A novel ADP-forming succinyl-CoA synthetase in Thermococcus kodakaraensis structurally related to the archaeal nucleoside diphosphate-forming acetyl-CoA synthetases

Abstract

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 (SCSTk) is a heteromeric enzyme (α2β 2) encoded by TK1880 (α-subunit) and TK0943 (β-subunit). Although both SCSTk 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 IPf and IIPf). 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 SCSTk is also a component of the presumed ACS II from T. kodakaraensis (ACS IITk). We coexpressed the α/β-genes of SCSTk (TK1880/TK0943) and of ACS II Tk (TK0139/TK0943). ACS IITk recognizes a broad range of hydrophobic/aromatic acid compounds, as is the case with ACS IIPf, whereas SCSTk 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 SCSTk and ACS IITk. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.