Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

Abstract

The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/ 4HB) carbon fixation cycle. This cycle adds two CO 2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining onethird is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyrylCoA dehydrogenase (Msed_0399), and acetoacetyl-CoA ␤-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO 2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO 2 -limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO 2 fixation and central metabolism in M. sedula. Metallosphaera sedula is an extremely thermoacidophilic archaeon (optimum growth temperature, 73°C, and optimum pH, 2.0) that grows heterotrophically on peptides and chemolithoautotrophically on metal sulfides or hydrogen gas (1). For chemolithotrophic growth, it uses a unique carbon fixation pathway known as the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle (2), so far found only in members of the order Sulfolobales. This cycle is one of two such cycles found exclusively in thermophilic archaea, the other being the dicarboxylate/4-hydroxybutyrate (DC/4HB) cycle present in the orders Desulfurococcales and Thermoproteales (3-5). In the first part of the 3HP/4HB cycle, acetyl coenzyme A (CoA) (C 2 ) is converted into succinylCoA (C 4 ) by two successive carboxylation steps (5-7). In the second half of this cycle, succinyl-CoA is then converted to 4HB, which is rearranged and cleaved to produce two molecules of acetyl-CoA. Both the 3HP/4HB and DC/4HB cycles use the same set of enzymes to convert succinyl-CoA to acetyl-CoA. However, the enzymes used in ...

Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO 2 fixation

“…Third, the oxygen stability of 4-hydroxybutyryl-CoA dehydratase that until now has been assumed to require anoxic conditions because of its radical mechanism (33) is a prerequisite for the aerobic operation of the thaumarchaeal HP/HB cycle, lowering enzyme maintenance and turnover costs further. Interestingly, 4-hydroxybutyryl-CoA dehydratase from M. sedula also is robust in the presence of oxygen, as reported during revision of this work (34).…”

Reaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea

“…Third, it was shown that the 3HP/4HB cycle can drive rapid autotrophic growth with a doubling time of less than five hours (Hawkins et al, 2013), suggesting the potential for fast pathway kinetics. Components of the 3HP/4HB cycle can be identified in genomes within the crenarchaeal order Sulfolobales (Kockelkorn and Fuchs, 2009), and has been studied most intensively in the extremely thermoacidophilic archaeon Metallosphaera sedula (T opt = 73°C; pH opt = 2.0) (Auernik and Kelly, 2010; Berg et al, 2010; Hawkins et al, 2014). The cycle can be divided into three sub-pathways to track the reduction of CO 2 into acetyl-CoA.…”

Malonic Semialdehyde Reductase from the Archaeon Nitrosopumilus maritimus Is Involved in the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

“…All genes encoding the enzymes of the crenarchaeal HP/HB cycle were identified in M. sedula, and the corresponding proteins were biochemically characterized (19,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). In contrast, only four enzymes involved in the thaumarchaeal HP/HB cycle were biochemically characterized (13).…”