title: Role of 4-hydroxybutyrate-CoA synthetase in the CO2 fixation cycle in thermoacidophilic archaea.
abstract: 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 H(2)-CO(2) 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 (K(m) 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
authors.forename: Aaron S
authors.initials: AS
authors.lastname: Hawkins
authors.forename: Yejun
authors.initials: Y
authors.lastname: Han
authors.forename: Robert K
authors.initials: RK
authors.lastname: Bennett
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D015256:Coenzyme A-Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020745:Genome, Archaeal / Q000502:physiology; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism*; D016943:Sulfolobaceae / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D006885:Hydroxybutyrates; D002245:Carbon Dioxide; C111420:4-hydroxybutyric acid; C072231:4-hydroxybutyrate CoA-transferase; D015256:Coenzyme A-Transferases