The arginine synthetase pathway is responsible for carbamoyl phosphate synthesis in Thermococcus kodakarensis

Carbamoyl phosphate is considered a precursor for arginine and pyrimidine nucleotide biosynthesis in all domains of life. Carbamoyl phosphate is formed from bicarbonate and ammonia by carbamoyl phosphate synthetase (CPS) at the expense of two ATPs. Although CPS homologs are widespread across archaea, many members of Thermococcales, including Thermococcus kodakarensis, do not harbor these homologs, leaving the source of carbamoyl phosphate unresolved. Here we show that in T. kodakarensis, carbamoyl phosphate is supplied from arginine via the recently identified arginine synthetase pathway. Disruption of arcE, encoding arginine synthetase, or arcB, encoding ornithine transcarbamoylase, caused severe growth defects in pyrimidine-free medium, whereas disruption of arcC, encoding carbamate kinase, had little effect. Under growth conditions dependent on hydrogenase activity, arcC disruption again had little effect, whereas growth of the arcB disruption strain was not observed. Growth was partially restored by heterologous expression of a CPS homolog from Pyrococcus chitonophagus. The same construct also partially restored growth of arcE and arcB disruption strains under pyrimidine-free conditions. Comparative genomic analysis revealed that while pyrimidine biosynthesis and [NiFe]-hydrogenases are universally conserved in Thermococcales, canonical carbamoyl phosphate synthetase is absent from most members, implying that the arginine synthetase pathway serves as the principal source of carbamoyl phosphate in this archaeal order.