A Novel ADP-forming Succinyl-CoA Synthetase in Thermococcus kodakaraensis Structurally Related to the Archaeal Nucleoside Diphosphate-forming Acetyl-CoA Synthetases*

We have identified and characterized a structurally novel succinyl-CoA synthetase (SCS) from the hyperthermophilic Archaea Thermococcus kodakaraensis. The presence of an SCS completes the metabolic pathway from glutamate to succinate in Thermococcales, which had not been clarified because of the absence of classical SCS homologs on their genomes. The SCS from T. kodakaraensis (SCS_Tk) is a heteromeric enzyme (α₂β₂) encoded by TK1880 (α-subunit) and TK0943 (β-subunit). Although both SCS_Tk and classical SCSs harbor the five domains present in enzymes of the acyl-CoA synthetase (nucleoside diphosphate-forming) superfamily, the domain order and distribution among subunits in SCS_Tk (α-subunit, domains 1-2-5; β-subunit, domains 3-4) are distinct from those of classical SCSs (α-subunit, domains 1-2; β-subunit, domains 3-4-5) and instead resemble the acetyl-CoA synthetases from Pyrococcus furiosus (ACSs I_Pf and II_Pf). Comparison of the four Thermococcales genomes revealed that each strain harbors five α- and two β-subunit homologs. Sequence similarity suggests that the β-subunit of SCS_Tk is also a component of the presumed ACS II from T. kodakaraensis (ACS II_Tk). We coexpressed the α/β-genes of SCS_Tk (TK1880/TK0943) and of ACS II_Tk (TK0139/TK0943). ACS II_Tk recognizes a broad range of hydrophobic/aromatic acid compounds, as is the case with ACS II_Pf, whereas SCS_Tk displays a distinct and relatively strict substrate specificity for several acids, including succinate. This indicates that the α-subunits are responsible for the distinct substrate specificities of SCS_Tk and ACS II_Tk.