AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Abstract

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.

Figure 1.

Phylogeny of ACS sequences. A phylogeny of putative short and medium chain acyl-CoA synthetases from the finished genome sequences available at NCBI was constructed using the neighbor joining algorithm of MEGA (Kumar et al. 1994). Only the major clade containing the proven ACSs is shown here. For most genera, sequences from only one species were used in constructing the phylogeny for brevity and readability. Eukaryotic sequences are indicated in black, bacterial sequences in red and archaeal sequences in blue.

Figure 2.

Temperature optima for MT1-ACS and AF-ACS2. Enzyme reactions were performed at the indicated temperatures in triplicate. Activities are reported as a percentage of the maximum activity determined for each enzyme. Symbols: filled circles = MT1-ACS; and open circles = AF-ACS2.

Figure 3.

Divalent metal specificity for MT1-ACS and AF-ACS2. Enzyme reactions were performed in triplicate at 65 °C in the presence of 20 mM metal (as the chloride salt) +20 mM ATP. Activities are reported as a percentage of the maximum activity determined for each enzyme with Mg²⁺ as the metal substrate.

Figure 4.

Alignment and ConSurf analysis of ACS sequences. The M. thermauto­trophicus MT-ACS1 and A. ful­gidus AF-ACS2 amino acid sequences were aligned with the S. enterica ACS sequence (SE-ACS) and the ACS sequences from P. aerophilum (PA-ACS), Sulfolobus tokodaii (ST-ACS1 and ACS2), Sulfolobus solfataricus (SS-­ACS), and Sulfolobus acido­caldarius (SA-ACS) using Clustal X (Thompson et al. 1997). A partial alignment is shown here. Residues of the S. enterica ACS found to have high evolutionary conservation scores by ConSurf analysis (Armon et al. 2001, Glaser et al. 2003, Landau et al. 2005) (http://consurf.tau.ac.il) are shaded, as are residues in the other ACS sequences that are identical or among the alternative residues listed for each of these highly conserved positions. Asterisks indicate those positions that are identical in all nine sequences. The acetate binding pocket residues are boldfaced and numbered above the aligned sequences according to their position within MT-ACS1. Those residues at positions with high ConSurf scores that differ in AF-ACS2, PA-ACS, and the Sulfolobus sequences from the three ACS sequences representing enzymes with “traditional” characteristics are indicated in red.

Figure 5.

The acetate binding pocket of MT-ACS1 and AF-ACS2. (A) MT-ACS1 and (B) AF-ACS2 were modeled on the S. enterica ACS structure (PDB: 1PG4) using Accelrys DS Modeler 1.1 and the stereo image of the putative active site acetate binding pocket was created using DS ViewerPro 5.0. Residues within a 10 Å sphere of the propyl moiety of the propyl­phosphate group of the adeno­sine-5’-propylphosphate mimic of the acetyl-adenylate intermediate are shown. The acetate pocket residues (Ile³¹², Thr³¹³, Val³⁸⁸, and Trp⁴¹⁶ of MT-ACS1 and Ile³²⁹, Thr³³⁰, Val⁴⁰⁵, and Trp⁴³³ of AF-ACS2) are colored and labeled. Those residues with high ConSurf scores that differ between MT-ACS1 and AF-ACS2 are shown in orange. Residues discussed in the text are labeled and shown in dark green. The propyl­phos­phate group is shown in aqua.