Role of 4-Hydroxybutyrate-CoA Synthetase in the CO2 Fixation Cycle in Thermoacidophilic Archaea

• Published in: The Journal of biological chemistry Vol. 288; no. 6; pp. 4012 - 4022
• Main Authors: Hawkins, Aaron S., Han, Yejun, Bennett, Robert K., Adams, Michael W.W., Kelly, Robert M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08-02-2013; American Society for Biochemistry and Molecular Biology
• Subjects: Archaea; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Bioenergy; Biofuel; Biotechnology; Carbon Dioxide - metabolism; Catalysis; Catalytic Domain; Coenzyme A-Transferases - chemistry; Coenzyme A-Transferases - genetics; Coenzyme A-Transferases - metabolism; Extreme Thermophiles; Genome, Archaeal - physiology; Hydroxybutyrates - chemistry; Hydroxybutyrates - metabolism; Microbiology; Sulfolobaceae - enzymology; Sulfolobaceae - genetics; Biotechnology; Extreme Thermophiles; Bioenergy; Biofuel; Microbiology; Archaea
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.413195

Metallosphaera sedula is an extremely thermoacidophilic archaeon that grows heterotrophically on peptides and chemolithoautotrophically on hydrogen, sulfur, or reduced metals as energy sources. During autotrophic growth, carbon dioxide is incorporated into cellular carbon via the 3-hydroxypropionate/4-hydroxybutyrate cycle (3HP/4HB). To date, all of the steps in the pathway have been connected to enzymes encoded in specific genes, except for the one responsible for ligation of coenzyme A (CoA) to 4HB. Although several candidates for this step have been identified through bioinformatic analysis of the M. sedula genome, none have been shown to catalyze this biotransformation. In this report, transcriptomic analysis of cells grown under strict H2-CO2 autotrophy was consistent with the involvement of Msed_0406 and Msed_0394. Recombinant versions of these enzymes catalyzed the ligation of CoA to 4HB, with similar affinities for 4HB (Km values of 1.9 and 1.5 mm for Msed_0406 and Msed_0394, respectively) but with different rates (1.69 and 0.22 μmol × min−1 × mg−1 for Msed_0406 and Msed_0394, respectively). Neither Msed_0406 nor Msed_0394 have close homologs in other Sulfolobales, although low sequence similarity is not unusual for acyl-adenylate-forming enzymes. The capacity of these two enzymes to use 4HB as a substrate may have arisen from simple modifications to acyl-adenylate-forming enzymes. For example, a single amino acid substitution (W424G) in the active site of the acetate/propionate synthetase (Msed_1353), an enzyme that is highly conserved among the Sulfolobales, changed its substrate specificity to include 4HB. The identification of the 4-HB CoA synthetase now completes the set of enzymes comprising the 3HP/4HB cycle.
Background: Thermoacidophilic Sulfolobales contain a novel CO2 fixation pathway; all enzymes but one have been accounted for in Metallosphaera sedula.
Results: Enzymes encoded in Msed_0394 and Msed_0406 each exhibit 4-hydroxybutyrate-CoA synthetase activity, consistent with transcriptomic evidence.
Conclusion: Msed_0406 is likely the physiologically relevant enzyme in the cycle.
Significance: All enzymes are now accounted for in the CO2 fixation cycle of M. sedula.