Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation

Abstract

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.

Importance: Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

FIG 1

Abundance of transcripts and peptides of potential candidates for ATP synthesis by substrate-level phosphorylation in S. aciditrophicus grown under different conditions. (A) Transcript abundance in percentage of total RNA sequences detected in S. aciditrophicus grown in coculture with M. hungatei on benzoate, cyclohexane-1-carboxylate, and crotonate. (B) Peptide abundance in percentage of total peptide sequences detected in S. aciditrophicus grown in coculture with M. hungatei on benzoate, crotonate, and cyclohexane-1-carboxylate and in pure culture on crotonate. (Inset) Peptide abundance in percentage of total peptide sequences detected of annotated phosphate acetyltransferase and acetate kinase gene products in S. wolfei grown on crotonate and butyrate. The accession number for each gene is listed in parentheses after the gene locus tag number as follows: phosphate acetyl-/butyryltransferases, SYN_00653 (WP_011417962.1), SYN_00654 (WP_011417963.1), SYN_01211 (WP_011418341.1), and SYN_01212 (WP_011418342.1); acetate/butyrate kinase, SYN_03090 (WP_011417964.1) and SYN_01210 (WP_011418340.1); acetyl-CoA synthetase (ADP forming), SYN_00049 (WP_011418090.1), SYN_00646 (WP_011417955.1), SYN_00647 (WP_011417956.1), SYN_01949 (WP_011416704.1), SYN_02607 (WP_011416366.1), SYN_02609 (WP_011416368.1), SYN_00748 (WP_011418068.1), SYN_2112 (WP_011416964.1), and SYN_02878 (WP_011417834.1); acetyl-CoA synthetase (AMP forming), SYN_02635 (WP_011418543.1) and SYN_01223 (WP_011418354.1)

FIG 2

Gel filtration chromatography of the acetyl-CoA synthetase activity from cell extracts of S. aciditrophicus grown in pure culture on crotonate. There were two independent cultures (run 1 and run 2). (A) Specific activities of acetyl-CoA synthetases (units per microgram of protein): circles (closed, run 1; open, run 2), AMP-forming, acetyl-CoA synthetase in the acetyl-CoA-forming direction; squares (closed, run 1; open, run 2), AMP-forming, acetyl-CoA synthetase in the acetate-forming direction. (Inset) Specific activities of ADP-dependent, acetyl-CoA synthetase in the acetyl-CoA-forming direction (units per microgram of protein): triangles (closed, run 1; open, run 2). (B) Protein concentration: diamonds (closed, run 1; open, run 2). The amount of acetyl-CoA synthetase activity loaded on the column was 4.2 and 6.8 U, and the total amount of acetyl-CoA synthetase activity after chromatography was 4.8 and 5.6 U for run 1 and run 2, respectively.

FIG 3

Importance of pyrophosphate cycling and Acs1 in the bioenergetics of S. aciditrophicus. Pyrophosphate formed during substrate activation or during biosynthesis is used to make ATP by Acs1. Additional pyrophosphate needed for the Acs1 reaction can be made by membrane-bound pyrophosphatases (red) using ion gradients formed by glutaconyl-CoA decarboxylase (blue) or ATP synthase (yellow).