title: The smallest active carbamoyl phosphate synthetase was identified in the human gut archaeon Methanobrevibacter smithii.
abstract: The genome of the major intestinal archaeon Methanobrevibacter smithii contains a complex gene system coding for carbamoyl phosphate synthetase (CPSase) composed of both full-length and reduced-size synthetase subunits. These ammonia-metabolizing enzymes could play a key role in controlling ammonia assimilation in M. smithii, affecting the metabolism of gut bacterial microbiota, with an impact on host obesity. In this study, we isolated and characterized the small (41 kDa) CPSase homolog from M. smithii. The gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was purified in one step. Chemical cross-linking and size exclusion chromatography indicated a homodimeric/tetrameric structure, in accordance with a dimer-based CPSase activity and reaction mechanism. This small enzyme, MS-s, synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia and catalyzed the same ATP-dependent partial reactions observed for full-length CPSases. Steady-state kinetics revealed a high apparent affinity for ATP and ammonia. Sequence comparisons, molecular modeling, and kinetic studies suggest that this enzyme corresponds to one of the two synthetase domains of the full-length CPSase that catalyze the ATP-dependent phosphorylations involved in the three-step synthesis of carbamoyl phosphate. This protein represents the smallest naturally occurring active CPSase characterized thus far. The small M. smithii CPSase appears to be specialized for carbamoyl phosphate metabolism in methanogens.
authors.affiliation: Department of Microbiology, Institute of Biology Bucharest, Romanian Academy, Bucharest, Romania.
authors.forename: Elena
authors.initials: E
authors.lastname: Popa
authors.forename: Nirosha
authors.initials: N
authors.lastname: Perera
authors.forename: Csaba Z
authors.initials: CZ
authors.lastname: Kibédi-Szabó
authors.forename: Hedeel
authors.initials: H
authors.lastname: Guy-Evans
authors.forename: David R
authors.initials: DR
authors.lastname: Evans
authors.forename: Cristina
authors.initials: C
authors.lastname: Purcarea
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism; D020134:Catalytic Domain; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D041981:Gastrointestinal Tract / Q000382:microbiology; D019766:Genes, Archaeal; D006801:Humans; D041581:Methanobrevibacter / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010802:Phylogeny; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013045:Species Specificity
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D002221:Carbamyl Phosphate; D000641:Ammonia; D000255:Adenosine Triphosphate; D002222:Carbamoyl-Phosphate Synthase (Ammonia)