acs (Starai, V. J.; Escalante-Semerena, J. C. 2004)

Synthesis of acetyl-CoA by AMP-forming acetyl-CoA synthetase

Acs catalyzes the synthesis of Ac-CoA through a Bi Uni Uni Bi ping-pong mechanism. The enzyme first binds ATP and then acetate, which results in the formation of enzyme-bound acetyl-AMP and the release of pyrophosphate. Ac-CoA is formed after CoA combines with Acs. The reaction ends with the sequential release of Ac-CoA and AMP. People's understanding of how Acs synthesizes Ac-CoA is still incomplete. Through kinetic and structural analysis of wild-type and mutant Acs and other AMP-forming enzymes isolated from a variety of organisms, the role of this enzyme is gradually revealed.

Regulation of acs gene expression

The transcriptional regulation of acs in the Gram-negative bacterium Escherichia coli is best understood. In E. coli, the acs expression is triggered in the mid-exponential phase, reaches the maximum when the cells enter the stationary phase, and decreases with the increase of culture age. The transcription of acs is inhibited by glucose because it is activated by the Crp (catabolite inhibitory protein) protein in response to rising cyclic AMP (cAMP). In this bacterium, acs is the promoter proximal gene of the three-gene operon, which includes an acetate transporter (encoded by actP) and a gene with unknown function, yjcH. The ActP function is relevant only when the acetate concentration in the environment is very low. The activation of acs by Crp is carried out through a synergistic type III mechanism, in which Crp concentrates the housekeeper s⁷⁰-RNA polymerase on acsP2 to generate a highly efficient open complex. The acs regulatory region contains a proximal (CrpI, center at –69.5) and a distal (CrpII, center at –122.5) Crp binding site. The high-affinity CrpI site is necessary for acs expression, but to achieve optimal expression, the coordinated activation of the two sites is required. Three binding sites for the Fis (factor for inversion stimulation; FisI-III) and Ihf (integration host factor; IhfI-III) proteins are also present in the acs regulatory region.

Posttranslational regulation of Acs activity

The research on the post-translational regulation of Acs has only made progress in recent years. Interestingly, the Sir2 (sirtuin)-dependent protein acetylation/deacetylation system (SDPADS), which is responsible for controlling Acs activity, is also involved in the control of eukaryotic gene expression. From the perspective of metabolism, the most important feature of SDPADS is that it consumes the cofactor NAD⁺, so its activity must be strictly controlled by the cell. Acs is the first metabolic enzyme controlled by SDPADS in prokaryotes and eukaryotes. There is a link between Acs activity (carbon metabolism) and NAD⁺ biosynthesis (energy production). It is speculated that too low NAD⁺ levels may send low-energy charges to cells, leading to a down-regulation of energy expenditure response (such as Acs response). Currently, our understanding of how acetylases and deacetylases interact with their substrates is limited. Since the enzyme is well-characterized in terms of kinetics and the conformation that catalyzes the two half-reactions is also known, Acs can be used as a good model system to learn more about these interactions.