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.
Highlights
Acetyl-CoA plays a central role in carbon metabolism in the Bacteria, Archaea and Eukarya as an essential intermediate at the junction of various anabolic and catabolic pathways
We report here the biochemical and kinetic characterization of two acetyl-CoA synthetase (ACS) from the archaea Methanothermobacter thermautotrophicus (MT-ACS1) and Archaeoglobus fulgidus (AFACS2)
Analysis of the genome sequence of M. thermautotrophicus ΔH revealed the presence of two putative ACSs, designated here as MTΔH-ACS1 and MTΔH-ACS2
Summary
Acetyl-CoA plays a central role in carbon metabolism in the Bacteria, Archaea and Eukarya as an essential intermediate at the junction of various anabolic and catabolic pathways. The first step of the reaction, which requires acetate and ATP, but not CoA, involves formation of the acetyl-AMP intermediate and release of pyrophosphate (PPi). The ACS is a member of the acyl-adenylate forming enzyme superfamily in which all members undergo a similar two-step reaction mechanism with an enzyme-bound acyl-adenylate intermediate formed in the first step of the reaction Members of this superfamily all catalyze mechanistically similar reactions, they share little identity and similarity in amino acid sequence with the exception of a few signature motifs and conserved core sequence motifs (Babbitt et al 1992, Kleinkauf and Von Dohren 1996, Chang et al 1997, Marahiel et al 1997). Structures for several members of this family have been determined (Conti et al 1996, Conti et al 1997, May et al 2002), but provide little information regarding the active site and catalytic mechanism of ACS, because they catalyze unrelated reactions in which the intermediates serve different functions and share too little homology to allow structural modeling of ACS
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