Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs

Abstract

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO₂, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (V_max of 8.4 μmol mg^-1 min^-1 and K_m of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (V_max of 0.4 μmol mg^-1 min^-1 and K_m of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar V_max values for both directions (V_max,acetate of 0.9 μmol mg^-1 min^-1 versus V_{max,acetyl-CoA} of 0.3 μmol mg^-1 min^-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with ¹³C₂-labeled acetate produced 3 μmol of [¹³C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]^-1 min^-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Keywords: ANME; acetate ligase; acetate metabolism; aceticlastic methanogenesis; acetyl-CoA synthetase.

FIG 1

Phylogenetic tree based on the protein sequences of acetyl-CoA synthetases (ACSs), for which the characteristics are summarized in Table 1. The tree was generated with MegaX using the maximum likelihood method and the Jones-Taylor-Thornton (JTT) matrix-based model (1,000 ultrafast bootstraps). *, Methanosaeta thermophila is currently known as Methanothrix thermoacetophila.

FIG 2

(A to D) Michaelis-Menten kinetics for acetyl-CoA synthetase (ACS) purified from ANME-2a at the optimal temperature of 50°C for acetate (A) and acetyl-CoA (B) as well as for ACS from ANME-2d at the optimal temperature of 55°C for acetate (C) and acetyl-CoA (D). (E) Michaelis-Menten curve for acetate ligase (ACD) from ANME-2d at the optimal temperature of 60°C for acetyl-CoA conversion. For the reverse reaction using acetate as a substrate, the activity was not detectable using up to 200 μg mL⁻¹ enzyme. The data are shown as averages, with error bars depicting the distributions from two biological replicates. For ACS from ANME-2d, 100 μg enzyme was used; for all other enzymes, 50 μg was used. A curve was fitted through the data using Michaelis-Menten kinetics {V = V_max × [S]/(K_m + [S])} in Excel, from which the K_m and V_max values were calculated (K_m,A = 0.68 mM, V_max,A = 8.4 μmol mg⁻¹ min⁻¹, K_m,B = 0.2 mM, V_max,B = 0.4 μmol mg⁻¹ min⁻¹, K_m,C = 0.2 mM, V_max,C = 0.9 μmol mg⁻¹ min⁻¹, K_m,D = 0.03 mM, V_max,D = 0.3 μmol mg⁻¹ min⁻¹, K_m,E = 0.1 mM, and V_max,E = 0.3 μmol mg⁻¹ min⁻¹).

FIG 3

Batch incubations of an enrichment culture dominated by “Ca. Methanoperedens.” (A) ¹³CH₄ produced under two conditions, with ¹³C-labeled acetate added to the medium (circles) and a negative control without the substrate added (crosses). (B) Total methane concentrations (in micromoles) produced during the experiment shown in panel A. Circles, acetate added; crosses, no acetate added. (C) Total methane (in micromoles) consumed by a “Ca. Methanoperedens” enrichment culture supplied with methane and nitrate as a positive control to validate the activity of the methanotrophic culture.